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Sample records for auto-luminescent genetically-encoded ratiometric

  1. Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.

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    Kenta Saito

    Full Text Available BACKGROUND: Efficient bioluminescence resonance energy transfer (BRET from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. METHODOLOGY/PRINCIPAL FINDINGS: We applied BRET to develop an autoluminescent Ca(2+ indicator, BRAC, which is composed of Ca(2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca(2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca(2+-independent signal drifts due to change in cell shape, focus shift, etc. CONCLUSIONS/SIGNIFICANCE: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca(2+ imaging not only in single live cells but also in small living subjects.

  2. pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.

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    Yunfei Zhang

    Full Text Available We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+ specific; neither Ca(++, Mg(++, Na(+, nor K(+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

  3. Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe▿ †

    OpenAIRE

    Bagar, Tanja; Altenbach, Kirsten; Read, Nick D.; Benčina, Mojca

    2009-01-01

    A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin...

  4. Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator

    OpenAIRE

    Minderer, M; Liu, W.; Sumanovski, L. T.; Kügler, S; Helmchen, F; Margolis, D. J.

    2012-01-01

    Abstract  In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal ...

  5. Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator.

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    Minderer, Matthias; Liu, Wenrui; Sumanovski, Lazar T; Kügler, Sebastian; Helmchen, Fritjof; Margolis, David J

    2012-01-01

    In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal precision, and furthermore, can be measured repeatedly in separate imaging sessions over multiple weeks. This method opens new possibilities for the longitudinal study of stability and plasticity of cortical sensory representations.

  6. Genetically Encoded Voltage Indicators in Circulation Research.

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    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-09-08

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

  7. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

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    Mojca Benčina

    2013-12-01

    Full Text Available Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.

  8. Toward Better Genetically Encoded Sensors of Membrane Potential.

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    Storace, Douglas; Sepehri Rad, Masoud; Kang, BokEum; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J

    2016-05-01

    Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions. Optical signals of membrane potential are useful because membrane potential changes are a direct sign of both synaptic and action potentials. Here we describe recent improvements in the in vitro and in vivo signal size and kinetics of genetically encoded voltage indicators (GEVIs) and discuss their relationship to alternative sensors of neural activity.

  9. A genetically encoded sensor for H2O2 with expanded dynamic range.

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    Markvicheva, Kseniya N; Bilan, Dmitry S; Mishina, Natalia M; Gorokhovatsky, Andrey Yu; Vinokurov, Leonid M; Lukyanov, Sergey; Belousov, Vsevolod V

    2011-02-01

    Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.

  10. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

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    Hanna, David A; Harvey, Raven M; Martinez-Guzman, Osiris; Yuan, Xiaojing; Chandrasekharan, Bindu; Raju, Gheevarghese; Outten, F Wayne; Hamza, Iqbal; Reddi, Amit R

    2016-07-01

    Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines. PMID:27247412

  11. Calibration and functional analysis of three genetically encoded Cl−/pH sensors

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    Marat eMukhtarov

    2013-04-01

    Full Text Available Monitoring of the intracellular concentrations of Cl− and H+ requires sensitive probes that allow reliable quantitative measurements without perturbation of cell functioning. For these purposes the most promising are genetically encoded fluorescent biosensors, which have become powerful tools for non-invasive intracellular monitoring of ions, molecules and enzymatic activity. A ratiometric CFP/YFP-based construct with a relatively good sensitivity to Cl− has been developed (Markova et al., 2008; Waseem et al., 2010. Recently, a combined Cl−/pH sensor (ClopHensor opened the way for simultaneous ratiometric measurement of these two ions (Arosio et al., 2010. ClopHensor was obtained by fusion of a red-fluorescent protein (DsRed-monomer to the E2GFP variant that contains a specific Cl−-binding site. This construct possesses pKa = 6.8 for H+ and Kd in the 40-50 mM range for Cl− at physiological pH (~7.3 As in the majority of cell types the intracellular Cl− concentration ([Cl−]i is about 10 mM, the development of sensors with higher sensitivity is highly desirable. Here we report the intracellular calibration and functional characterization of ClopHensor and its two derivatives: the membrane targeting PalmPalm-ClopHensor and the H148G/V224L mutant with improved Cl− affinity, reduced pH dependence and pKa shifted to more alkaline values. For functional analysis, constructs were expressed in CHO cells and [Cl−]i was changed by using pipettes with different Cl− concentrations during whole-cell recordings. Kd values for Cl− measured at 33°C and pH ~ 7.3 were, respectively, 39 mM, 47 mM and 21 mM for ClopHensor, PalmPalm-ClopHensor and the H148G/V224L mutant. PalmPalm-ClopHensor resolved responses to activation of Cl−-selective glycine receptor channels better than did ClopHensor. Our observations indicate that these different ClopHensor constructs are promising tools for non-invasive measurement of [Cl−]i in various living

  12. Genetically encoded cleavable protein photo-cross-linker.

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    Lin, Shixian; He, Dan; Long, Teng; Zhang, Shuai; Meng, Rong; Chen, Peng R

    2014-08-27

    We have developed a genetically encoded, selenium-based cleavable photo-cross-linker that allows for the separation of bait and prey proteins after protein photo-cross-linking. We have further demonstrated the efficient capture of the in situ generated selenenic acid on the cleaved prey proteins. Our strategy involves tagging the selenenic acid with an alkyne-containing dimethoxyaniline molecule and subsequently labeling with an azide-bearing fluorophore or biotin probe. This cleavage-and-capture after protein photo-cross-linking strategy allows for the efficient capture of prey proteins that are readily accessible by two-dimensional gel-based proteomics and mass spectrometry analysis.

  13. Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids.

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    Hu, C; Wang, J

    2016-01-01

    We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First, we considered the unnatural amino acids and the protein scaffold separately. The scaffold is designed by traditional protein design methods. The unnatural amino acids are inspired by natural structure and organic chemistry methods, and synthesized by either organic chemistry methods or enzymatic conversion. With the increasing number of published unnatural amino acids with various functions, we described an unnatural amino acids toolkit containing metal chelators, redox mediators, and click chemistry reagents. These efforts enable a researcher to search the toolkit for appropriate unnatural amino acids for the study, rather than design and synthesize the unnatural amino acids from the beginning. After the first step, the model enzyme was optimized by computational methods and directed evolution. Lastly, we describe a general method for evolving aminoacyl-tRNA synthetase and expressing unnatural amino acids incorporated into a protein. PMID:27586330

  14. Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

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    Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang; Johnson, Carl Hirschie

    2016-01-01

    "pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator. PMID:27424899

  15. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

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    Potzkei Janko

    2012-03-01

    Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.

  16. Genetically encoded reactive oxygen species (ROS) and redox indicators.

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    Pouvreau, Sandrine

    2014-02-01

    Redox processes are increasingly being recognized as key elements in the regulation of cellular signaling cascades. They are frequently encountered at the frontier between physiological functions and pathological events. The biological relevance of intracellular redox changes depends on the subcellular origin, the spatio-temporal distribution and the redox couple involved. Thus, a key task in the elucidation of the role of redox reactions is the specific and quantitative measurement of redox conditions with high spatio-temporal resolution. Unfortunately, until recently, our ability to perform such measurements was limited by the lack of adequate technology. Over the last 10 years, promising imaging tools have been developed from fluorescent proteins. Genetically encoded reactive oxygen species (ROS) and redox indicators (GERRIs) have the potential to allow real-time and pseudo-quantitative monitoring of specific ROS and thiol redox state in subcellular compartments or live organisms. Redox-sensitive yellow fluorescent proteins (rxYFP family), redox-sensitive green fluorescent proteins (roGFP family), HyPer (a probe designed to measure H2 O2 ), circularly permuted YFP and others have been used in several models and sufficient information has been collected to highlight their main characteristics. This review is intended to be a tour guide of the main types of GERRIs, their origins, properties, advantages and pitfalls.

  17. Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage Indicators

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    Nakajima, Ryuichi; Jung, Arong; Yoon, Bong-June; Baker, Bradley J.

    2016-01-01

    The age of genetically encoded voltage indicators (GEVIs) has matured to the point that changes in membrane potential can now be observed optically in vivo. Improving the signal size and speed of these voltage sensors has been the primary driving forces during this maturation process. As a result, there is a wide range of probes using different voltage detecting mechanisms and fluorescent reporters. As the use of these probes transitions from optically reporting membrane potential in single, cultured cells to imaging populations of cells in slice and/or in vivo, a new challenge emerges—optically resolving the different types of neuronal activity. While improvements in speed and signal size are still needed, optimizing the voltage range and the subcellular expression (i.e., soma only) of the probe are becoming more important. In this review, we will examine the ability of recently developed probes to report synaptic activity in slice and in vivo. The voltage-sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the rhodopsin family of probes are all good at reporting changes in membrane potential, but all have difficulty distinguishing subthreshold depolarizations from action potentials and detecting neuronal inhibition when imaging populations of cells. Finally, we will offer a few possible ways to improve the optical resolution of the various types of neuronal activities. PMID:27547183

  18. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

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    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  19. Genetically encoded molecular tools for light-driven silencing of targeted neurons

    OpenAIRE

    Chow, Brian Y.; Han, Xue; Boyden, Edward S.

    2012-01-01

    The ability to silence, in a temporally precise fashion, the electrical activity of specific neurons embedded within intact brain tissue, is important for understanding the role that those neurons play in behaviors, brain disorders, and neural computations. “Optogenetic” silencers, genetically encoded molecules that, when expressed in targeted cells within neural networks, enable their electrical activity to be quieted in response to pulses of light, are enabling these kinds of causal circuit...

  20. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

    OpenAIRE

    Akerboom, Jasper; Carreras Calderón, Nicole; Tian, Lin; Wabnig, Sebastian; Prigge, Matthias; Tolö, Johan; Gordus, Andrew; Orger, Michael B; Severi, Kristen E.; Macklin, John J.; Patel, Ronak; Pulver, Stefan R.; Trevor J Wardill; Fischer, Elisabeth; Schüler, Christina

    2013-01-01

    This work was supported by Goethe University Frankfurt; Deutsche Forschungsgemeinschaft (DFG), grant EXC115; European Union FP7 grant “EUTrigTreat and German Research Council (DFG) Grant MI 685/2-1. Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red senso...

  1. Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters

    OpenAIRE

    Germond, Arno; Fujita, Hideaki; Ichimura, Taro; Watanabe, Tomonobu M.

    2016-01-01

    Over the past decades many researchers have made major contributions towards the development of genetically encoded (GE) fluorescent sensors derived from fluorescent proteins. GE sensors are now used to study biological phenomena by facilitating the measurement of biochemical behaviors at various scales, ranging from single molecules to single cells or even whole animals. Here, we review the historical development of GE fluorescent sensors and report on their current status. We specifically f...

  2. Photoswitching-free FRAP analysis with a genetically encoded fluorescent tag.

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    Morisaki, Tatsuya; McNally, James G

    2014-01-01

    Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring protein dynamics in live cells that has provided many important biological insights. Although FRAP presumes that the conversion of a fluorophore from a bright to a dark state is irreversible, GFP as well as other genetically encoded fluorescent proteins now in common use can also exhibit a reversible conversion known as photoswitching. Various studies have shown how photoswitching can cause at least four different artifacts in FRAP, leading to false conclusions about various biological phenomena, including the erroneous identification of anomalous diffusion or the overestimation of the freely diffusible fraction of a cellular protein. Unfortunately, identifying and then correcting these artifacts is difficult. Here we report a new characteristic of an organic fluorophore tetramethylrhodamine bound to the HaloTag protein (TMR-HaloTag), which like GFP can be genetically encoded, but which directly and simply overcomes the artifacts caused by photoswitching in FRAP. We show that TMR exhibits virtually no photoswitching in live cells under typical imaging conditions for FRAP. We also demonstrate that TMR eliminates all of the four reported photoswitching artifacts in FRAP. Finally, we apply this photoswitching-free FRAP with TMR to show that the chromatin decondensation following UV irradiation does not involve loss of nucleosomes from the damaged DNA. In sum, we demonstrate that the TMR Halo label provides a genetically encoded fluorescent tag very well suited for accurate FRAP experiments. PMID:25233348

  3. Genetically encoded voltage indicators for large scale cortical imaging come of age.

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    Knöpfel, Thomas; Gallero-Salas, Yasir; Song, Chenchen

    2015-08-01

    Electrical signals are fundamental to cellular sensing, communication and motility. In the nervous system, information is represented as receptor, synaptic and action potentials. Understanding how brain functions emerge from these electrical signals is one of the ultimate challenges in neuroscience and requires a methodology to monitor membrane voltage transients from large numbers of cells at high spatio-temporal resolution. Optical voltage imaging holds longstanding promises to achieve this, and has gained a fresh powerful momentum with the development of genetically encoded voltage indicators (GEVIs). With a focus on neuroimaging studies on intact mouse brains, we highlight recent advances in this field. PMID:26115448

  4. Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses.

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    Jackson, Rachel E; Burrone, Juan

    2016-01-01

    Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses. PMID:27507942

  5. FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

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    Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

    2015-10-01

    Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

  6. FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

    Science.gov (United States)

    Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

    2015-10-01

    Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging. PMID:26184603

  7. Development and properties of genetically encoded pH sensors in plants.

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière

    2013-12-01

    Full Text Available Fluorescent proteins (FPs have given access to a large choice of live imaging techniques and has thereby profoundly modified our view of plant cells. Together with technological improvement of imaging, they have opened the possibility to monitor physico-chemical changes within cells. For this purpose, a new generation of fluorescent proteins has been engineered. For instance, pHluorin, a point mutated version of GFP, allows to get local pH estimates. In this paper, we will describe how genetically encoded sensors can be used to measure pH in the microenvironment of living tissues and subsequently discuss the role of pH in (i exocytosis, (ii ion uptake by plant roots, (iii cell growth and (iv protein trafficking.

  8. A Genetically Encodable System for Sequence-Specific Detection of RNAs in Two Colors.

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    Kellermann, Stefanie J; Rentmeister, Andrea

    2016-05-17

    Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non-fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths. PMID:26919688

  9. KillerRed and miniSOG as genetically encoded photosensitizers for photodynamic therapy of cancer

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    Shirmanova, Marina V.; Serebrovskaya, Ekaterina O.; Snopova, Ludmila B.; Kuznetsova, Maria M.; Ryumina, Alina P.; Turchin, Ilya V.; Sergeeva, Ekaterina A.; Ignatova, Nadezhda I.; Klementieva, Natalia V.; Lukyanov, Konstantin A.; Lukyanov, Sergey A.; Zagaynova, Elena V.

    2013-06-01

    Despite of the success of photodynamic therapy (PDT) in cancer treatment, the problems of low selective accumulation of a photosensitizer in a tumor and skin phototoxicity have not resolved yet. The idea of encoding of a photosensitizer in genome of cancer cells is attractive, particularly because it can provide highly selective light induced cell killing. This work is aimed at the development of new approach to PDT of cancer, namely to using genetically encoded photosensitizers. A phototoxicity of red fluorescent GFP-like protein KillerRed and FMN-binding protein miniSOG was investigated on HeLa tumor xenografts in nude mice. The tumors were generated by subcutaneous injection of HeLa cells stably expressing the phototoxic proteins. The tumors were irradiated with 594 nm or 473 nm laser at 150 mW/cm2 for 20 or 30 min, repeatedly. Fluorescence intensity of the tumors was measured in vivo before and after each treatment procedure. Detailed pathomorphological analysis was performed 24 h after the therapy. On the epi-fluorescence images in vivo photobleaching of both proteins was observed indicating photodynamic reaction. Substantial pathomorphological abnormalities were found in the treated KillerRed-expressing tumor tissue, such as vacuolization of cytoplasm, cellular and nuclear membrane destruction, activation of apoptosis. In contrast, miniSOG-expressing tumors displayed no reaction to PDT, presumably due to the lack of FMN cofactor needed for fluorescence recovery of the flavoprotein. The results are of interest for photodynamic therapy as a proof of possibility to induce photodamages in cancer cells in vivo using genetically encoded photosensitizers.

  10. Controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

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    Zheng, Shun; Kwon, Inchan

    2013-09-01

    Enzyme inhibition plays an important role in drug development, metabolic pathway regulation, and biocatalysis with product inhibition. When an inhibitor has high structural similarities to the substrate of an enzyme, controlling inhibitor binding without affecting enzyme substrate binding is often challenging and requires fine-tuning of the active site. We hypothesize that an extended set of genetically encoded amino acids can be used to design an enzyme active site that reduces enzyme inhibitor binding without compromising substrate binding. As a model case, we chose murine dihydrofolate reductase (mDHFR), substrate dihydrofolate, and inhibitor methotrexate. Structural models of mDHFR variants containing non-natural amino acids complexed with each ligand were constructed to identify a key residue for inhibitor binding and non-natural amino acids to replace the key residue. Then, we discovered that replacing the key phenylalanine residue with two phenylalanine analogs (p-bromophenylalanine (pBrF) and L-2-naphthylalanine (2Nal)) enhances binding affinity toward the substrate dihydrofolate over the inhibitor by 4.0 and 5.8-fold, respectively. Such an enhanced selectivity is mainly due to a reduced inhibitor binding affinity by 2.1 and 4.3-fold, respectively. The catalytic efficiency of the mDHFR variant containing pBrF is comparable to that of wild-type mDHFR, whereas the mDHFR variant containing 2Nal exhibits a moderate decrease in the catalytic efficiency. The work described here clearly demonstrates the feasibility of selectively controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

  11. Reversibly switchable photoacoustic tomography using a genetically encoded near-infrared phytochrome

    Science.gov (United States)

    Yao, Junjie; Kaberniuk, Andrii A.; Li, Lei; Shcherbakova, Daria M.; Zhang, Ruiying; Wang, Lidai; Li, Guo; Verkhusha, Vladislav V.; Wang, Lihong V.

    2016-03-01

    Optical imaging of genetically encoded probes has revolutionized biomedical studies by providing valuable information about targeted biological processes. Here, we report a novel imaging technique, termed reversibly switchable photoacoustic tomography (RS-PAT), which exhibits large penetration depth, high detection sensitivity, and super-resolution. RS-PAT combines advanced photoacoustic imaging techniques with, for the first time, a nonfluorescent photoswitchable bacterial phytochrome. This bacterial phytochrome is the most near-infrared shifted genetically encoded probe reported so far. Moreover, this bacterial phytochrome is reversibly photoconvertible between its far-red and near-infrared light absorption states. Taking maximum advantage of the powerful imaging capability of PAT and the unique photochemical properties of the phytochrome, RS-PAT has broken through both the optical diffusion limit for deep-tissue imaging and the optical diffraction limit for super-resolution photoacoustic microscopy. Specifically, with RS-PAT we have achieved an unprecedented detection sensitivity of ~2 μM, or as few as ~20 tumor cells, at a centimeter depth. Such high sensitivity is fully demonstrated in our study by monitoring tumor growth and metastasis at whole-body level with ~100 μm resolution. Moreover, our microscopic implementation of RS-PAT is capable of imaging mammalian cells with a sub-diffraction lateral resolution of ~140 nm and axial resolution of ~400 nm, which are respectively ~2-fold and ~75-fold finer than those of our conventional photoacoustic microscopy. Overall, RS-PAT is a new and promising imaging technology for studying biological processes at different length scales.

  12. Ratiometric Temperature Sensing with Semiconducting Polymer Dots

    OpenAIRE

    Ye, Fangmao; Wu, Changfeng; Jin, Yuhui; Chan, Yang-Hsiang; Zhang, Xuanjun; Chiu, Daniel T.

    2011-01-01

    This communication describes ultra-bright single-nanoparticle ratiometric temperature sensors based on semiconducting polymer dots (Pdots). We attached the temperature sensitive dye—Rhodamine B (RhB), whose emission intensity decreases with increasing temperature—within the matrix of Pdots. The as-prepared Pdot-RhB nanoparticle showed excellent temperature sensitivity and high brightness because it took advantage of the light harvesting and amplified energy transfer capability of Pdots. More ...

  13. Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements

    Directory of Open Access Journals (Sweden)

    Pauline Vandame

    2014-01-01

    Full Text Available Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i fluorophores and (ii linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.

  14. Dissecting T Cell Contraction In Vivo Using a Genetically Encoded Reporter of Apoptosis

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    Kym R. Garrod

    2012-11-01

    Full Text Available Contraction is a critical phase of immunity whereby the vast majority of effector T cells die by apoptosis, sparing a population of long-lived memory cells. Where, when, and why contraction occurs has been difficult to address directly due in large part to the rapid clearance of apoptotic T cells in vivo. To circumvent this issue, we introduced a genetically encoded reporter for caspase-3 activity into naive T cells to identify cells entering the contraction phase. Using two-photon imaging, we found that caspase-3 activity in T cells was maximal at the peak of the response and was associated with loss of motility followed minutes later by cell death. We demonstrated that contraction is a widespread process occurring uniformly in all organs tested and targeting phenotypically diverse T cells. Importantly, we identified a critical window of time during which antigen encounters act to antagonize T cell apoptosis, supporting a causal link between antigen clearance and T cell contraction. Our results offer insight into a poorly explored phase of immunity and provide a versatile methodology to study apoptosis during the development or function of a variety of immune cells in vivo.

  15. Optogenetic measurement of presynaptic calcium transients using conditional genetically encoded calcium indicator expression in dopaminergic neurons.

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    Carmelo Sgobio

    Full Text Available Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.

  16. Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS.

    Science.gov (United States)

    Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A; Moore, Christina A; Vella, Stephen A; Hortua Triana, Miryam A; Liu, Jing; Garcia, Celia R S; Pace, Douglas A; Moreno, Silvia N J

    2015-11-01

    Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait.

  17. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    Science.gov (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. PMID:26908592

  18. Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

    Science.gov (United States)

    Lee, Sungmoo; Piao, Hong Hua; Sepheri-Rad, Masoud; Jung, Arong; Sung, Uhna; Song, Yoon-Kyu; Baker, Bradley J

    2016-02-04

    Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

  19. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.

    Directory of Open Access Journals (Sweden)

    Xiaokun Shu

    2011-04-01

    Full Text Available Electron microscopy (EM achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator, a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

  20. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    Science.gov (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

  1. A neuron-based screening platform for optimizing genetically-encoded calcium indicators.

    Directory of Open Access Journals (Sweden)

    Trevor J Wardill

    Full Text Available Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc. are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs. Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

  2. EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels.

    Science.gov (United States)

    Schmidt, Moritz J; Fedoseev, Artem; Bücker, Dennis; Borbas, Julia; Peter, Christine; Drescher, Malte; Summerer, Daniel

    2015-12-18

    The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.

  3. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.

    Science.gov (United States)

    Gee, J Michael; Gibbons, Meredith B; Taheri, Marsa; Palumbos, Sierra; Morris, S Craig; Smeal, Roy M; Flynn, Katherine F; Economo, Michael N; Cizek, Christian G; Capecchi, Mario R; Tvrdik, Petr; Wilcox, Karen S; White, John A

    2015-01-01

    Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain. PMID:25926768

  4. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer C Ewald

    Full Text Available Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET. We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

  5. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain

    Directory of Open Access Journals (Sweden)

    Yukiko eMishina

    2014-09-01

    Full Text Available Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviours. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP prototypical design or on the voltage dependent state transitions of microbial opsins.We recently introduced a new VSFP design in which the voltage-sensing domain (VSD is sandwiched between a FRET pair of fluorescent proteins (termed VSFP-Butterflies and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

  6. Genetically encoded Ca2+ indicators; expanded affinity range, color hue and compatibility with optogenetics

    Directory of Open Access Journals (Sweden)

    Takeharu eNagai

    2014-11-01

    Full Text Available Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs such as derivatives of yellow cameleons (YCs and GCaMPs/pericams (Miyawaki et al., 1997; Nakai et al., 2001; Nagai et al., 2001 are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photo-stability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca2+ affinity and Hill constant, have also contributed to increase the detectability of Ca2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.

  7. Genetically-encoded tools for cAMP probing and modulation in living systems.

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov

    2015-09-01

    Full Text Available Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming - all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells, underpin the ensuing limitations of the conventional cAMP assays: 1 genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; 2 inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control – something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.

  8. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

    Directory of Open Access Journals (Sweden)

    Jasper eAkerboom

    2013-03-01

    Full Text Available Genetically encoded calcium indicators (GECIs are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, RCaMPs, engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+] and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2 or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

  9. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation

    Directory of Open Access Journals (Sweden)

    J. Michael eGee

    2015-04-01

    Full Text Available Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb. In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal

  10. Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors.

    Science.gov (United States)

    Hessels, Anne M; Taylor, Kathryn M; Merkx, Maarten

    2016-02-01

    The Zn(2+)-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn(2+) from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn(2+) release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn(2+) concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn(2+), eZinCh-2 (Kd = 1 nM at pH 7.1) and eCALWY-4 (Kd = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn(2+) and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn(2+) in both cytosol and ER, suggesting that Zn(2+) was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn(2+) levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn(2+), nor in experiments in which cytosolic and ER Zn(2+) were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF-ionomycin treatment does not result in significant changes in cytosolic Zn(2+) levels as a result from Zn(2+) release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn(2+) dyes. PMID:26739447

  11. Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells.

    Science.gov (United States)

    Araki, Nobukazu; Ikeda, Yuka; Kato, Takuma; Kawai, Katsuhisa; Egami, Youhei; Miyake, Katsuya; Tsurumaki, Nobuhide; Yamaguchi, Mitsunari

    2014-06-01

    Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

  12. A highly sensitive colorimetric and ratiometric sensor for fluoride ion

    Institute of Scientific and Technical Information of China (English)

    Zhao Wu Xu; Jin Tang; He Tian

    2008-01-01

    A new benzoimidazole-naphthalimide derivative 4 was synthesized and its photophysical properties were studied.This compound showed highly selectively and sensitive colorimetric and ratiometric sensing ability for fluoride anion.

  13. Targeted silver nanoparticles for ratiometric cell phenotyping

    Science.gov (United States)

    Willmore, Anne-Mari A.; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N.; Ruoslahti, Erkki; Braun, Gary B.; Teesalu, Tambet

    2016-04-01

    Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The

  14. Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements

    DEFF Research Database (Denmark)

    Lundby, Alicia; Mutoh, Hiroki; Dimitrov, Dimitar;

    2008-01-01

    Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a g...... of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs....

  15. Imaging voltage in zebrafish as a route to characterizing a vertebrate functional connectome: promises and pitfalls of genetically encoded indicators.

    Science.gov (United States)

    Kibat, Caroline; Krishnan, Seetha; Ramaswamy, Mahathi; Baker, Bradley J; Jesuthasan, Suresh

    2016-06-01

    Neural circuits are non-linear dynamical systems that transform information based on the pattern of input, current state and functional connectivity. To understand how a given stimulus is processed, one would ideally record neural activity across the entire brain of a behaving animal, at cellular or even subcellular resolution, in addition to characterizing anatomical connectivity. Given their transparency and relatively small size, larval zebrafish provide a powerful system for brain-wide monitoring of neural activity. Genetically encoded calcium indicators have been used for this purpose, but cannot directly report hyperpolarization or sub-threshold activity. Voltage indicators, in contrast, have this capability. Here, we test whether two different genetically encoded voltage reporters, ASAP1 and Bongwoori, can be expressed and report activity in the zebrafish brain, using widefield, two-photon and light sheet microscopy. We were unable to express ASAP1 in neurons. Bongwoori, in contrast expressed well, and because of its membrane localization, allowed visualization of axon trajectories in 3D. Bongwoori displayed stimulus-evoked changes in fluorescence, which could be detected in single trials. However, under high laser illumination, puncta on neural membranes underwent spontaneous fluctuations in intensity, suggesting that the probe is susceptible to blinking artefacts. These data indicate that larval zebrafish can be used to image electrical activity in the brain of an intact vertebrate at high resolution, although care is needed in imaging and analysis. Recording activity across the whole brain will benefit from further developments in imaging hardware and indicators. PMID:27328843

  16. Ratiometric Imaging of Extracellular pH in Dental Biofilms

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Dige, Irene

    2016-01-01

    allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6...... of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms...

  17. Resolution improvement of a ratiometric wavelength measurement system by using an optical microfibre coupler

    OpenAIRE

    Wang, Pengfei; Ding, M.; Brambilla, G; Semenova, Y.; Wu, G; Farrell, G.

    2012-01-01

    The application of microfibre couplers in a comb-filter-based ratiometric wavelength measurement system is discussed. The fabrication of microfibre coupler is presented and its temperature-dependent performance is investigated. The resolution of the ratiometric wavelength measurement system with a microfiber coupler is significantly improved in comparison with a conventional ratiometric system to better than 4 pm maintaining the potential for high measurement speed and wide measurable wavelen...

  18. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    Science.gov (United States)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  19. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

    Directory of Open Access Journals (Sweden)

    Alexander S. Goryashchenko

    2015-07-01

    Full Text Available This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds, pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

  20. Ratiometric Fluorescent pH Probes Based on Glycopolymers.

    Science.gov (United States)

    Li, Zhiyun; Zhang, Pengshan; Lu, Wei; Peng, Lun; Zhao, Yun; Chen, Gaojian

    2016-09-01

    Effectively detecting pH changes plays a critical role in exploring cellular functions and determining physiological and pathological processes. A novel ratiometric pH probe based on a glycopolymer, armored with properties of serum-stability, tumor-targeting, and pH monitoring, is designed. Random copolymers of 2-(methacrylamido) glucopyranose and fluorescein O-methacrylate are first synthesized by reversible addition fragmentation chain transfer polymerization. Acryloxyethyl thiocarbamoyl rhodamine B is then attached to the polymer chain to prepare ratiometric fluorescent pH probes via a thiol-ene reaction. The synthesized polymeric probes are characterized by NMR, gel permeation chromatography, UV-vis spectroscopy, and transmission electron microscopy, and the fluorescence responses are examined in phosphate buffer at different pHs. The cytotoxicity and confocal imaging experiments of the probes are detected using HeLa cells, demonstrating a low toxicity and superior biocompatibility for detecting pH changes in bioapplications. PMID:27439338

  1. A ratiometric fluorescent sensor for the mitochondrial copper pool.

    Science.gov (United States)

    Shen, Clara; Kolanowski, Jacek L; Tran, Carmen M-N; Kaur, Amandeep; Akerfeldt, Mia C; Rahme, Matthew S; Hambley, Trevor W; New, Elizabeth J

    2016-09-01

    Copper plays a key role in the modulation of cellular function, defence, and growth. Here we present InCCu1, a ratiometric fluorescent sensor for mitochondrial copper, which changes from red to blue emission in the presence of Cu(i). Employing this probe in microscopy and flow cytometry, we show that cisplatin-treated cells have an impaired ability to accumulate copper in the mitochondria. PMID:27550322

  2. Fluorescent Peptide Beacons for the Selective Ratiometric Detection of Heparin.

    Science.gov (United States)

    Maity, Debabrata; Schmuck, Carsten

    2016-09-01

    Heparin is extensively used as an anticoagulant drug during surgery. Two fluorophore-functionalized cationic oligopeptides HS 1 and HS 2 were developed to monitor heparin ratiometrically in aqueous media. Upon binding to heparin, HS 1 and HS 2 undergo a conformational change from an open form to a folded form, which leads to a distinct change in the fluorescence properties. HS 1 switches from pyrene monomer emission to an excimer emission. For HS 2, a fluorescence resonance energy transfer (FRET) process is enabled between a naphthalene donor and a dansyl acceptor. This method is highly selective for heparin relative to other similar biological analytes such as hyaluronic acid or chondroitin sulfate. HS 1 and HS 2 could also detect heparin ratiometrically in diluted bovine serum. The strong ratiometric emission color change can also be observed by the naked eye. Addition of the polycationic protein protamine releases both HS 1 and HS 2 from their heparin complex, which simultaneously restores pyrene monomer emission for the first case and decreases the FRET process for the latter case, respectively. Dynamic light scattering (DLS) and AFM studies confirm aggregate formation of heparin with HS 1 and HS 2. PMID:27534383

  3. Visual and fluorescent detection of tyrosinase activity by using a dual-emission ratiometric fluorescence probe.

    Science.gov (United States)

    Yan, Xu; Li, Hongxia; Zheng, Weishi; Su, Xingguang

    2015-09-01

    In this work, we designed a dual-emission ratiometric fluorescence probe by hybridizing two differently colored quantum dots (QDs), which possess a built-in correction that eliminates the environmental effects and increases sensor accuracy. Red emissive QDs were embedded in the silica nanoparticle as reference while the green emissive QDs were covalently linked to the silica nanoparticle surface to form ratiometric fluorescence probes (RF-QDs). Dopamine (DA) was then conjugated to the surface of RF-QDs via covalent bonding. The ratiometric fluorescence probe functionalized with dopamine (DA) was highly reactive toward tyrosinase (TYR), which can catalyze the oxidization of DA to dopamine quinine and therefore quenched the fluorescence of the green QDs on the surface of ratiometric fluorescence probe. With the addition of different amounts of TYR, the ratiometric fluorescence intensity of the probe continually varied, leading to color changes from yellow-green to red. So the ratiometric fluorescence probe could be utilized for sensitive and selective detection of TYR activity. There was a good linear relationship between the ratiometric fluorescence intensity and TYR concentration in the range of 0.05-5.0 μg mL(-1), with the detection limit of 0.02 μg mL(-1). Significantly, the ratiometric fluorescence probe has been used to fabricate paper-based test strips for visual detection of TYR activity, which validates the potential on-site application. PMID:26249217

  4. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice

    Directory of Open Access Journals (Sweden)

    Henry Lütcke

    2010-04-01

    Full Text Available Fluorescent calcium (Ca2+ indicator proteins (FCIPs are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60 in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 - in combination with various optical techniques - thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.

  5. Facile synthesis of a ratiometric oxygen nanosensor for cellular imaging.

    Science.gov (United States)

    Lu, Sisi; Xu, Wei; Zhang, Jinliang; Chen, Yiying; Xie, Lei; Yao, Qiuhong; Jiang, Yaqi; Wang, Yiru; Chen, Xi

    2016-12-15

    A new type of cell-penetrating ratiometric fluorescence oxygen sensing nanoparticle was prepared through a facile co-precipitation method. Amphiphilic polymer poly (styrene-co-maleic anhydride) (PSMA) was firstly cooperated with polystyrene (PS) to envelop the highly photostable phosphorescent oxygen indicator, platinum(II)-tetrakis(pentafluorophenyl)porphyrin (PtTFPP, emission at 648nm), and the reference fluorophore, poly(9, 9-dioctylfluorene) (PFO, emission at 440nm ), via hydrophobic interaction in aqueous solution. To improve the sensor biocompatibility, the biomacromolecule poly-l-lysine (PLL) was selected to act as a shell via electrostatic forces. The as-prepared PtTFPP doped core-shell nanoparticles (called PPMA/PLL NPs) exhibited an excellent ratiometric luminescence response to O2 content with high quenching efficiency and full reversibility in the oxygen sensing. More importantly, these oxygen nanosensors passed across the cell membrane after co-incubation without external force. Labeled cells exhibited high brightness in the matching blue and red channels of a digital camera. And most nanosensors were found locating in cytoplasm rather than being trapped in endosomes. PMID:27372571

  6. Synthesis of a new ratiometric emission Ca2+ indicator for in vivo bioimaging

    Institute of Scientific and Technical Information of China (English)

    Qiao-Ling Liu; Meng Fan; Wei Bian; Shao-Min Shuang; Chuan Dong

    2013-01-01

    A novel fluorescent calcium indicator with a 490/582 nm ratiometric emission has been designed and synthesized.The indicator exhibits a highly selective ratiometric emission response to Ca2+ over other metal cations and a large Stokes shift of 202 nm.Moreover,its practical cell imaging capability for intracellular Ca2+ in the resting-and dynamic-state has been demonstrated in human umbilical vein endothelial cells using a confocal laser scanning microscope.

  7. Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

    Science.gov (United States)

    Shirmanova, Marina; Yuzhakova, Diana; Snopova, Ludmila; Perelman, Gregory; Serebrovskaya, Ekaterina; Lukyanov, Konstantin; Turchin, Ilya; Subochev, Pavel; Lukyanov, Sergey; Kamensky, Vladislav; Zagaynova, Elena

    2015-01-01

    The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation. PMID:26657001

  8. Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

    Directory of Open Access Journals (Sweden)

    Marina Shirmanova

    Full Text Available The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm and pulsed laser (584 nm, 10 Hz, 18 ns modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

  9. Ratiometric fluorescence signalling of fluoride ions by an amidophthalimide derivative

    Indian Academy of Sciences (India)

    Moloy Sarkar; Raghavendra Yellampalli; Bhaswati Bhattacharya; Ravi Kumar Kanaparthi; Anunay Samanta

    2007-03-01

    Fluorescence behaviour of 4-benzoylamido-N-methylphthalimide (1), designed and developed for selective detection of fluoride ions, is reported. 1 displays F--induced colour change that allows its detection with the naked eye. The F- specificity of the sensor system is evident from the fact that unlike F-, other halides do not affect the absorption characteristics of 1. Apart from the colorimetric response, the fluorescence output of 1 is also modulated by F- in a manner that permits ratiometric fluorescence signalling of F- as well. It is found that the system can detect F- in the concentration range of 10- 60 M. The results of the experiments and theoretical calculations unambiguously suggest that the changes of the electronic absorption and fluorescence behaviour of 1, which have been exploited for signalling purpose, are due to F--induced deprotonation of the 4-amido moiety of the sensor system.

  10. A Ratiometric Fluorescence Imaging System for Surgical Guidance

    Directory of Open Access Journals (Sweden)

    Brian C. Wilson

    2008-10-01

    Full Text Available A 3-chip CCD imaging system has been developed for quantitative in vivo fluorescence imaging. This incorporates a ratiometric algorithm to correct for the effects of tissue optical absorption and scattering, imaging “geometry” and tissue autofluorescence background. The performance was characterized, and the algorithm was validated in tissue-simulating optical phantoms for quantitative measurement of the fluorescent molecule protoporphyrin IX (PpIX. The technical feasibility to use this system for fluorescence-guided surgical resection of malignant brain tumor tissue was assessed in an animal model in which PpIX was induced exogenously in the tumor cells by systemic administration of aminolevulinic acid (ALA.

  11. Ratiometric Imaging of Extracellular pH in Dental Biofilms.

    Science.gov (United States)

    Schlafer, Sebastian; Dige, Irene

    2016-03-09

    The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

  12. Ratiometric optical fiber sensor for dual sensing of copper ion and dissolved oxygen.

    Science.gov (United States)

    Chu, Cheng-Shane; Chuang, Chih-Yung

    2015-12-20

    This paper develops a new ratiometric optical dual sensor for Cu2+ ions and dissolved oxygen (DO) incorporating a sol-gel matrix doped with palladium tetrakis pentafluorophenyl porphine as the oxygen-sensitive material, CdSe quantum dots as the Cu2+ ion-sensing material, and 7-amino-4-trifluoromethyl coumarin as the Cu2+ /DO practically independent fluorescent dye. The feasibility of coating an optical fiber with the sensing film to fabricate a ratiometric optical fiber dual sensor is investigated. Using an LED with a central wavelength of 405 nm as an excitation source, it is shown that the emission wavelengths of the Cu2+ ion-sensitive, DO-sensitive dye and the reference dye have no spectral overlap and therefore permit Cu2+ ion and DO concentration to be measured using a ratiometric-based method. The ratiometric optical fiber dual sensor has been tested with regard to monitoring different Cu2+ ion (0-10 μM) and DO concentrations (0-38 mg/L). The results show that the luminescence properties of the Cu2+ ion sensor are independent of the presence of the oxygen sensor and have a uniquely good linear response in the 0-10 μM range. The proposed ratiometric sensing approach presented in this study has the advantage of suppressing spurious fluctuations in the intensity of the excitation source. PMID:26837033

  13. Ratiometric fluorescence, electrochemiluminescence, and photoelectrochemical chemo/biosensing based on semiconductor quantum dots

    Science.gov (United States)

    Wu, Peng; Hou, Xiandeng; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-04-01

    Ratiometric fluorescent sensors, which can provide built-in self-calibration for correction of a variety of analyte-independent factors, have attracted particular attention for analytical sensing and optical imaging with the potential to provide a precise and quantitative analysis. A wide variety of ratiometric sensing probes using small fluorescent molecules have been developed. Compared with organic dyes, exploiting semiconductor quantum dots (QDs) in ratiometric fluorescence sensing is even more intriguing, owing to their unique optical and photophysical properties that offer significant advantages over organic dyes. In this review, the main photophysical mechanism for generating dual-emission from QDs for ratiometry is discussed and categorized in detail. Typically, dual-emission can be obtained either with energy transfer from QDs to dyes or with independent dual fluorophores of QDs and dye/QDs. The recent discovery of intrinsic dual-emission from Mn-doped QDs offers new opportunities for ratiometric sensing. Particularly, the signal transduction of QDs is not restricted to fluorescence, and electrochemiluminescence and photoelectrochemistry from QDs are also promising for sensing, which can be made ratiometric for correction of interferences typically encountered in electrochemistry. All these unique photophysical properties of QDs lead to a new avenue of ratiometry, and the recent progress in this area is addressed and summarized here. Several interesting applications of QD-based ratiometry are presented for the determination of metal ions, temperature, and biomolecules, with specific emphasis on the design principles and photophysical mechanisms of these probes.

  14. Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

    Science.gov (United States)

    Mondal, Samsuzzoha; Rakshit, Ananya; Pal, Suranjana; Datta, Ankona

    2016-07-15

    Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.

  15. Economical wireless optical ratiometric pH sensor

    Science.gov (United States)

    Vuppu, Sandeep; Kostov, Yordan; Rao, Govind

    2009-04-01

    The development and application of a portable, wireless fluorescence-based optical pH sensor is presented. The design incorporates the MSP430 microcontroller as the control unit, an RF transceiver for wireless communication, digital filters and amplifiers and a USB-based communication module for data transmission. The pH sensor is based on ratiometric fluorescence detection from pH sensitive dye incorporated in a peel-and-stick patch. The ability of the instrument to detect the pH of the solution with contact only between the sensor patch and the solution makes it partially non-invasive. The instrument also has the ability to transmit data wirelessly, enabling its use in processes that entail stringent temperature control and sterility. The use of the microcontroller makes it a reliable, low-cost and low-power device. The luminous intensity of the light source can be digitally controlled to maximize the sensitivity of the instrument. It has a resolution of 0.05 pH. The sensor is accurate and reversible over the pH range of 6.5-9.

  16. Ratiometric fluorescent nanosensors for selective detecting cysteine with upconversion luminescence.

    Science.gov (United States)

    Guan, Yunlong; Qu, Songnan; Li, Bin; Zhang, Liming; Ma, Heping; Zhang, Ligong

    2016-03-15

    Fluorescent sensors based on upconversion (UC) luminescence have been considered as a promising strategy to detect bio-analyte due to their advantages in deep penetration, minimum autofluorescence, and ratiometric fluorescent output. A prototype of nanosensors combined with mesoporous silica coated upconversion nanoparticles (UCNPs) and a fluorescein-based fluorescent probe loaded in pores was therefore designed to detect cysteine (Cys). The silica shell provided loading space for the probe and enabled the nanosensors to disperse in water. In the presence of Cys, the fluorescent probe was transformed into 5(6)-carboxyfluorescein with an emission band centering at 518 nm which was secondarily excited by the light at around 475 nm from NaYF4:Yb(3+), Tm(3+) UCNPs driven by 980 nm near-infrared (NIR) laser. The intensity ratio between green and blue luminescence (I518/I475) grew exponentially with increasing concentrations of Cys over a range of 20-200 μmolL(-1). The response of the nanosensors towards Cys was recognizable with naked eyes by luminescence color change. Evidences suggest that these nanosensors are capable of sensing Cys in aqueous solution and distinguishing Cys from homocysteine (Hcy) with kinetically-controlled selectivity. The system was further employed to detect Cys in human serum and the result was in agreement with it tested by high performance liquid chromatography with acceptable recovery. PMID:26402589

  17. Economical wireless optical ratiometric pH sensor

    International Nuclear Information System (INIS)

    The development and application of a portable, wireless fluorescence-based optical pH sensor is presented. The design incorporates the MSP430 microcontroller as the control unit, an RF transceiver for wireless communication, digital filters and amplifiers and a USB-based communication module for data transmission. The pH sensor is based on ratiometric fluorescence detection from pH sensitive dye incorporated in a peel-and-stick patch. The ability of the instrument to detect the pH of the solution with contact only between the sensor patch and the solution makes it partially non-invasive. The instrument also has the ability to transmit data wirelessly, enabling its use in processes that entail stringent temperature control and sterility. The use of the microcontroller makes it a reliable, low-cost and low-power device. The luminous intensity of the light source can be digitally controlled to maximize the sensitivity of the instrument. It has a resolution of 0.05 pH. The sensor is accurate and reversible over the pH range of 6.5–9

  18. Fluorescence Ratiometric Properties Induced by Nanoparticle Plasmonics and Nanoscale Dye Dynamics

    Directory of Open Access Journals (Sweden)

    Aron Hakonen

    2013-01-01

    Full Text Available Nanoscale transport of merocyanine 540 within/near the plasmon field of gold nanoparticles was recognized as an effective inducer of single-excitation dual-emission ratiometric properties. With a high concentration of the signal transducer (ammonium, a 700% increase in fluorescence was observed at the new red-shifted emission maximum, compared to a nanoparticle free sensor membrane. A previously nonrecognized isosbestic point is demonstrated at  nm. The mechanism can be utilized for enhanced and simplified ratiometric optical chemical sensors and potentially for thin film engineering to make solar cells more effective and stable by a broader and more regulated absorption.

  19. Structural analysis of the PSD-95 cluster by electron tomography and CEMOVIS: a proposal for the application of the genetically encoded metallothionein tag.

    Science.gov (United States)

    Hirabayashi, Ai; Fukunaga, Yuko; Miyazawa, Atsuo

    2014-06-01

    Postsynaptic density-95 (PSD-95) accumulates at excitatory postsynapses and plays important roles in the clustering and anchoring of numerous proteins at the PSD. However, a detailed ultrastructural analysis of clusters exclusively consisting of PSD-95 has never been performed. Here, we employed a genetically encoded tag, three tandem repeats of metallothionein (3MT), to study the structure of PSD-95 clusters in cells by electron tomography and cryo-electron microscopy of vitreous sections. We also performed conventional transmission electron microscopy (TEM). Cultured hippocampal neurons expressing a fusion protein of PSD-95 coupled to 3MT (PDS-95-3MT) were incubated with CdCl2 to result in the formation of Cd-bound PSD-95-3MT. Two types of electron-dense deposits composed of Cd-bound PSD-95-3MT were observed in these cells by TEM, as reported previously. Electron tomography revealed the presence of membrane-shaped structures representing PSD-95 clusters at the PSD and an ellipsoidal structure located in the non-synaptic cytoplasm. By TEM, the PSD-95 clusters appeared to be composed of a number of dense cores. In frozen hydrated sections, these dense cores were also found beneath the postsynaptic membrane. Taken together, our findings suggest that dense cores of PSD-95 aggregate to form the larger clusters present in the PSD and the non-synaptic cytoplasm.

  20. Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display.

    Science.gov (United States)

    Guntas, Gurkan; Lewis, Steven M; Mulvaney, Kathleen M; Cloer, Erica W; Tripathy, Ashutosh; Lane, Thomas R; Major, Michael B; Kuhlman, Brian

    2016-01-01

    In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.

  1. Design of modular "plug-and-play" expression platforms derived from natural riboswitches for engineering novel genetically encodable RNA regulatory devices.

    Science.gov (United States)

    Trausch, Jeremiah J; Batey, Robert T

    2015-01-01

    Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA leaders and regulating their expression. These regulatory elements are generally composed of two domains: an aptamer that binds a specific effector molecule and an expression platform that informs the transcriptional or translational machinery. While it was long established that riboswitch aptamers are modular and portable, capable of directing different output domains including ribozymes, switches, and fluorophore-binding modules, the same has not been demonstrated until recently for expression platforms. We have engineered and validated a set of expression platforms that regulate transcription through a secondary structural switch that can host a variety of different aptamers, including those derived through in vitro selection methods, to create novel chimeric riboswitches. These synthetic switches are capable of a highly specific regulatory response both in vitro and in vivo. Here we present the methodology for the design and engineering of chimeric switches using biological expression platforms. PMID:25605380

  2. Unconventional ratiometric-enhanced optical sensing of oxygen by mixed-phase TiO2

    Science.gov (United States)

    Lettieri, S.; Pallotti, D. K.; Gesuele, F.; Maddalena, P.

    2016-07-01

    We show that mixed-phase titanium dioxide (TiO2) can be effectively employed as an unconventional, inorganic, dual-emitting, and ratiometric optical sensor of O2. Simultaneous availability of rutile and anatase TiO2 photoluminescence (PL) and their peculiar "anti-correlated" PL responses to O2 allow using their ratio as a measurement parameter associated with the O2 concentration, leading to an experimental responsivity being by construction larger than the one obtainable for single-phase PL detection. A proof of this concept is given, showing a two-fold enhancement of the optical responsivity provided by the ratiometric approach. Besides the peculiar ratiometric-enhanced responsivity, other characteristics of mixed phase TiO2 can be envisaged as favorable for O2 optical probing, namely (a) low production costs, (b) absence of heterogeneous components, and (c) self-supporting properties. These characteristics encourage experimenting with its use for applications requiring high indicator quantities at a competitive price, possibly also tackling the need to develop supporting matrixes that carry the luminescent probes and avoiding issues related to the use of different components for ratiometric sensing.

  3. A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity.

    Science.gov (United States)

    Mallick, Suman; Chandra, Falguni; Koner, Apurba L

    2016-02-01

    An ultrasensitive ratiometric fluorescent sensor made of an N,N-dimethylaminonaphthalene anhydride moiety for detection of aliphatic primary amines is reported. Biogenic amines at nanomolar concentration is detected with the additional ability to discriminate between primary, secondary and tertiary amines by using both UV-Visible and fluorescence spectroscopy. PMID:26734688

  4. Ratiometric electrochemiluminescent strategy regulated by electrocatalysis of palladium nanocluster for immunosensing.

    Science.gov (United States)

    Huang, Yin; Lei, Jianping; Cheng, Yan; Ju, Huangxian

    2016-03-15

    This work designed a novel ratiometric electrochemiluminescence (ECL) immunosensing approach based on two different ECL emitters: CdS quantum dots (QDs) as cathodic emitter and luminol as anodic emitter. The ECL immunosensor was constructed by a layer-by-layer modification of CdS QDs, Au nanoparticles and capture antibody on a glassy carbon electrode. With hydrogen peroxide as ECL coreactant, the immunosensor showed a cathodic ECL emission of CdS QDs at -1.5 V (vs Ag/AgCl) in air-saturated pH 8.0 buffer. Upon the formation of sandwich immunoassay, the lumiol/palladium nanoclusters (Pd NCs)@graphene oxide probe was introduced to the electrode. Therefore, the cathodic ECL intensity decreased and luminol anodic ECL emission was appeared at +0.3 V (vs Ag/AgCl) owing to the competition of the coreactant of hydrogen peroxide. Using carcino-embryonic antigen as model, this ratiometric ECL strategy could be used for immunoassay with a linear range of 1.0-100 pg mL(-1) and a detection limit of 0.62 pg mL(-1). The enhanced ratiometric ECL signal resulted from the high density and excellent electrocatalysis of the loaded Pd NCs. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing a great promising for clinical application. This electrocatalysis-regulated ratiometric ECL provides a new concept for ECL measurement, and could be conveniently extended for detection of other protein biomarkers. PMID:26499869

  5. Preassembly-driven ratiometric sensing of H2PO4(-) anions in organic and aqueous environments.

    Science.gov (United States)

    Gong, Wei-tao; Na, Duo; Fang, Le; Mehdi, Hassan; Ning, Gui-ling

    2015-02-21

    Gemini surfactant-like receptor is designed and synthesized. The special preassembly phenomenon of in a nonpolar solvent facilitates the novel ratiometric fluorescence sensing of H2PO4(-)via an anion-induced reassembly process in organic solvents and an anion-induced disassembly process in water. PMID:25563510

  6. Ratiometric fluorescence sensing of sugars via a reversible disassembly and assembly of the peptide aggregates mediated by sugars.

    Science.gov (United States)

    Neupane, Lok Nath; Han, Song Yee; Lee, Keun-Hyeung

    2014-06-01

    An amphiphilic dipeptide (1) bearing pyrene and phenylboronic acid was demonstrated as a unique example of a ratiometric sensing system for sugars by reversibly converting the peptide aggregates into the monomer form of the complex with sugars in aqueous solutions.

  7. Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution

    KAUST Repository

    Quitterer, Felix

    2012-12-01

    The second step in the biosynthesis of the 22nd genetically encoded amino acid pyrrolysine (Pyl) is catalyzed by PylC that forms the pseudopeptide l-lysine-Nε-3R-methyl-d-ornithine. Here, we present six crystal structures of the monomeric active ligase in complex with substrates, reaction intermediates, and products including ATP, the non-hydrolyzable ATP analogue 5′-adenylyl-β-γ-imidodiphosphate, ADP, d-ornithine (d-Orn), l-lysine (Lys), phosphorylated d-Orn, l-lysine-Nε-d-ornithine, inorganic phosphate, carbonate, and Mg2 +. The overall structure of PylC reveals similarities to the superfamily of ATP-grasp enzymes; however, there exist unique structural and functional features for a topological control of successive substrate entry and product release. Furthermore, the presented high-resolution structures provide detailed insights into the reaction mechanism of isopeptide bond formation starting with phosphorylation of d-Orn by transfer of a phosphate moiety from activated ATP. The binding of Lys to the enzyme complex is then followed by an SN2 reaction resulting in l-lysine-Nε-d-ornithine and inorganic phosphate. Surprisingly, PylC harbors two adenine nucleotides bound at the active site, what has not been observed in any ATP-grasp protein analyzed to date. Whereas one ATP molecule is involved in catalysis, the second adenine nucleotide functions as a selective anchor for the C- and N-terminus of the Lys substrate and is responsible for protein stability as shown by mutagenesis. © 2012 Elsevier Ltd.

  8. Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

    Science.gov (United States)

    Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

    2014-01-01

    The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

  9. A near-Infrared Fluorescent Chemodosimeter for Ratiometric Detecting Fluoride Based on Desilylation Reaction.

    Science.gov (United States)

    Xie, Puhui; Guo, Fengqi; Gao, Guangqin; Fan, Wei; Yang, Guoyu; Xie, Lixia

    2016-09-01

    A new chemodosimeter based on dicyanomethylene-4H-chromene chromophore (probe 1) was developed as a ratiometric fluorescent probe in near-infrared range for F(-) with good selectivity in acetonitrile. Probe 1 could be used to directly visualize F(-) by the naked eye and showed more than 621-fold fluorescence enhancement at 715 nm upon reaction with F(-) upon excitation at 625 nm. The recognition of probe 1 to fluoride was featured by F(-)-induced red-shifts of both absorption (185 nm) and fluorescence peaks (132 nm) based on internal charge transfer (ICT) in acetonitrile. The desilylation reaction of 1 by F(-) was proposed for its dual absorption and emission ratiometric detection of fluoride. PMID:27365125

  10. Ratiometric and near-infrared molecular probes for the detection and imaging of zinc ions.

    Science.gov (United States)

    Carol, Priya; Sreejith, Sivaramapanicker; Ajayaghosh, Ayyappanpillai

    2007-03-01

    The detection and imaging of Zn2+ in biological samples are of paramount interest owing to the role of this cation in physiological functions. This is possible only with molecular probes that specifically bind to Zn2+ and result in changes in emission properties. A "turn-on" emission or shift in the emission color upon binding to Zn2+ should be ideal for in vivo imaging. In this context, ratiometric and near-IR probes are of particular interest. Therefore, in the area of chemosensors or molecular probes, the design of fluorophores that allow ratiometric sensing or imaging in the near-IR region is attracting the attention of chemists. The purpose of this Focus Review is to highlight recent developments in this area and stress the importance of further research for future applications.

  11. Ratiometric Imaging of Extracellular pH in Dental Biofilms Using C-SNARF-4

    DEFF Research Database (Denmark)

    Dige, Irene

    pH in dental biofilms plays a central role for the development of caries lesions. For decades, pH measurements in biofilms have been limited to recording pH with electrodes/microelectrodes that do not permit monitoring horizontal pH gradients in biofilms in real-time. Quantitative fluorescent...... microscopy can overcome these problems. Objective: The aim of this demonstration study was to monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4 in in-situ-grown dental biofilms. Methods: Using confocal microscopy, the dye C-SNARF-4 was employed both as p......H-sensitive ratiometric dye and as a bacterial stain. We tested the method on natural 48-h in-situ-grown dental biofilms from two individuals. Four biofilms per person were collected on standardized glass slabs mounted in intra-oral appliances. Digital image analysis was employed to remove the bacterial biomass from...

  12. A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform.

    Science.gov (United States)

    Duan, Yu-Wei; Yang, Xiao-Feng; Zhong, Yaogang; Guo, Yuan; Li, Zheng; Li, Hua

    2015-02-15

    A ratiometric fluorescent probe for H2S was developed based on a coumarin- benzopyrylium platform. The ratiometric sensing is realized by a selective conversion of acyl azide to the corresponding amide, which subsequently undergoes an intramolecular spirocyclization to alter the large π-conjugated system of CB fluorophore. Compared with the traditional azide-based H2S probes, the proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid response (∼1min) towards H2S, which is superior to most of the azide-based H2S probes. Preliminary fluorescence imaging experiments show that probe 1 has potential to track H2S in living cells.

  13. Trihydroxytrioxatriangulene - An Extended Fluorescein and a Ratiometric pH Sensor

    DEFF Research Database (Denmark)

    Westerlund, Fredrik; Hildebrandt, Christoffer Boli; Sørensen, Thomas Just;

    2010-01-01

    Fluorescein ver. 2.0: A new, highly fluorescent, pH-sensitive trihydroxytrioxatriangulenium dye (H-TOTA) has been synthesised and characterised. The dye is closely related to fluorescein and may be considered to be a two-dimensional extended version. This new dye can exist in four different proto...... protonation states (see graphic) depending on the pH, and its use as a sensitive fluorescent ratiometric pH probe in a physiological buffer is demonstrated....

  14. Peptide-Based, Two-Fluorophore, Ratiometric Probe for Quantifying Mobile Zinc in Biological Solutions

    OpenAIRE

    Zhang, Daniel Y.; Azrad, Maria; Demark-Wahnefried, Wendy; Frederickson, Christopher J.; Lippard, Stephen J.; Radford, Robert J.

    2014-01-01

    Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe...

  15. A simple ratiometric and colorimetric chemosensor for the selective detection of fluoride in DMSO buffered solution

    Science.gov (United States)

    Niu, Hu; Shu, Qinghai; Jin, Shaohua; Li, Bingjun; Zhu, Jiaping; Li, Lijie; Chen, Shusen

    2016-01-01

    A derivative of squaramide (cyclobuta[b]quinoxaline-1, 2(3H, 8H)-dione) has been synthesized for the ratiometric and colorimetric sensing of F- in aqueous solution in competitive fashion. With F-, probe 1 showed a highly selective naked-eye detectable color change along with a characteristic UV-Vis absorbance over other tested ions, which probably originates from the deprotonation occurred between 1 and F-, as proved by the 1H NMR titration experiments and DFT calculations.

  16. A quinoline based pH sensitive ratiometric fluorescent sensor: Structure and spectroscopy

    Indian Academy of Sciences (India)

    Soma Mukherjee; Amit Kumar Paul; Helen Stoeckli-Evans

    2015-09-01

    A new quinoline based hydrazone was synthesized via a condensation reaction and characterized by NMR, mass and single crystal X-ray diffraction studies. It was investigated for suitability as a reversible ratiometric fluorescent pH sensor in acidic pH region. The sensor exhibits intramolecular charge transfer (ICT) type photophysical changes upon protonation of the quinoline ring. No significant interference on emission behavior was observed in the presence of various metal ions.

  17. A highly sensitive ratiometric fluorescent probe for the detection of cytoplasmic and nuclear hydrogen peroxide.

    Science.gov (United States)

    Wen, Ying; Liu, Keyin; Yang, Huiran; Li, Yi; Lan, Haichuang; Liu, Yi; Zhang, Xinyu; Yi, Tao

    2014-10-01

    As a marker for oxidative stress and a second messenger in signal transduction, hydrogen peroxide (H2O2) plays an important role in living systems. It is thus critical to monitor the changes in H2O2 in cells and tissues. Here, we developed a highly sensitive and versatile ratiometric H2O2 fluorescent probe (NP1) based on 1,8-naphthalimide and boric acid ester. In response to H2O2, the ratio of its fluorescent intensities at 555 and 403 nm changed 1020-fold within 200 min. The detecting limit of NP1 toward H2O2 is estimated as 0.17 μM. It was capable of imaging endogenous H2O2 generated in live RAW 264.7 macrophages as a cellular inflammation response, and especially, it was able to detect H2O2 produced as a signaling molecule in A431 human epidermoid carcinoma cells through stimulation by epidermal growth factor. This probe contains an azide group and thus has the potential to be linked to various molecules via the click reaction. After binding to a Nuclear Localization Signal peptide, the peptide-based combination probe (pep-NP1) was successfully targeted to nuclei and was capable of ratiometrically detecting nuclear H2O2 in living cells. These results indicated that NP1 was a highly sensitive ratiometric H2O2 dye with promising biological applications.

  18. A novel, cell-permeable, fluorescent probe for ratiometric imaging of zinc ion.

    Science.gov (United States)

    Maruyama, Satoko; Kikuchi, Kazuya; Hirano, Tomoya; Urano, Yasuteru; Nagano, Tetsuo

    2002-09-11

    Zn(2+) plays important roles in various biological systems; as a result, the development of tools that can visualize chelatable Zn(2+) has attracted much attention recently. We report here newly synthesized fluorescent sensors for Zn(2+), ZnAF-Rs, whose excitation maximum is shifted by Zn(2+) under physiological conditions. Thus, these sensors enable ratiometric imaging, which is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a probe. Ratiometric measurement can provide precise data, and some probes allow quantitative detection. ZnAF-Rs are the first ratiometric fluorescent sensors for Zn(2+) that enable quantitative analysis under physiological conditions. ZnAF-Rs also possess suitable K(d) for applications, and high selectivity against other biologically relevant cations, especially Ca(2+). Using these probes, changes of intracellular Zn(2+) concentration in cultured cells were monitored successfully. We believe that these probes will be extremely useful in studies on the biological functions of Zn(2+).

  19. Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

    Science.gov (United States)

    Huynh Nhat, Kim Phuong; Watanabe, Takayoshi; Yoshikoshi, Kensuke; Hohsaka, Takahiro

    2016-08-01

    Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules. PMID:26896314

  20. An effective colorimetric and ratiometric fluorescent probe for bisulfite in aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Xi [Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Zhang, Tao [Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100 (China); Du, Zhi-Fang; Cao, Xiang-Jian; Chen, Ming-Yu [Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Taishan College, Shandong University, Jinan 250100 (China); Hu, Sheng-Wen [Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Miao, Jun-Ying, E-mail: miaojy@sdu.edu.cn [Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100 (China); Zhao, Bao-Xiang, E-mail: bxzhao@sdu.edu.cn [Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-08-12

    We have developed the first two-photon colorimetric and ratiometric fluorescent probe, BICO, for the detection of bisulfite (HSO{sub 3}{sup −}) in aqueous solution. The probe contains coumarin and benzimidazole moieties and can detect HSO{sub 3}{sup −} based on the Michael addition reaction with a limit of detection 5.3 × 10{sup −8} M in phosphate-buffered saline solution. The probe was used to detect bisulfite in tap water, sugar and dry white wine. Moreover, test strips were made and used easily. We successfully applied the probe to image living cells, using one-photon fluorescence imaging. BICO overcomes the limitations in sensitivity of previously reported probes and the solvation effect of bisulfite, which demonstrates its excellent value in practical application. - Highlights: • A colorimetric and ratiometric fluorescent probe was developed. • The probe could detect bisulfite in PBS buffer solution and real samples. • Bisulfite test paper was made to naked-eye detect bisulfite. • This probe successfully used to living cell imaging in ratiometric manner.

  1. A novel ratiometric two-photon fluorescent probe for imaging of Pd(2+) ions in living cells and tissues.

    Science.gov (United States)

    Zhou, Liyi; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd(2+) in biosystems, herein, we reported a ratiometric two-photon fluorescent probe, termed as Np-Pd, which based on a D-π-A-structure two-photon fluorophore of the naphthalimide derivative and deprotection of aryl propargyl ethers by palladium species. The probe Np-Pd displayed a more than 25-fold enhancement towards palladium species with high sensitivity and selectivity. Additionally, the probe Np-Pd was further used for fluorescence imaging of Pd(2+) ions in living cells and tissues under two-photon excitation (820nm), which showed large tissue-imaging depth (19.6-184.6μm), and a high resolution for ratiometric imaging. PMID:27203231

  2. Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

    Directory of Open Access Journals (Sweden)

    Xinyue Zhu

    2015-01-01

    Full Text Available A reaction-based two-photon (TP ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM.

  3. A novel ratiometric two-photon fluorescent probe for imaging of Pd2 + ions in living cells and tissues

    Science.gov (United States)

    Zhou, Liyi; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd2 + in biosystems, herein, we reported a ratiometric two-photon fluorescent probe, termed as Np-Pd, which based on a D-π-A-structure two-photon fluorophore of the naphthalimide derivative and deprotection of aryl propargyl ethers by palladium species. The probe Np-Pd displayed a more than 25-fold enhancement towards palladium species with high sensitivity and selectivity. Additionally, the probe Np-Pd was further used for fluorescence imaging of Pd2 + ions in living cells and tissues under two-photon excitation (820 nm), which showed large tissue-imaging depth (19.6-184.6 μm), and a high resolution for ratiometric imaging.

  4. A Three-Photon Active Organic Fluorophore for Deep Tissue Ratiometric Imaging of Intracellular Divalent Zinc.

    Science.gov (United States)

    Philips, Divya Susan; Sreejith, Sivaramapanicker; He, Tingchao; Menon, Nishanth Venugopal; Anees, Palapuravan; Mathew, Jomon; Sajikumar, Sreedharan; Kang, Yuejun; Stuparu, Mihaiela Corina; Sun, Handong; Zhao, Yanli; Ajayaghosh, Ayyappanpillai

    2016-05-20

    Deep tissue bioimaging with three-photon (3P) excitation using near-infrared (NIR) light in the second IR window (1.0-1.4 μm) could provide high resolution images with an improved signal-to-noise ratio. Herein, we report a photostable and nontoxic 3P excitable donor-π-acceptor system (GMP) having 3P cross-section (σ3 ) of 1.78×10(-80)  cm(6)  s(2)  photon(-2) and action cross-section (σ3 η3 ) of 2.31×10(-81)  cm(6)  s(2)  photon(-2) , which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn(2+) binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ3 of 1.85×10(-80)  cm(6)  s(2)  photon(-2) and σ3 η3 of 3.33×10(-81)  cm(6)  s(2)  photon(-2) . The application of this probe is demonstrated for ratiometric 3P imaging of Zn(2+) in vitro using HuH-7 cell lines. Furthermore, the Zn(2+) concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn(2+) ion imaging. PMID:26991763

  5. Synthesis and characterization of ratiometric nanosensors for pH quantification: a mixed micelle approach

    DEFF Research Database (Denmark)

    Ek, Pramod Kumar; Almdal, Kristoffer; Andresen, Thomas Lars

    2012-01-01

    Optical nanoparticle pH sensors designed for ratiometric measurements have previously been synthesized using post-functionalization approaches to introduce sensor molecules and to modify nanoparticle surface chemistry. This strategy often results in low control of the nanoparticle surface chemistry...... and is prone to batch-to-batch variations, which is undesirable for succeeding sensor calibrations and cellular measurements. Here we provide a new synthetic approach for preparing nanoparticle pH sensors based on self-organization principles, which in comparison to earlier strategies offers a much higher...... design flexibility and high control of particle size, morphology and surface chemistry....

  6. Dual core quantum dots for highly quantitative ratiometric detection of trypsin activity in cystic fibrosis patients

    Science.gov (United States)

    Castelló Serrano, Iván; Stoica, Georgiana; Matas Adams, Alba; Palomares, Emilio

    2014-10-01

    We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help.We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for

  7. Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

    Science.gov (United States)

    Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

    2016-08-01

    Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels. PMID:27334762

  8. A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues.

    Science.gov (United States)

    Jin, Qiang; Feng, Lei; Wang, Dan-Dan; Dai, Zi-Ru; Wang, Ping; Zou, Li-Wei; Liu, Zhi-Hong; Wang, Jia-Yue; Yu, Yang; Ge, Guang-Bo; Cui, Jing-Nan; Yang, Ling

    2015-12-30

    In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

  9. Dual excitation ratiometric fluorescent pH sensor for noninvasive bioprocess monitoring: development and application.

    Science.gov (United States)

    Kermis, Haley R; Kostov, Yordan; Harms, Peter; Rao, Govind

    2002-01-01

    The development and application of a fluorescent excitation-ratiometric, noninvasive pH sensor for continuous on-line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion-exchange resin, which was subsequently entrapped into a proton-permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6-9. The sensor responded rapidly (line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off-line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed-loop feedback control systems. PMID:12363356

  10. An efficient ratiometric fluorescent probe for tracking dynamic changes in lysosomal pH.

    Science.gov (United States)

    Wang, Qianqian; Zhou, Liyi; Qiu, Liping; Lu, Danqing; Wu, Yongxiang; Zhang, Xiao-Bing

    2015-08-21

    Lysosomes are acidic organelles (approximately pH 4.5-5.5) and tracking the changes in lysosomal pH is of great biological importance. To address this issue, quite a few of fluorescent probes have been developed. However, few of these probes can realize the tracking of dynamic changes in lysosomal pH. Herein, we report a new lysosome-targeted ratiometric fluorescent probe (FR-Lys) by hybridizing morpholine with a xanthane derivative and an o-hydroxy benzoxazole group. In this probe, the morpholine group serves as a targeting unit for lysosome, the xanthane derivative exhibits a pH-modulated open/close reaction of the spirocycle, while the o-hydroxy benzoxazole moiety shows a pH modulated excited-state intramolecular proton transfer (ESIPT) process. Such a design affords the probe a ratiometric fluorescence response towards pH with pH values ranging from 4.0 to 6.3. The response of the probe to pH was fast and reversible with high selectivity. Moreover, this probe possesses further advantages such as easy synthesis, high photostability and low cytotoxicity. These features are favorable for tracking dynamic pH changes in biosystems. It was then applied for dynamic imaging pH changes in lysosomes with satisfactory results. PMID:26107774

  11. Ratiometric QD-FRET Sensing of Aqueous H2S in Vitro.

    Science.gov (United States)

    Shamirian, Armen; Samareh Afsari, Hamid; Wu, Donghui; Miller, Lawrence W; Snee, Preston T

    2016-06-01

    We report a platform for the ratiometric fluorescent sensing of endogenously generated gaseous transmitter H2S in its aqueous form (bisulfide or hydrogen sulfide anion) based on the alteration of Förster resonance energy transfer from an emissive semiconductor quantum dot (QD) donor to a dithiol-linked organic dye acceptor. The disulfide bridge between the two chromophores is cleaved upon exposure to bisulfide, resulting in termination of FRET as the dye diffuses away from the QD. This results in enhanced QD emission and dye quenching. The resulting ratiometric response can be correlated quantitatively to the concentration of bisulfide and was found to have a detection limit as low as 1.36 ± 0.03 μM. The potential for use in biological applications was demonstrated by measuring the response of the QD-based FRET sensor microinjected into live HeLa cells upon extracellular exposure to bisulfide. The methodology used here is built upon a highly multifunctional platform that offers numerous advantages, such as low detection limit, enhanced photochemical stability, and sensing ability within a biological milieu. PMID:27156947

  12. A cyclization-induced emission enhancement (CIEE)-based ratiometric fluorogenic and chromogenic probe for the facile detection of a nerve agent simulant DCP.

    Science.gov (United States)

    Mahapatra, Ajit Kumar; Maiti, Kalipada; Manna, Saikat Kumar; Maji, Rajkishor; Mondal, Sanchita; Das Mukhopadhyay, Chitrangada; Sahoo, Prithidipa; Mandal, Debasish

    2015-06-14

    The first ratiometric fluorescent probe for the detection of a nerve agent simulant was developed based on tandem phosphorylation and intramolecular cyclization, by which high sensitivity as well as large emission shift could be achieved. PMID:25980383

  13. A cyclization-induced emission enhancement (CIEE)-based ratiometric fluorogenic and chromogenic probe for the facile detection of a nerve agent simulant DCP.

    Science.gov (United States)

    Mahapatra, Ajit Kumar; Maiti, Kalipada; Manna, Saikat Kumar; Maji, Rajkishor; Mondal, Sanchita; Das Mukhopadhyay, Chitrangada; Sahoo, Prithidipa; Mandal, Debasish

    2015-06-14

    The first ratiometric fluorescent probe for the detection of a nerve agent simulant was developed based on tandem phosphorylation and intramolecular cyclization, by which high sensitivity as well as large emission shift could be achieved.

  14. Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein-Ligand Interactions.

    Science.gov (United States)

    Guo, Yuan; Sakonsinsiri, Chadamas; Nehlmeier, Inga; Fascione, Martin A; Zhang, Haiyan; Wang, Weili; Pöhlmann, Stefan; Turnbull, W Bruce; Zhou, Dejian

    2016-04-01

    A highly efficient cap-exchange approach for preparing compact, dense polyvalent mannose-capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC-SIGN and DC-SIGNR (collectively termed as DC-SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC-SIGN, but not its closely related receptor DC-SIGNR, which is further confirmed by its specific blocking of DC-SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC-SIGN binds more efficiently to densely packed mannosides. A FRET-based thermodynamic study reveals that the binding is enthalpy-driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein-ligand interactions. PMID:26990806

  15. Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein–Ligand Interactions

    Science.gov (United States)

    Sakonsinsiri, Chadamas; Nehlmeier, Inga; Fascione, Martin A.; Zhang, Haiyan; Wang, Weili; Pöhlmann, Stefan; Turnbull, W. Bruce

    2016-01-01

    Abstract A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

  16. Fluorescent Ratiometric Indicators Based on Cu(II-Induced Changes in Poly(NIPAM Microparticle Volume

    Directory of Open Access Journals (Sweden)

    Aaron M. Jones

    2013-01-01

    Full Text Available Microparticles consisting of the thermal responsive polymer N-isopropyl acrylamide (polyNIPAM, a metal ion-binding ligand and a fluorophore pair that undergoes fluorescence resonance energy transfer (FRET have been prepared and characterized. Upon the addition of Cu(II, the microparticles swell or contract depending on whether charge is introduced or neutralized on the polymer backbone. The variation in microparticle morphology is translated into changes in emission of each fluorophore in the FRET pair. By measuring the emission intensity ratio between the FRET pair upon Cu(II addition, the concentration of metal ion in solution can be quantified. This ratiometric fluorescent indicator is the newest technique in an ongoing effort to use emission spectroscopy to monitor Cu(II thermodynamic activity in environmental water samples.

  17. A FRET-based ratiometric fluorescent and colorimetric probe for the facile detection of organophosphonate nerve agent mimic DCP.

    Science.gov (United States)

    Xuan, Weimin; Cao, Yanting; Zhou, Jiahong; Wang, Wei

    2013-11-18

    A FRET ratiometric fluorescent probe enabling a fast and highly sensitive response to OP nerve agent mimic DCP within 1 min and with as low as 0.17 ppm concentration detection limit has been developed. Moreover, the probe exhibits noticeable color changes under UV light and even with the naked eye. It is also demonstrated that it can detect both liquid and gas nerve agents. PMID:24080856

  18. Analysis of Temperature Dependence for a Ratiometric Wavelength Measurement System Using SMS Fiber Structure Based Edged Filters

    OpenAIRE

    Hatta, Agus; Semenova, Yuliya; Rajan, Ginu; Wang, Pengfei; Zheng, J; Farrell, Gerald

    2009-01-01

    Temperature dependence of an edge filter based on singlemode-multimodesinglemode (SMS) fiber structure is investigated numerically and experimentally. The experimental results and numerical results are in good agreement within an operational temperature range from 10 to 40 oC. It is found that the thermo-optic coefficient (TOC) has a more significant effect on the temperature dependence of an SMS edge filter compared to the thermal expansion coefficient (TEC). In the ratiometric wavelength me...

  19. A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

    Science.gov (United States)

    Maity, Debabrata; Jiang, Juanjuan; Ehlers, Martin; Wu, Junchen; Schmuck, Carsten

    2016-05-01

    A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells. PMID:27071707

  20. A FRET-based ratiometric fluorescent and colorimetric probe for the facile detection of organophosphonate nerve agent mimic DCP.

    Science.gov (United States)

    Xuan, Weimin; Cao, Yanting; Zhou, Jiahong; Wang, Wei

    2013-11-18

    A FRET ratiometric fluorescent probe enabling a fast and highly sensitive response to OP nerve agent mimic DCP within 1 min and with as low as 0.17 ppm concentration detection limit has been developed. Moreover, the probe exhibits noticeable color changes under UV light and even with the naked eye. It is also demonstrated that it can detect both liquid and gas nerve agents.

  1. A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

    Science.gov (United States)

    Maity, Debabrata; Jiang, Juanjuan; Ehlers, Martin; Wu, Junchen; Schmuck, Carsten

    2016-05-01

    A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells.

  2. Construction of single fluorophore ratiometric pH sensors using dual-emission Mn(2+)-doped quantum dots.

    Science.gov (United States)

    Pratiwi, Feby Wijaya; Hsia, Chih-Hao; Kuo, Chiung Wen; Yang, Shun-Min; Hwu, Yeu-Kuang; Chen, Peilin

    2016-10-15

    We present a novel ratiometric pH sensor design using water-soluble, dual-emission, Mn(2+)-doped quantum dots (Qdots) decorated with D-penicillamine (DPA-MnQdots). In contrast to more commonly used ratiometric pH sensors that rely on the coupling of two fluorophores, our design uses only a single emitter, which simplifies ratiometric sensing and broadens the applications of the sensor. Our single-emitter DPA-MnQdots exhibit two emission bands, at 510nm (green) and 610nm (red), which are, respectively, attributable to exciton recombination and emission of the Mn(2+) dopants. The emission intensity ratio (I510/I610) of the DPA-MnQdots depends linearly on surrounding pH values within physiological conditions (from pH 4.5 to 8.5). Moreover, the biocompatible DPA-MnQdots were used for long-term monitoring of local pH values in HeLa cells. PMID:26852157

  3. A tautomeric zinc sensor for ratiometric fluorescence imaging: application to nitric oxide-induced release of intracellular zinc.

    Science.gov (United States)

    Chang, Christopher J; Jaworski, Jacek; Nolan, Elizabeth M; Sheng, Morgan; Lippard, Stephen J

    2004-02-01

    Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn(2+) homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn(2+) physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn(2+) concentrations with high spatial and temporal fidelity. To address this critical need, we have synthesized, characterized, and applied an intracellular fluorescent probe for the ratiometric imaging of Zn(2+) based on a tautomeric seminaphthofluorescein platform. Zin-naphthopyr 1 (ZNP1) affords single-excitation, dual-emission ratiometric detection of intracellular Zn(2+) through Zn(2+)-controlled switching between fluorescein and naphthofluorescein tautomeric forms. The probe features visible excitation and emission profiles, excellent selectivity responses for Zn(2+) over competing Ca(2+) and Mg(2+) ions at intracellular concentrations, a dissociation constant (K(d)) for Zn(2+) of zinc binding. We demonstrate the value of the ZNP1 platform for biological applications by imaging changes in intracellular [Zn(2+)] in living mammalian cells. Included is the ratiometric detection of endogenous pools of intracellular Zn(2+) after NO-induced release of Zn(2+) from cellular metalloproteins. We anticipate that ZNP1 and related probes should find utility for interrogating the biology of Zn(2+).

  4. MPPT pattern search tracking control of photovoltaic system based on genetic encoding%基于遗传编码的光伏MPPT模式搜索跟踪控制

    Institute of Scientific and Technical Information of China (English)

    陈艳; 周林; 刘强; 武剑

    2011-01-01

    In order to solve the maximum power point tracking (MPPT) problem in photovoltaic system, a new MPPT method based on both genetic encoding and pattern search was proposed. The genetic encoding has excellent flexibility, randomicity and parallelism, and can not be influenced by the continuously differentiable performance of fitness function. The globle search range can be adjusted quickly due to the self-adaptability of the genetic algorithm when the environment conditions change. Based on the global search, the local search was enhanced by the pattern search in order to improve the search performance from search efficiency and precision aspects. The simulated and experimental results show that the proposed method can overcome the power loss caused by MPP oscillating phenomenon and improve the tracking efficiency, compared with the traditional hill climbing method and single genetic algorithm control. When the environment conditions change, the tracking effect is still efficient, stable and accurate.%为解决当前太阳能光伏发电系统中最大功率点跟踪(MPPT)问题,采用遗传算法编码技术与模式搜索相结合的MPPT算法,利用遗传编码灵活、随机、采用并行搜索机制且不受适应度函数连续可微影响的特点进行粗搜索.当外界条件发生变化时,其自适应的特点使得粗搜索的范围迅速调整,在此基础上采用模式搜索增强细搜索功能,旨在从速度和精度两方面提高算法搜索性能.仿真及实验表明,与传统爬山法和单一遗传算法控制相比,该方法不但能够克服MPP振荡带来的功率损失,而且能够提高跟踪效率,当外界条件改变时,仍然能够表现出快速稳定而且准确的跟踪效果.

  5. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

    Science.gov (United States)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-07-01

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag+ ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the

  6. Highly-sensitive Eu3+ ratiometric thermometers based on excited state absorption with predictable calibration

    Science.gov (United States)

    Souza, Adelmo S.; Nunes, Luiz A. O.; Silva, Ivan G. N.; Oliveira, Fernando A. M.; da Luz, Leonis L.; Brito, Hermi F.; Felinto, Maria C. F. C.; Ferreira, Rute A. S.; Júnior, Severino A.; Carlos, Luís D.; Malta, Oscar L.

    2016-02-01

    Temperature measurements ranging from a few degrees to a few hundreds of Kelvin are of great interest in the fields of nanomedicine and nanotechnology. Here, we report a new ratiometric luminescent thermometer using thermally excited state absorption of the Eu3+ ion. The thermometer is based on the simple Eu3+ energy level structure and can operate between 180 and 323 K with a relative sensitivity ranging from 0.7 to 1.7% K-1. The thermometric parameter is defined as the ratio between the emission intensities of the 5D0 --> 7F4 transition when the 5D0 emitting level is excited through the 7F2 (physiological range) or 7F1 (down to 180 K) level. Nano and microcrystals of Y2O3:Eu3+ were chosen as a proof of concept of the operational principles in which both excitation and detection are within the first biological transparent window. A novel and of paramount importance aspect is that the calibration factor can be calculated from the Eu3+ emission spectrum avoiding the need for new calibration procedures whenever the thermometer operates in different media.Temperature measurements ranging from a few degrees to a few hundreds of Kelvin are of great interest in the fields of nanomedicine and nanotechnology. Here, we report a new ratiometric luminescent thermometer using thermally excited state absorption of the Eu3+ ion. The thermometer is based on the simple Eu3+ energy level structure and can operate between 180 and 323 K with a relative sensitivity ranging from 0.7 to 1.7% K-1. The thermometric parameter is defined as the ratio between the emission intensities of the 5D0 --> 7F4 transition when the 5D0 emitting level is excited through the 7F2 (physiological range) or 7F1 (down to 180 K) level. Nano and microcrystals of Y2O3:Eu3+ were chosen as a proof of concept of the operational principles in which both excitation and detection are within the first biological transparent window. A novel and of paramount importance aspect is that the calibration factor can be

  7. Development and application of an excitation ratiometric optical pH sensor for bioprocess monitoring.

    Science.gov (United States)

    Badugu, Ramachandram; Kostov, Yordan; Rao, Govind; Tolosa, Leah

    2008-01-01

    The development of a fluorescent excitation ratiometric pH sensor (AHQ-PEG) using a novel allylhydroxyquinolinium (AHQ) derivative copolymerized with polyethylene glycol dimethacrylate (PEG) is described. The AHQ-PEG sensor film is shown to be suitable for real-time, noninvasive, continuous, online pH monitoring of bioprocesses. Optical ratiometric measurements are generally more reliable, robust, inexpensive, and insensitive to experimental errors such as fluctuations in the source intensity and fluorophore photobleaching. The sensor AHQ-PEG in deionized water was shown to exhibit two excitation maxima at 375 and 425 nm with a single emission peak at 520 nm. Excitation spectra of AHQ-PEG show a decrease in emission at the 360 nm excitation and an increase at the 420 nm excitation with increasing pH. Accordingly, the ratio of emission at 420:360 nm excitation showed a maximum change between pH 5 and 8 with an apparent pK(a) of 6.40. The low pK(a) value is suitable for monitoring the fermentation of most industrially important microorganisms. Additionally, the AHQ-PEG sensor was shown to have minimal sensitivity to ionic strength and temperature. Because AHQ is covalently attached to PEG, the film shows no probe leaching and is sterilizable by steam and alcohol. It shows rapid (approximately 2 min) and reversible response to pH over many cycles without any photobleaching. Subsequently, the AHQ-PEG sensor film was tested for its suitability in monitoring the pH of S. cereviseae (yeast) fermentation. The observed pH using AHQ-PEG film is in agreement with a conventional glass pH electrode. However, unlike the glass electrode, the present sensor is easily adaptable to noninvasive monitoring of sterilized, closed bioprocess environments without the awkward wire connections that electrodes require. In addition, the AHQ-PEG sensor is easily miniaturized to fit in microwell plates and microbioreactors for high-throughput cell culture applications. PMID:19194954

  8. Polycation-induced benzoperylene probe excimer formation and the ratiometric detection of heparin and heparinase.

    Science.gov (United States)

    Yang, Meiding; Chen, Jian; Zhou, Huipeng; Li, Wenying; Wang, Yan; Li, Juanmin; Zhang, Cuiyun; Zhou, Chuibei; Yu, Cong

    2016-01-15

    A benzoperylene probe excimer emission in an aqueous buffer solution is observed for the first time, and a novel ratiometric fluorescence method based on the probe excimer emission for the sensitive detection of heparin and heparinase is demonstrated. A negatively charged benzoperylene derivative, 6-(benzo[ghi]perylene-1,2-dicarboxylic imide-yl)hexanoic acid (BPDI), was employed. A polycation, poly(diallyldimethylammonium) chloride (poly-DDA), could induce aggregation of BPDI through noncovalent interactions. A decrease of BPDI monomer emission and a simultaneous increase of BPDI excimer emission were observed. Upon the addition of heparin, the strong binding between heparin and poly-DDA caused release of BPDI monomer molecules, and an excimer-monomer emission signal transition was detected. However, after the enzymatic hydrolysis of heparin by heparinase, heparin was hydrolyzed into small fragments, which weakened the competitive binding of heparin to poly-DDA. Poly-DDA induced aggregation of BPDI, and a monomer-excimer emission signal transition was detected. Our assay is simple, rapid, inexpensive, sensitive and selective, which could facilitate the heparin and heparinase related biochemical and biomedical research.

  9. Polycation-induced benzoperylene probe excimer formation and the ratiometric detection of heparin and heparinase.

    Science.gov (United States)

    Yang, Meiding; Chen, Jian; Zhou, Huipeng; Li, Wenying; Wang, Yan; Li, Juanmin; Zhang, Cuiyun; Zhou, Chuibei; Yu, Cong

    2016-01-15

    A benzoperylene probe excimer emission in an aqueous buffer solution is observed for the first time, and a novel ratiometric fluorescence method based on the probe excimer emission for the sensitive detection of heparin and heparinase is demonstrated. A negatively charged benzoperylene derivative, 6-(benzo[ghi]perylene-1,2-dicarboxylic imide-yl)hexanoic acid (BPDI), was employed. A polycation, poly(diallyldimethylammonium) chloride (poly-DDA), could induce aggregation of BPDI through noncovalent interactions. A decrease of BPDI monomer emission and a simultaneous increase of BPDI excimer emission were observed. Upon the addition of heparin, the strong binding between heparin and poly-DDA caused release of BPDI monomer molecules, and an excimer-monomer emission signal transition was detected. However, after the enzymatic hydrolysis of heparin by heparinase, heparin was hydrolyzed into small fragments, which weakened the competitive binding of heparin to poly-DDA. Poly-DDA induced aggregation of BPDI, and a monomer-excimer emission signal transition was detected. Our assay is simple, rapid, inexpensive, sensitive and selective, which could facilitate the heparin and heparinase related biochemical and biomedical research. PMID:26344903

  10. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters.

    Science.gov (United States)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-08-14

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag(+) ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection. PMID:27406901

  11. Molecular imprinting ratiometric fluorescence sensor for highly selective and sensitive detection of phycocyanin.

    Science.gov (United States)

    Wang, Xiaoyan; Yu, Jialuo; Kang, Qi; Shen, Dazhong; Li, Jinhua; Chen, Lingxin

    2016-03-15

    A facile strategy was developed to prepare molecular imprinting ratiometric fluorescence sensor for highly selective and sensitive detection of phycocyanin (PC) based on fluorescence resonance energy transfer (FRET), via a sol-gel polymerization process using nitrobenzoxadiazole (NBD) as fluorescent signal source. The ratio of two fluorescence peak emission intensities of NBD and PC was utilized to determine the concentration of PC, which could effectively reduce the background interference and fluctuation of diverse conditions. As a result, this sensor obtained high sensitivity with a low detection limit of 0.14 nM within 6 min, and excellent recognition specificity for PC over its analogues with a high imprinting factor of 9.1. Furthermore, the sensor attained high recoveries in the range of 93.8-110.2% at three spiking levels of PC, with precisions below 4.7% in seawater and lake water samples. The developed sensor strategy demonstrated simplicity, reliability, rapidity, high selectivity and high sensitivity, proving to be a feasible way to develop high efficient fluorescence sensors and thus potentially applicable for ultratrace analysis of complicated matrices. PMID:26485176

  12. Optical tweezers and non-ratiometric fluorescent-dye-based studies of respiration in sperm mitochondria

    International Nuclear Information System (INIS)

    The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and a non-ratiometric fluorescent probe, DiOC6(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of 450 mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force from the laser tweezers were measured. Human (Homo sapiens), dog (Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated with DiOC6(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Sperm from all three species exhibited an increase in fluorescence when treated with the DiOC6(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all three species exhibited a reduction in fluorescence to pre-dye levels. With respect to VCL and escape force, the CCCP had no effect on dog or human sperm, suggesting a major reliance upon anaerobic respiration (glycolysis) for ATP in these two species. Based on the preliminary study on drill sperm, CCCP caused a drop in the VCL, suggesting potential reliance on both glycolysis and aerobic respiration for motility. The results demonstrate that optical trapping in combination with DiOC6(3) is an effective way to study sperm motility and energetics

  13. Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions.

    Science.gov (United States)

    Zhang, Daniel Y; Azrad, Maria; Demark-Wahnefried, Wendy; Frederickson, Christopher J; Lippard, Stephen J; Radford, Robert J

    2015-02-20

    Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson's coefficient of 0.73.

  14. A Highly Copper-Selective Ratiometric Fluorescent Sensor Based on BODIPY

    Institute of Scientific and Technical Information of China (English)

    黄金龙; 王波; 叶建国; 刘斌; 邱化玉; 尹守春

    2012-01-01

    A new ratiometric fluorescent sensor (1) for Cu2+ based on 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) with di(2-picolyl)amine (DPA) as ion recognition subunit has been synthesized and investigated in this work. The binding abilities of 1 towards different metal ions such as alkali and alkaline earth metal ions (Na+, K+, Mg2+, Ca2+) and other metal ions ( Ba2+, Zn2+, Cd2+, Fe2+, Fe3+, Pb2+, Ni2+, Co2+, Hg2+, Ag+) have been examined by UV-vis and fluorescence spectroscopies. 1 displays high selectivity for Cu2+ among all test metal ions and a ~10-fold fluorescence enhancement in I582/I558 upon excitation at visible excitation wavelength. The binding mode of 1 and Cu2+ is a 1:1 stoichiometry determined via studies of Job plot, the nonlinear fitting of the fluorometric titration and ESI mass.

  15. Quadruple labelled dual oxygen and pH-sensitive ratiometric nanosensors

    Directory of Open Access Journals (Sweden)

    Veeren M. Chauhan

    2016-05-01

    Full Text Available Nanosensors capable of simultaneously measuring dissolved oxygen concentrations from 0 to 100% saturation and pH over the full physiological range, from pH 3.5 to 7.5, that advance the methods towards understanding of key biological gradients, were synthesised. A library of water soluble oxygen-sensitive porphyrins, with three substituted charged functional groups and a chemically flexible carboxylate functional group were spectroscopically analysed to assess their sensitivity to changes in dissolved oxygen concentrations as free species in solution and in suspension as nanoparticle conjugates. A platinum cationic porphyrin was taken forward to fabricate ratiometric oxygen-sensitive nanosensors, using 5-(and-6-carboxytetramethylrhodamine (TAMRA as internal standard. In addition, quadruple labelled dual oxygen and pH-sensitive nanosensors were synthesised using the cationic Pt porphyrin, pH-sensitive fluorescein dyes, carboxyfluorescein (FAM and Oregon Green (OG, in a 1:1 ratio, and TAMRA. We envisage the dual oxygen and pH nanosensors will find broad utility in the characterisation of diverse microenvironments, where there are complex interactions between molecular oxygen and pH.

  16. A ratiometric fluorescent quantum dots based biosensor for organophosphorus pesticides detection by inner-filter effect.

    Science.gov (United States)

    Yan, Xu; Li, Hongxia; Han, Xiaosong; Su, Xingguang

    2015-12-15

    In this work, we develop a novel and sensitive sensor for the detection of organophosphorus pesticides based on the inner-filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent quantum dots (RF-QDs). The RF-QDs has been designed by hybridizing two differently colored CdTe QDs, in which the red emissive QDs entrapped in the silica sphere acting as the reference signal, and the green emissive QDs covalently attached on the silica surface serving as the response signal.The fluorescence of RF-QDs could be quenched by AuNPs based on IFE. Protamine could effectively turn on the fluorescence due to the electrostatic attraction between protamine and AuNPs. Trypsin can easily hydrolyze protamine, leading to the quench of the fluorescence. Then, the fluorescence could be recovered again by the addition of parathion-methyl (PM) which could inhibit the activity of trypsin. By measuring the fluorescence of RF-QDs, the inhibition efficiency of PM to trypsin activity was evaluated. Under the optimized conditions, the inhibition efficiency was proportional to the logarithm of PM concentration in the range of 0.04-400 ng mL(-1), with a detection limit of 0.018 ng mL(-1). Furthermore, the simple and convenient method had been used for PM detection in environmental and agricultural samples with satisfactory results. PMID:26143468

  17. Colorimetric sensing of anions in water using ratiometric indicator-displacement assay.

    Science.gov (United States)

    Feng, Liang; Li, Hui; Li, Xiao; Chen, Liang; Shen, Zheng; Guan, Yafeng

    2012-09-19

    The analysis of anions in water presents a difficult challenge due to their low charge-to-radius ratio, and the ability to discriminate among similar anions often remains problematic. The use of a 3×6 ratiometric indicator-displacement assay (RIDA) array for the colorimetric detection and identification of ten anions in water is reported. The sensor array consists of different combinations of colorimetric indicators and metal cations. The colorimetric indicators chelate with metal cations, forming the color changes. Upon the addition of anions, anions compete with the indicator ligands according to solubility product constants (K(sp)). The indicator-metal chelate compound changes color back dramatically when the competition of anions wins. The color changes of the RIDA array were used as a digital representation of the array response and analyzed with standard statistical methods, including principal component analysis and hierarchical clustering analysis. No confusion or errors in classification by hierarchical clustering analysis were observed in 44 trials. The limit of detection was calculated approximately, and most limits of detections of anions are well below μM level using our RIDA array. The pH effect, temperature influence, interfering anions were also investigated, and the RIDA array shows the feasibility of real sample testing.

  18. New highly fluorescent pH indicator for ratiometric RGB imaging of pCO2

    International Nuclear Information System (INIS)

    A new diketo-pyrrolo-pyrrole (DPP) indicator dye for optical sensing of carbon dioxide is prepared via a simple one step synthesis from commercially available low cost ‘Pigment Orange 73’. The pigment is modified via alkylation of one of the lactam nitrogens with a tert-butylbenzyl group. The indicator dye is highly soluble in organic solvents and in polymers and shows pH-dependent absorption (λmax 501 and 572 nm for the protonated and deprotonated forms, respectively) and emission spectra (λmax 524 and 605 nm for the protonated and deprotonated forms, respectively). Both the protonated and the deprotonated forms show high fluorescence quantum yields (Φprot 0.86; Φdeprot 0.66). Hence, colorimetric read-out and ratiometric fluorescence intensity measurements are possible. The emission of the two forms of the indicator excellently matches the response of the green and the red channels of an RGB camera. This enables imaging of carbon dioxide distribution with a simple and low cost optical set-up. The sensor based on the new DPP dye shows very high sensitivity and is particularly promising for monitoring atmospheric levels of carbon dioxide. (paper)

  19. Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring

    Directory of Open Access Journals (Sweden)

    Kline Richard

    2004-03-01

    Full Text Available Abstract Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. Methods PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs. Results DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks. Conclusion Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.

  20. Genetically encoded fluorescent coumarin amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiangyun (San Diego, CA); Xie, Jianming (San Diego, CA); Schultz, Peter G. (La Jolla, CA)

    2012-06-05

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

  1. Genetically encoded fluorescent coumarin amino acids

    Science.gov (United States)

    Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.

    2010-10-05

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

  2. Ratiometric optical fiber dissolved oxygen sensor based on metalloporphyrin and CdSe quantum dots embedded in sol–gel matrix

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Cheng-Shane, E-mail: cschu@mail.mcut.edu.tw; Chuang, Chih-Yung

    2015-11-15

    A simple, low cost technique to fabricate a ratiometric optical fiber dissolved oxygen sensor has been presented. The ratiometric optical fiber dissolved oxygen sensor comprising a plastic optical fiber coated at one end with Pd(II)/CdSe QDs or Pt(II)/CdSe QDs embedded in sol–gel matrix. Using an LED with a central wavelength of 405 nm as an excitation source, it is shown that the emission wavelengths of the oxygen-sensitive dye (PdTFPP, PdTCPP, PtTFPP and PtOEP) and the reference CdSe QDs have no spectral overlap and therefore permit the dissolved oxygen concentration to be measured using a ratiometric-based method. The sensitivity of optical fiber dissolved oxygen sensor is quantified in terms of the ratio I{sub 0}/I{sub 100}, where I{sub 0} and I{sub 100} represent the detected luminescence intensities in fully-deoxygenated and fully-oxygenated water, respectively. The experimental results show that the sensitivities of the ratiometric optical fiber dissolved oxygen sensors are estimated to be 21.7 for PdTFPP-doped sensor, 7.4 for PdTCPP-doped sensor, 6.5 for PtTFPP-doped sensor and 9.2 for PtOEP-doped sensor. The ratiometric sensing approach presented in this study has the advantage of suppressing the effects of spurious fluctuations in the intensity of the excitation source and optical transmission properties of the optic fiber. - Highlights: • A simple, low cost technique to fabricate a ratiometric optical fiber DO sensor. • Optical fiber coated with Pd(II) or Pt(II)/CdSe QDs embedded in sol-gel matrix. • Using an LED with a central wavelength of 405 nm as an excitation source. • Suppressing the effects of spurious fluctuations in the intensity.

  3. Ratiometric optical fiber dissolved oxygen sensor based on metalloporphyrin and CdSe quantum dots embedded in sol–gel matrix

    International Nuclear Information System (INIS)

    A simple, low cost technique to fabricate a ratiometric optical fiber dissolved oxygen sensor has been presented. The ratiometric optical fiber dissolved oxygen sensor comprising a plastic optical fiber coated at one end with Pd(II)/CdSe QDs or Pt(II)/CdSe QDs embedded in sol–gel matrix. Using an LED with a central wavelength of 405 nm as an excitation source, it is shown that the emission wavelengths of the oxygen-sensitive dye (PdTFPP, PdTCPP, PtTFPP and PtOEP) and the reference CdSe QDs have no spectral overlap and therefore permit the dissolved oxygen concentration to be measured using a ratiometric-based method. The sensitivity of optical fiber dissolved oxygen sensor is quantified in terms of the ratio I0/I100, where I0 and I100 represent the detected luminescence intensities in fully-deoxygenated and fully-oxygenated water, respectively. The experimental results show that the sensitivities of the ratiometric optical fiber dissolved oxygen sensors are estimated to be 21.7 for PdTFPP-doped sensor, 7.4 for PdTCPP-doped sensor, 6.5 for PtTFPP-doped sensor and 9.2 for PtOEP-doped sensor. The ratiometric sensing approach presented in this study has the advantage of suppressing the effects of spurious fluctuations in the intensity of the excitation source and optical transmission properties of the optic fiber. - Highlights: • A simple, low cost technique to fabricate a ratiometric optical fiber DO sensor. • Optical fiber coated with Pd(II) or Pt(II)/CdSe QDs embedded in sol-gel matrix. • Using an LED with a central wavelength of 405 nm as an excitation source. • Suppressing the effects of spurious fluctuations in the intensity

  4. Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mt00257e Click here for additional data file.

    Science.gov (United States)

    Hessels, Anne M.; Taylor, Kathryn M.

    2016-01-01

    The Zn2+-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn2+ from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn2+ release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn2+ concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn2+, eZinCh-2 (K d = 1 nM at pH 7.1) and eCALWY-4 (K d = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn2+ and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn2+ in both cytosol and ER, suggesting that Zn2+ was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn2+ levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn2+, nor in experiments in which cytosolic and ER Zn2+ were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF–ionomycin treatment does not result in significant changes in cytosolic Zn2+ levels as a result from Zn2+ release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn2+ dyes. PMID:26739447

  5. Ratiometric fluorescent paper sensor utilizing hybrid carbon dots-quantum dots for the visual determination of copper ions

    Science.gov (United States)

    Wang, Yahui; Zhang, Cheng; Chen, Xiaochun; Yang, Bo; Yang, Liang; Jiang, Changlong; Zhang, Zhongping

    2016-03-01

    A simple and effective ratiometric fluorescence nanosensor for the selective detection of Cu2+ has been developed by covalently connecting the carboxyl-modified red fluorescent cadmium telluride (CdTe) quantum dots (QDs) to the amino-functionalized blue fluorescent carbon nanodots (CDs). The sensor exhibits the dual-emissions peaked at 437 and 654 nm, under a single excitation wavelength of 340 nm. The red fluorescence can be selectively quenched by Cu2+, while the blue fluorescence is a internal reference, resulting in a distinguishable fluorescence color change from pink to blue under a UV lamp. The detection limit of this highly sensitive ratiometric probe is as low as 0.36 nM, which is lower than the U.S. Environmental Protection Agency (EPA) defined limit (20 μM). Moreover, a paper-based sensor has been prepared by printing the hybrid carbon dots-quantum dots probe on a microporous membrane, which provides a convenient and simple approach for the visual detection of Cu2+. Therefore, the as-synthesized probe shows great potential application for the determination of Cu2+ in real samples.A simple and effective ratiometric fluorescence nanosensor for the selective detection of Cu2+ has been developed by covalently connecting the carboxyl-modified red fluorescent cadmium telluride (CdTe) quantum dots (QDs) to the amino-functionalized blue fluorescent carbon nanodots (CDs). The sensor exhibits the dual-emissions peaked at 437 and 654 nm, under a single excitation wavelength of 340 nm. The red fluorescence can be selectively quenched by Cu2+, while the blue fluorescence is a internal reference, resulting in a distinguishable fluorescence color change from pink to blue under a UV lamp. The detection limit of this highly sensitive ratiometric probe is as low as 0.36 nM, which is lower than the U.S. Environmental Protection Agency (EPA) defined limit (20 μM). Moreover, a paper-based sensor has been prepared by printing the hybrid carbon dots-quantum dots probe on a

  6. Water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit for ratiometric sensing of oxygen levels in living cells.

    Science.gov (United States)

    Hara, Daiki; Komatsu, Hirokazu; Son, Aoi; Nishimoto, Sei-Ichi; Tanabe, Kazuhito

    2015-04-15

    Dual emission was applied to a molecular probe for the ratiometric sensing of oxygen concentration in a living system. We prepared ruthenium complexes possessing a coumarin unit (Ru-Cou), in which the (3)MLCT phosphorescence of the ruthenium complex was efficiently quenched by molecular oxygen, whereas the coumarin unit emitted constant fluorescence independent of the oxygen concentration. The oxygen status could be determined precisely from the ratio of phosphorescence to fluorescence. We achieved the molecular imaging of cellular oxygen levels using Ru-Cou possessing an alkyl chain, which provided appropriate lipophilicity to increase cellular uptake. PMID:25848851

  7. Magnetic core-shell fluorescent pH ratiometric nanosensor using a Stoeber coating method

    Energy Technology Data Exchange (ETDEWEB)

    Lapresta-Fernandez, A., E-mail: lapresta@ugr.es [Institute of Physical Chemistry, Friedrich-Schiller-University Jena, Lessingstrasse 10, 07743 Jena (Germany); Instituto de Ciencia de Materiales de Sevilla, centro mixto CSIC-Univ. Sevilla, Avda. Americo Vespucio 49, 41092 Sevilla (Spain); Doussineau, T. [Institute of Physical Chemistry, Friedrich-Schiller-University Jena, Lessingstrasse 10, 07743 Jena (Germany); Universite Lyon 1, CNRS, UMR 5579, LASIM, F-69622 Villeurbanne (France); Moro, A.J. [Institute of Physical Chemistry, Friedrich-Schiller-University Jena, Lessingstrasse 10, 07743 Jena (Germany); REQUIMTE, Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Dutz, S. [Institute of Photonic Technology, Department of Nano Biophotonics, Jena (Germany); Steiniger, F. [Center for Electron Microscopy of the Medical Faculty, Jena (Germany); Mohr, G.J. [Fraunhofer Research Institution for Modular Solid State Technologies, Department of Polytronic Systems, Workgroup Sensor Materials, Josef-Engert-Strasse 13, D-93053 Regensburg (Germany)

    2011-11-30

    Highlights: Black-Right-Pointing-Pointer Architecture combination of magnetic core with two fluorescence silica shells. Black-Right-Pointing-Pointer Both shells properly functionalized which develops ratiometric pH measurements. Black-Right-Pointing-Pointer Reference dye does not change significantly ({approx}1.9%) by modifying the pH. Black-Right-Pointing-Pointer Sensitivity range between 2.0% and 4.9% and a few seconds of response time. Black-Right-Pointing-Pointer One month stability with a signal variation of 4.3%. - Abstract: We describe the use of a modified Stoeber method for coating maghemite ({gamma}-Fe{sub 2}O{sub 3}) nanocrystals with silica shells in order to built magnetic fluorescent sensor nanoparticles in the 50-70 nm diameter range. In detail, the magnetic cores were coated by two successive silica shells embedding two fluorophores (two different silylated dye derivatives), which allows for ratiometric pH-measurements in the pH range 5-8. Silica coated magnetic nanoparticles were prepared using maghemite nanocrystals as cores (5-10 nm in diameter) coated by tetraethoxyorthosilicate via hydrolysis/condensation in ethanol, catalyzed by ammonia. In the inner shell was covalently attached a sulforhodamine B, which was used as a reference dye; while a pH-sensitive fluorescein was incorporated into the outer shell. Once synthesized, the particles were characterized in terms of morphology, size, composition and magnetization, using dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD) and vibrating sample magnetometry (VSM). TEM analysis showed the nanoparticles to be very uniform in size. Wide-angle X-ray diffractograms showed, for uncoated as well as coated nanoparticles, typical peaks for the spinel structure of maghemite at the same diffraction angle, with no structural changes after coating. When using VSM, we obtained the magnetization curves of the resulting nanoparticles and the typical magnetization

  8. Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4.

    Science.gov (United States)

    Schlafer, Sebastian; Garcia, Javier E; Greve, Matilde; Raarup, Merete K; Nyvad, Bente; Dige, Irene

    2015-02-01

    pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.

  9. Magnetically assisted fluorescence ratiometric assays for adenosine deaminase using water-soluble conjusated polymers

    Institute of Scientific and Technical Information of China (English)

    HE Fang; YU MingHui; WANG Shu

    2009-01-01

    A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deami-nase assays with high sensitivity using water-soluble cationic conjugated polymers (CCPs).The assay contains three elements:a biotin-labeled aptamer of adenosine (biotin-aptamer),a signaling probe single-stranded DNA-tagged fiuorescein at terminus (ssDNA-FI) and a CCP.The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-FI unhybridized,and the ssDNA-FI is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field.In this case,after the addition of CCP to the magnetic beads solution,the fluo-rescence resonance energy transfer (FRET) from CCP to fluorescein is inefficient.Upon adding adenosine deaminase,the adenosine is converted into inosine,and the biotin-aptamer is hybridized with ssDNA-FI to form doubled stranded DNA (biotin-dsDNA-FI).The ssONA-FI is attached to the mag-netic beads at the separation step,and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein.Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions.The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.

  10. Magnetic and fluorescent core-shell nanoparticles for ratiometric pH sensing

    Science.gov (United States)

    Lapresta-Fernández, Alejandro; Doussineau, Tristan; Dutz, Silvio; Steiniger, Frank; Moro, Artur J.; Mohr, Gerhard J.

    2011-10-01

    This paper describes the preparation of nanoparticles composed of a magnetic core surrounded by two successive silica shells embedding two fluorophores, showing uniform nanoparticle size (50-60 nm in diameter) and shape, which allow ratiometric pH measurements in the pH range 5-8. Uncoated iron oxide magnetic nanoparticles (~10 nm in diameter) were formed by the coprecipitation reaction of ferrous and ferric salts. Then, they were added to a water-in-oil microemulsion where the hydrophilic silica shells were obtained through hydrolysis and condensation of tetraethoxyorthosilicate together with the corresponding silylated dye derivatives—a sulforhodamine was embedded in the inner silica shell and used as the reference dye while a pH-sensitive fluorescein was incorporated in the outer shell as the pH indicator. The magnetic nanoparticles were characterized using vibrating sample magnetometry, dynamic light scattering, transmission electron microscopy, x-ray diffraction and Fourier transform infrared spectroscopy. The relationship between the analytical parameter, that is, the ratio of fluorescence between the sensing and reference dyes versus the pH was adjusted to a sigmoidal fit using a Boltzmann type equation giving an apparent pKa value of 6.8. The fluorescence intensity of the reference dye did not change significantly (~3.0%) on modifying the pH of the nanoparticle dispersion. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% of horse serum, indicating that there are no significant statistical differences at a 95% confidence level.

  11. Magnetic and fluorescent core-shell nanoparticles for ratiometric pH sensing

    Energy Technology Data Exchange (ETDEWEB)

    Lapresta-Fernandez, Alejandro; Doussineau, Tristan; Moro, Artur J [Institute of Physical Chemistry, Friedrich-Schiller-University Jena, Lessingstrasse 10, 07743 Jena (Germany); Dutz, Silvio [Institute of Photonic Technology, Department of Nano-Biophotonics, Jena (Germany); Steiniger, Frank [Centre for Electron Microscopy of the Medical Faculty, Jena (Germany); Mohr, Gerhard J, E-mail: lapresta@ugr.es [Fraunhofer Research Institution for Modular Solid State Technologies, Department of Polytronic Systems, Workgroup Sensor Materials, Josef-Engert-Strasse 9, D-93053 Regensburg (Germany)

    2011-10-14

    This paper describes the preparation of nanoparticles composed of a magnetic core surrounded by two successive silica shells embedding two fluorophores, showing uniform nanoparticle size (50-60 nm in diameter) and shape, which allow ratiometric pH measurements in the pH range 5-8. Uncoated iron oxide magnetic nanoparticles ({approx}10 nm in diameter) were formed by the coprecipitation reaction of ferrous and ferric salts. Then, they were added to a water-in-oil microemulsion where the hydrophilic silica shells were obtained through hydrolysis and condensation of tetraethoxyorthosilicate together with the corresponding silylated dye derivatives-a sulforhodamine was embedded in the inner silica shell and used as the reference dye while a pH-sensitive fluorescein was incorporated in the outer shell as the pH indicator. The magnetic nanoparticles were characterized using vibrating sample magnetometry, dynamic light scattering, transmission electron microscopy, x-ray diffraction and Fourier transform infrared spectroscopy. The relationship between the analytical parameter, that is, the ratio of fluorescence between the sensing and reference dyes versus the pH was adjusted to a sigmoidal fit using a Boltzmann type equation giving an apparent pK{sub a} value of 6.8. The fluorescence intensity of the reference dye did not change significantly ({approx}3.0%) on modifying the pH of the nanoparticle dispersion. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% of horse serum, indicating that there are no significant statistical differences at a 95% confidence level.

  12. Real-Time Tracking and In Vivo Visualization of β-Galactosidase Activity in Colorectal Tumor with a Ratiometric Near-Infrared Fluorescent Probe.

    Science.gov (United States)

    Gu, Kaizhi; Xu, Yisheng; Li, Hui; Guo, Zhiqian; Zhu, Shaojia; Zhu, Shiqin; Shi, Ping; James, Tony D; Tian, He; Zhu, Wei-Hong

    2016-04-27

    Development of "smart" noninvasive bioimaging probes for trapping specific enzyme activities is highly desirable for cancer therapy in vivo. Given that β-galactosidase (β-gal) is an important biomarker for cell senescence and primary ovarian cancers, we design an enzyme-activatable ratiometric near-infrared (NIR) probe (DCM-βgal) for the real-time fluorescent quantification and trapping of β-gal activity in vivo and in situ. DCM-βgal manifests significantly ratiometric and turn-on NIR fluorescent signals simultaneously in response to β-gal concentration, which makes it favorable for monitoring dynamic β-gal activity in vivo with self-calibration in fluorescent mode. We exemplify DCM-βgal for the ratiometric tracking of endogenously overexpressed β-gal distribution in living 293T cells via the lacZ gene transfection method and OVCAR-3 cells, and further realize real-time in vivo bioimaging of β-gal activity in colorectal tumor-bearing nude mice. Advantages of our system include light-up ratiometric NIR fluorescence with large Stokes shift, high photostability, and pH independency under the physiological range, allowing for the in vivo real-time evaluation of β-gal activity at the tumor site with high-resolution three-dimensional bioimaging for the first time. Our work provides a potential tool for in vivo real-time tracking enzyme activity in preclinical applications.

  13. A TP-FRET-based two-photon fluorescent probe for ratiometric visualization of endogenous sulfur dioxide derivatives in mitochondria of living cells and tissues.

    Science.gov (United States)

    Yang, Xiaoguang; Zhou, Yibo; Zhang, Xiufang; Yang, Sheng; Chen, Yun; Guo, Jingru; Li, Xiaoxuan; Qing, Zhihe; Yang, Ronghua

    2016-08-11

    A ratiometric two-photon fluorescent probe for SO2 derivatives was first proposed based on acedan-merocyanine dyads via a TP-FRET strategy. It was successfully applied to visualization of the fluctuations of enzymatically generated SO2 derivatives in the mitochondria of HepG2 cells and rat liver tissues using two-photon fluorescence microscopy imaging. PMID:27469474

  14. A ratiometric fluorescent probe for hyaluronidase detection via hyaluronan-induced formation of red-light emitting excimers.

    Science.gov (United States)

    Hu, Qinghua; Zeng, Fang; Wu, Shuizhu

    2016-05-15

    Hyaluronidase (HAase), which is involved in various physiological and pathological processes, can selectively degrade hyaluronan (HA) into small fragments, and it has been reported as a diagnostic and prognostic biomarker for bladder cancer. Herein, a facile ratiometric fluorescent sensing system for HAase has been developed, which is based on hyaluronan-induced formation of red-light emitting excimers and can realize sensitive detection of HAase with a detection limit of 0.007 U/mL. A positively-charged pyrene analog (N-Py) has been synthesized and then mixed with the negatively-charged HA, due to electrostatic interaction between the two components, aggregation along with the N-Py excimers readily form which emits red light. While in the presence of HAase, the enzyme catalyzes the hydrolysis of HA into small fragments, which in turn triggers disassembly of excimers; consequently the N-Py excimer emission turns into monomer emission. The emission ratio resulted from the excimer-monomer transition can be used as the sensing signal for detecting HAase. The probe features visible-light excitation and red light emission (excimer), which is conducive to reducing possible interference from autofluorescence of biological samples. Furthermore, the assay system can be successfully used to determine HAase in human urine samples with satisfactory accuracy. This strategy may provide a suitable sensitive and accurate assay for HAase as well as an effective approach for developing fluorescent ratiometric assays for other enzymes. PMID:26774093

  15. A Simple and Effective Ratiometric Fluorescent Probe for the Selective Detection of Cysteine and Homocysteine in Aqueous Media

    Directory of Open Access Journals (Sweden)

    Risong Na

    2016-08-01

    Full Text Available Biothiols such as cysteine (Cys and homocysteine (Hcy are essential biomolecules participating in molecular and physiological processes in an organism. However, their selective detection remains challenging. In this study, ethyl 2-(3-formyl-4-hydroxyphenyl-4-methylthiazole-5-carboxylate (NL was synthesized as a ratiometric fluorescent probe for the rapid and selective detection of Cys and Hcy over glutathione (GSH and other amino acids. The fluorescence intensity of the probe in the presence of Cys/Hcy increased about 3-fold at a concentration of 20 equiv. of the probe, compared with that in the absence of these chemicals in aqueous media. The limits of detection of the fluorescent assay were 0.911 μM and 0.828 μM of Cys and Hcy, respectively. 1H-NMR and MS analyses indicated that an excited-state intramolecular proton transfer is the mechanism of fluorescence sensing. This ratiometric probe is structurally simple and highly selective. The results suggest that it has useful applications in analytical chemistry and diagnostics.

  16. Rapid and facile ratiometric detection of an anthrax biomarker by regulating energy transfer process in bio-metal-organic framework.

    Science.gov (United States)

    Zhang, Yihe; Li, Bin; Ma, Heping; Zhang, Liming; Zheng, Youxuan

    2016-11-15

    A ratiometric fluorescent sensor based on luminescent bio-metal-organic framework was prepared by exchanging both Tb(3+) and Eu(3+) cations into anionic bio-MOF-1. Due to a highly efficient energy transfer from Tb(3+) to Eu(3+) (>89%), emission color of Tb/Eu@bio-MOF-1 was orange-red even though Tb(3+) was the dominant content in this Tb/Eu co-doping material. More interestingly, this energy transfer process could be modulated by dipicolinic acid (DPA), an unique biomarker for bacillus spores. With DPA addition, corresponding DPA-to-Tb(3+) energy transfer was gradually enhanced while the energy transfer from Tb(3+) to Eu(3+) was significantly weakened. By regulating the energy transfer process in Tb/Eu@bio-MOF-1, visual colorimetric sensing of DPA in porous MOF was realized for the first time. Detection limit of Tb/Eu@bio-MOF-1 for DPA was 34nM, which was much lower than an infectious dosage of Bacillus anthracis spores (60μM) for human being. Besides, Tb/Eu@bio-MOF-1 showed a remarkable selectivity over other aromatic ligands and amino acids. More importantly, this porous ratiometric sensor worked equally well in human serum. These particularly attractive features of Tb/Eu@bio-MOF-1 made the direct, rapid and naked-eye detection of DPA for practical application possible.

  17. A high-resolution mitochondria-targeting ratiometric fluorescent probe for detection of the endogenous hypochlorous acid

    Science.gov (United States)

    Zhou, Liyi; Lu, Dan-Qing; Wang, Qianqian; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Hypochlorite anion, one of the biologically important reactive oxygen species, plays an essential role in diverse normal biochemical functions and abnormal pathological processes. Herein, an efficient high-resolution mitochondria-targeting ratiometric fluorescent probe for hypochlorous acid detection has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (Cdbnd C) of a 2-(2-hydroxyphenyl) quinazolin-4(3H)-one fluorophore and a cyanine group (mitochondria-targeting), which made the whole molecular a large Stokes shift (210 nm) and the two well-resolved emission peaks separated by 140 nm. As a result, it is considered as a good candidate for high resolution hypochlorous acid imaging in live cells. The ratiometric fluorescent probe exhibited outstanding features of high sensitivity, high selectivity, rapid response time (within 50 s), and excellent mitochondria-targeting ability. Moreover, the probe can also be successfully applied to imaging endogenously hypochlorous acid in the mitochondria of living cells with low cytotoxicity, and high resolution.

  18. Combined SERS biotags (SBTs) and microfluidic platform for the quantitative ratiometric discrimination between noncancerous and cancerous cells in flow

    Science.gov (United States)

    Pallaoro, Alessia; Hoonejani, Mehran R.; Braun, Gary B.; Meinhart, Carl; Moskovits, Martin

    2012-10-01

    SERS biotags are made from polymer-encapsulated silver nanoparticle dimers infused with unique Raman reporter molecules, and carry peptides as cell recognition moieties. We demonstrate their potential use for early and rapid identification of malignant cells, a central goal in cancer research. SERS biotags (SBTs) can be routinely synthesized and simultaneously excited with a single, low intensity laser source, making the determination of the relative contribution of the individual SBTs to the overall spectrum tractable. Importantly for biomedical applications, SERS employs tissuepenetrating lasers in the red to near-infrared range resulting in low autofluorescence. We have previously described a multiplexed, ratiometric method that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro based on receptor overexpression. Here we present the progress towards the application of this quantitative methodology for the identification of cancer cells in a microfluidic flow-focusing device. Beads are used as cell mimics to characterize the devices. Cells (and beads) are simultaneously incubated with two sets of SBTs while in suspension (simulating cells' capture from blood), then injected into the device for laser interrogation under flow. Each cell event is characterized by a composite Raman spectrum, deconvoluted into its single components to ultimately determine their relative contribution. We show that using SBTs ratiometrically can provide cell identification insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, autofluorescence, and turbidity.

  19. A high-resolution mitochondria-targeting ratiometric fluorescent probe for detection of the endogenous hypochlorous acid.

    Science.gov (United States)

    Zhou, Liyi; Lu, Dan-Qing; Wang, Qianqian; Hu, Shunqin; Wang, Haifei; Sun, Hongyan; Zhang, Xiaobing

    2016-09-01

    Hypochlorite anion, one of the biologically important reactive oxygen species, plays an essential role in diverse normal biochemical functions and abnormal pathological processes. Herein, an efficient high-resolution mitochondria-targeting ratiometric fluorescent probe for hypochlorous acid detection has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (CC) of a 2-(2-hydroxyphenyl) quinazolin-4(3H)-one fluorophore and a cyanine group (mitochondria-targeting), which made the whole molecular a large Stokes shift (210nm) and the two well-resolved emission peaks separated by 140nm. As a result, it is considered as a good candidate for high resolution hypochlorous acid imaging in live cells. The ratiometric fluorescent probe exhibited outstanding features of high sensitivity, high selectivity, rapid response time (within 50s), and excellent mitochondria-targeting ability. Moreover, the probe can also be successfully applied to imaging endogenously hypochlorous acid in the mitochondria of living cells with low cytotoxicity, and high resolution. PMID:27236136

  20. FRET ratiometric probes reveal the chiral-sensitive cysteine-dependent H2S production and regulation in living cells

    Science.gov (United States)

    Wei, Lv; Yi, Long; Song, Fanbo; Wei, Chao; Wang, Bai-Fan; Xi, Zhen

    2014-04-01

    Hydrogen sulfide (H2S) is an endogenously produced gaseous signalling molecule with multiple biological functions. In order to visualize and quantify the endogenous in situ production of H2S in living cells, here we developed two new sulphide ratiometric probes (SR400 and SR550) based on fluorescence resonance energy transfer (FRET) strategy for live capture of H2S. The FRET-based probes show excellent selectivity toward H2S in a high thiol background under physiological buffer. The probe can be used to in situ visualize cysteine-dependent H2S production in a chiral-sensitive manner in living cells. The ratiometric imaging studies indicated that D-Cys induces more H2S production than that of L-Cys in mitochondria of human embryonic kidney 293 cells (HEK293). The cysteine mimics propargylglycine (PPG) has also been found to inhibit the cysteine-dependent endogenous H2S production in a chiral-sensitive manner in living cells. D-PPG inhibited D-Cys-dependent H2S production more efficiently than L-PPG, while, L-PPG inhibited L-Cys-dependent H2S production more efficiently than D-PPG. Our bioimaging studies support Kimura's discovery of H2S production from D-cysteine in mammalian cells and further highlight the potential of D-cysteine and its derivatives as an alternative strategy for classical H2S-releasing drugs.

  1. Rapid and facile ratiometric detection of an anthrax biomarker by regulating energy transfer process in bio-metal-organic framework.

    Science.gov (United States)

    Zhang, Yihe; Li, Bin; Ma, Heping; Zhang, Liming; Zheng, Youxuan

    2016-11-15

    A ratiometric fluorescent sensor based on luminescent bio-metal-organic framework was prepared by exchanging both Tb(3+) and Eu(3+) cations into anionic bio-MOF-1. Due to a highly efficient energy transfer from Tb(3+) to Eu(3+) (>89%), emission color of Tb/Eu@bio-MOF-1 was orange-red even though Tb(3+) was the dominant content in this Tb/Eu co-doping material. More interestingly, this energy transfer process could be modulated by dipicolinic acid (DPA), an unique biomarker for bacillus spores. With DPA addition, corresponding DPA-to-Tb(3+) energy transfer was gradually enhanced while the energy transfer from Tb(3+) to Eu(3+) was significantly weakened. By regulating the energy transfer process in Tb/Eu@bio-MOF-1, visual colorimetric sensing of DPA in porous MOF was realized for the first time. Detection limit of Tb/Eu@bio-MOF-1 for DPA was 34nM, which was much lower than an infectious dosage of Bacillus anthracis spores (60μM) for human being. Besides, Tb/Eu@bio-MOF-1 showed a remarkable selectivity over other aromatic ligands and amino acids. More importantly, this porous ratiometric sensor worked equally well in human serum. These particularly attractive features of Tb/Eu@bio-MOF-1 made the direct, rapid and naked-eye detection of DPA for practical application possible. PMID:27183278

  2. Selective detection of endogenous H2S in living cells and the mouse hippocampus using a ratiometric fluorescent probe

    Science.gov (United States)

    Zhang, Ling; Meng, Wen-Qi; Lu, Liang; Xue, Yun-Sheng; Li, Cheng; Zou, Fang; Liu, Yi; Zhao, Jing

    2014-07-01

    As one of three gasotransmitters, the fundamental signalling roles of hydrogen sulphide are receiving increasing attention. New tools for the accurate detection of hydrogen sulphide in cells and tissues are in demand to probe its biological functions. We report the p-nitrobenzyl-based ratiometric fluorescent probe RHP-2, which features a low detection limit, high selectivity and good photostability. The emission intensity ratios had a good linear relationship with the sulphide concentrations in PBS buffer and bovine serum. Our probe was applied to the ratiometric determination and imaging of endogenous H2S in living cells. Furthermore, RHP-2 was used as an effective tool to measure endogenous H2S in the mouse hippocampus. We observed a significant reduction in sulphide concentrations and downregulated expression of cystathionine β-synthetase (CBS) mRNA and CBS protein in the mouse hippocampus in a chronic unpredictable mild stress (CUMS)-induced depression model. These data suggested that decreased concentrations of endogenous H2S may be involved in the pathogenesis of chronic stress depression.

  3. A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells.

    Science.gov (United States)

    Xu, Wang; Teoh, Chai Lean; Peng, Juanjuan; Su, Dongdong; Yuan, Lin; Chang, Young-Tae

    2015-07-01

    Sulfur dioxide (SO2) can be endogenously produced by enzymes in mitochondria during oxidation of H2S or sulphur-containing amino acids, and plays important roles in several physiological processes. However, the design and synthesis of fluorescent probes which can detect mitochondrial SO2 and its derivatives in living cells still remain unresolved. Herein, we report the preparation of a lipophilic cationic dye 1 (Mito-Ratio-SO2), which targets the mitochondria in living cells and is sensitive to the presence of SO2 derivatives. The ratiometric probe Mito-Ratio-SO2 displays a 170 nm blue-shift in emission with two well-resolved emission bands upon addition of sulfite. Mechanistic studies indicate that three probe-SO2 adducts coexist after reaction, as supported by liquid chromatography and density function theory investigations. Importantly, the ratiometric probe is highly selective for sulfite over other bio-species including H2S. Fluorescence co-localization studies indicate that the probe localizes solely in the mitochondria of HeLa cells. Last but not least, fluorescent imaging of HeLa cells successfully demonstrates the detection of intrinsically generated intracellular SO2 derivatives in living cells.

  4. Use of genetically encoded calcium indicators (GECIs combined with advanced motion tracking techniques to examine the behavior of neurons and glia in the enteric nervous system of the intact murine colon

    Directory of Open Access Journals (Sweden)

    Grant Willem Hennig

    2015-11-01

    Full Text Available Genetically encoded Ca2+ indicators (GECIs have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We developed several GCaMP3 mice: 1 Wnt1-GCaMP3, all enteric neurons and glia; 2 GFAP-GCaMP3, enteric glia; 3 nNOS-GaMP3, enteric nitrergic neurons, and 4 ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC, using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100-1000 active cells through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural

  5. Theoretical investigation on ratiometric two-photon fluorescent probe for Zn2+ detection based on ICT mechanism

    Science.gov (United States)

    Huang, Shuang; Yang, Bao-Zhu; Ren, Ai-Min

    2016-06-01

    OPA (one-photon absorption), TPA (two-photon absorption) and fluorescence properties of a free ligand L upon coordination with Zn2+, and the regeneration with CN- were investigated in theory. According to our research, OPA spectra of ligand L show red-shift binding with Zn2+ while blue-shift with CN-. The fluorescence spectra and TPA wavelength are shifted in the same situation as those of OPA spectra. The value of TPA cross-section decreased at first, and then increased to 1813 GM for [L-Zn(CN)4]2-. Intramolecular charge transfer (ICT) mechanism was investigated by natural bond orbital (NBO) analysis. It demonstrates that L is hopeful to be a good ratiometric fluorescent probe for zinc ion detection in solution, and it can regenerate after CN- was introduced.

  6. Facile and high spatial resolution ratio-metric luminescence thermal mapping in microfluidics by near infrared excited upconversion nanoparticles

    Science.gov (United States)

    Wang, Yu; Cao, Wenbin; Li, Shunbo; Wen, Weijia

    2016-02-01

    A local area temperature monitor is important for precise control of chemical and biological processes in microfluidics. In this work, we developed a facile method to realize micron spatial resolution of temperature mapping in a microfluidic channel quickly and cost effectively. Based on the temperature dependent fluorescence emission of NaYF4:Yb3+, Er3+ upconversion nanoparticles (UCNPs) under near-infrared irradiation, ratio-metric imaging of UCNPs doped polydimethylsiloxane can map detailed temperature distribution in the channel. Unlike some reported strategies that utilize temperature sensitive organic dye (such as Rhodamine) to achieve thermal sensing, our method is highly chemically inert and physically stable without any performance degradation in long term operation. Moreover, this method can be easily scaled up or down, since the spatial and temperature resolution is determined by an optical imaging system. Our method supplied a simple and efficient solution for temperature mapping on a heterogeneous surface where usage of an infrared thermal camera was limited.

  7. Highly Selective and Sensitive One- and Two-Photon Ratiometric Fluorescent Probe for Intracellular Hydrogen Polysulfide Sensing.

    Science.gov (United States)

    Han, Qingxin; Mou, Zuolin; Wang, Haihong; Tang, Xiaoliang; Dong, Zhe; Wang, Li; Dong, Xue; Liu, Weisheng

    2016-07-19

    Hydrogen polysulfide (H2Sn) has attracted increasing attention due to the fact that it is actually the key signaling molecule rather than hydrogen sulfide (H2S). Therefore, developing a sensitive and accurate assay to investigate the biosynthetic pathways of H2Sn is of physiological and pathological significance. In this work, based on the commonly used two-photon fluorophore, 1,8-naphthalimide, a new probe, NRT-HP, has been designed and synthesized that displayed both one- and two-photon ratiometric fluorescence changes toward H2Sn via H2Sn-mediated benzodithiolone formation. NRT-HP exhibits excellent pH stability, high selectivity and low detection limit (0.1 μM) in aqueous media. Furthermore, two-photon fluorescence microscopy experiments have demonstrated that NRT-HP could be used for the H2Sn detection in live cells as well as tissue slices. PMID:27312769

  8. Integrity of lipid nanocarriers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice.

    Science.gov (United States)

    Bouchaala, Redouane; Mercier, Luc; Andreiuk, Bohdan; Mély, Yves; Vandamme, Thierry; Anton, Nicolas; Goetz, Jacky G; Klymchenko, Andrey S

    2016-08-28

    Lipid nanocarriers are considered as promising candidates for drug delivery and cancer targeting because of their low toxicity, biodegradability and capacity to encapsulate drugs and/or contrasting agents. However, their biomedical applications are currently limited because of a poor understanding of their integrity in vivo. To address this problem, we report on fluorescent nano-emulsion droplets of 100nm size encapsulating lipophilic near-infrared cyanine 5.5 and 7.5 dyes with a help of bulky hydrophobic counterion tetraphenylborate. Excellent brightness and efficient Förster Resonance Energy Transfer (FRET) inside lipid NCs enabled for the first time quantitative fluorescence ratiometric imaging of NCs integrity directly in the blood circulation, liver and tumor xenografts of living mice using a whole-animal imaging set-up. This unique methodology revealed that the integrity of our FRET NCs in the blood circulation of healthy mice is preserved at 93% at 6h of post-administration, while it drops to 66% in the liver (half-life is 8.2h). Moreover, these NCs show fast and efficient accumulation in tumors, where they enter in nearly intact form (77% integrity at 2h) before losing their integrity to 40% at 6h (half-life is 4.4h). Thus, we propose a simple and robust methodology based on ratiometric FRET imaging in vivo to evaluate quantitatively nanocarrier integrity in small animals. We also demonstrate that nano-emulsion droplets are remarkably stable nano-objects that remain nearly intact in the blood circulation and release their content mainly after entering tumors. PMID:27327767

  9. Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as Ratiometric Biological Probes and Organelle-Selective Bioimaging Reagents.

    Science.gov (United States)

    Zhang, Kenneth Yin; Liu, Hua-Wei; Tang, Man-Chung; Choi, Alex Wing-Tat; Zhu, Nianyong; Wei, Xi-Guang; Lau, Kai-Chung; Lo, Kenneth Kam-Wing

    2015-07-01

    In this Article, we present a series of cyclometalated iridium(III) polypyridine complexes of the formula [Ir(N^C)2(N^N)](PF6) that showed dual emission under ambient conditions. The structures of the cyclometalating and diimine ligands were changed systematically to investigate the effects of the substituents on the dual-emission properties of the complexes. On the basis of the photophysical data, the high-energy (HE) and low-energy (LE) emission features of the complexes were assigned to triplet intraligand ((3)IL) and triplet charge-transfer ((3)CT) excited states, respectively. Time-dependent density functional theory (TD-DFT) calculations supported these assignments and indicated that the dual emission resulted from the interruption of the communication between the higher-lying (3)IL and the lower-lying (3)CT states by a triplet amine-to-ligand charge-transfer ((3)NLCT) state. Also, the avidin-binding properties of the biotin complexes were studied by emission titrations, and the results showed that the dual-emissive complexes can be utilized as ratiometric probes for avidin. Additionally, all the complexes exhibited efficient cellular uptake by live HeLa cells. The MTT and Annexin V assays confirmed that no cell death and early apoptosis occurred during the cell imaging experiments. Interestingly, laser-scanning confocal microscopy revealed that the complexes were selectively localized on the cell membrane, mitochondria, or both, depending on the nature of the substituents of the ligands. The results of this work will contribute to the future development of dual-emissive transition metal complexes as ratiometric probes and organelle-selective bioimaging reagents. PMID:26087119

  10. An efficient core-shell fluorescent silica nanoprobe for ratiometric fluorescence detection of pH in living cells.

    Science.gov (United States)

    Fu, Jingni; Ding, Changqin; Zhu, Anwei; Tian, Yang

    2016-08-01

    Intracellular pH plays a vital role in cell biology, including signal transduction, ion transport and homeostasis. Herein, a ratiometric fluorescent silica probe was developed to detect intracellular pH values. The pH sensitive dye fluorescein isothiocyanate isomer I (FITC), emitting green fluorescence, was hybridized with reference dye rhodamine B (RB), emitting red fluorescence, as a dual-emission fluorophore, in which RB was embedded in a silica core of ∼40 nm diameter. Moreover, to prevent fluorescence resonance energy transfer between FITC and RB, FITC was grafted onto the surface of core-shell silica colloidal particles with a shell thickness of 10-12 nm. The nanoprobe exhibited dual emission bands centered at 517 and 570 nm, under single wavelength excitation of 488 nm. RB encapsulated in silica was inert to pH change and only served as reference signals for providing built-in correction to avoid environmental effects. Moreover, FITC (λem = 517 nm) showed high selectivity toward H(+) against metal ions and amino acids, leading to fluorescence variation upon pH change. Consequently, variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor. The specific nanoprobe showed good linearity with pH variation in the range of 6.0-7.8. It can be noted that the fluorescent silica probe demonstrated good water dispersibility, high stability and low cytotoxicity. Accordingly, imaging and biosensing of pH variation was successfully achieved in HeLa cells. PMID:27291898

  11. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

    International Nuclear Information System (INIS)

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg2+. The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg2+, the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg2+ in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg2+ contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg2+ quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg2+ detection. • The Hg2+ content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg2+ detection. • The ratiometric probe is of good simplicity, low toxicity, and excellent stability

  12. Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring

    OpenAIRE

    Kline Richard; Sharma Rakesh

    2004-01-01

    Abstract Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. Methods PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining fo...

  13. Signal-Amplified Near-Infrared Ratiometric Electrochemiluminescence Aptasensor Based on Multiple Quenching and Enhancement Effect of Graphene/Gold Nanorods/G-Quadruplex.

    Science.gov (United States)

    Shao, Kang; Wang, Biru; Ye, Shiyi; Zuo, Yunpeng; Wu, Long; Li, Qin; Lu, Zhicheng; Tan, XueCai; Han, Heyou

    2016-08-16

    Dual-signaling ratiometric electrochemiluminescence (ECL) technology has attracted particular attention in analytical science due to its precise measurement to normalize variation in environmental changes. Creating new mated ECL report units with two emitting states and improving the detection sensitivity are major challenges for ratiometric ECL measurement. Here, we fabricate an ultrasensitive near-infrared ratiometric ECL aptasensor based on a dual-potential signal amplification strategy triggered by the quencher/enhancer [graphene/hemin/gold nanorods/G-quadruplex-hemin (rGO-H-AuNRs-G4H) composite]. The composite was initially prepared through three consecutive steps: the π-π stacking interaction between hemin and graphene, in-site growth of AuNRs, and surface ligand exchange. Dual ECL quenching of quantum dots (QDs) and multiple signal enhancement of luminol can be achieved simultaneously by the fabrication of the sandwich "thrombin aptamer I (TBA1)-TB-TBA2 (rGO-H-AuNRs-G4H)" mode: (i) the formation of three-dimensional G-quadruplex between aptamer and thrombin not only shortens the distance between the donor (QDs) and receptor (rGO-H and AuNRs) to trigger electrochemiluminescence energy transfer but also provides the place for intercalating hemin; (ii) the hemin intercalated into G4 structure and hemin connected onto rGO together with AuNRs/rGO nanomaterials can achieve the multiple peroxidase-like catalysis of H2O2 to greatly enhance the ECL of luminol. The ratiometric ECL aptasensor self-calibrated by the internal reference (luminol or QDs) exhibits ultrasensitive and accurate analytical performance toward thrombin (TB) with a linear detection range from 100 ng/mL to 0.5 pg/mL and a detection limit of 4.2 fg/mL [defined as signal-to-noise ratio (S/N) = 3]. PMID:27435830

  14. One-pot synthesis of mesoporous structured ratiometric fluorescence molecularly imprinted sensor for highly sensitive detection of melamine from milk samples.

    Science.gov (United States)

    Xu, Shoufang; Lu, Hongzhi

    2015-11-15

    A facile strategy was developed to prepare mesoporous structured ratiometric fluorescence molecularly imprinted sensor for highly sensitive and selective determination of melamine using CdTe QDs as target sensitive dye and hematoporphyrin as reference dyes. One-pot synthesis method was employed because it could simplify the imprinting process and shorten the experimental period. The as-prepared fluorescence MIPs sensor, which combined ratiometric fluorescence technique with mesoporous silica materials into one system, exhibited excellent selectivity and sensitivity. Under optimum conditions, these mesoporous structured ratiometric fluorescence MIP@QDs sensors showed detection limit as low as 38 nM, which was much lower than those non-mesoporous one. The recycling process was sustainable at least 10 times without obvious efficiency decrease. The feasibility of the developed method in real samples was successfully evaluated through the analysis of melamine in raw milk and milk powder samples with satisfactory recoveries of 92-101%. The developed method proposed in this work proved to be a convenient, rapid, reliable and practical way to prepared high sensitive and selective fluorescence sensors with potentially applicable for trace pollutants analysis in complicated samples. PMID:26057736

  15. Fluorescence Ratiometric Assay Strategy for Chemical Transmitter of Living Cells Using H2O2-Sensitive Conjugated Polymers.

    Science.gov (United States)

    Wang, Yunxia; Li, Shengliang; Feng, Liheng; Nie, Chenyao; Liu, Libing; Lv, Fengting; Wang, Shu

    2015-11-01

    A new water-soluble conjugated poly(fluorene-co-phenylene) derivative (PFP-FB) modified with boronate-protected fluorescein (peroxyfluor-1) via PEG linker has been designed and synthesized. In the presence of H2O2, the peroxyfluor-1 group can transform into green fluorescent fluorescein by deprotecting the boronate protecting groups. In this case, upon selective excitation of PFP-FB backbone at 380 nm, efficient fluorescence resonance energy transfer (FRET) from PFP-FB backbone to fluorescein occurs, and accordingly, the fluorescence color of PFP-FB changes from blue to green. Furthermore, the emission color of PFP-FB and the FRET ratio change in a concentration-dependent manner. By taking advantage of PFP-FB, ratiometric detection of choline and acetylcholine (ACh) through cascade enzymatic reactions and further dynamic monitoring of the choline consumption process of cancer cells have been successfully realized. Thus, this new polymer probe promotes the development of enzymatic biosensors and provides a simpler and more effective way for detecting the chemical transmitter of living cells. PMID:26451624

  16. A low cytotoxic and ratiometric fluorescent nanosensor based on carbon-dots for intracellular pH sensing and mapping

    Science.gov (United States)

    Du, Fangkai; Ming, Yunhao; Zeng, Fang; Yu, Changmin; Wu, Shuizhu

    2013-09-01

    Intracellular pH plays a critical role in the function of cells, and its regulation is essential for most cellular processes. In this study, we demonstrate a fluorescence resonance energy transfer (FRET)-based ratiometric pH nanosensor with carbon-dot (CD) as the carrier. The sensor was prepared by covalently linking a pH-sensitive fluorescent dye (fluorescein isothiocyanate, FITC) onto carbon-dot. As the FRET donor, the carbon-dot exhibits bright fluorescence emission as well as λex-dependent photoluminescence emission, and a suitable excitation wavelength for the donor (CD) can be chosen to match the energy acceptor (fluorescein moiety). The fluorescein moieties on a CD undergo structural and spectral conversion as the pH changes, affording the nanoplatform a FRET-based pH sensor. The CD-based system exhibits a significant change in fluorescence intensity ratio between pH 4 and 8 with a pKa value of 5.69. It also displays excellent water dispersibility, good spectral reversibility, satisfactory cell permeability and low cytotoxicity. Following the living cell uptake, this nanoplatform with dual-chromatic emissions can facilitate real-time visualization of the pH evolution involved in the endocytic pathway of the nanosensor. This reversible and low cytotoxic fluorescent nanoplatform may be highly valuable in a variety of biological studies, such as endocytic trafficking, endosome/lysosome maturation, and pH regulation in subcellular organelles.

  17. A portable fiberoptic ratiometric fluorescence analyzer provides rapid point-of-care determination of glomerular filtration rate in large animals.

    Science.gov (United States)

    Wang, Exing; Meier, Daniel J; Sandoval, Ruben M; Von Hendy-Willson, Vanessa E; Pressler, Barrak M; Bunch, Robert M; Alloosh, Mouhamad; Sturek, Michael S; Schwartz, George J; Molitoris, Bruce A

    2012-01-01

    Measurement of the glomerular filtration rate (GFR) is the gold standard for precise assessment of kidney function. A rapid, point-of-care determination of the GFR may provide advantages in the clinical setting over currently available assays. Here we demonstrate a proof of principle for such an approach in a pig and dogs, two species that approximate the vascular access and GFR results expected in humans. In both animal models, a sub-millimeter optical fiber that delivered excitation light and collected fluorescent emissions was inserted into a peripheral vein (dog) or central venous access (pig) by means of commercial intravenous catheters. A mixture of fluorescent chimeras of a small freely filterable reporter and large non-filterable plasma volume marker were infused as a bolus, excited by light-emitting diodes, and the in vivo signals detected and quantified by photomultiplier tubes in both species in less than 60 min. Concurrent standardized 6-h iohexol plasma kidney clearances validated the accuracy of our results for both physiologic and a chronic kidney disease setting. Thus, our ratiometric technique allows for both measurement of plasma vascular volume and highly accurate real-time GFR determinations, enabling clinical decision making in real time. PMID:21881552

  18. Mapping of healthy oral mucosal tissue using diffuse reflectance spectroscopy: ratiometric-based total hemoglobin comparative study.

    Science.gov (United States)

    Hafez, Razan; Hamadah, Omar; Bachir, Wesam

    2015-11-01

    The objective of this study is to clinically evaluate the diffuse reflectance spectroscopy (DRS) ratiometric method for differentiation of normal oral mucosal tissues with different histological natures and vascularizations in the oral cavity. Twenty-one healthy patients aged 20-44 years were diagnosed as healthy and probed with a portable DRS system. Diffuse reflectance spectra were recorded in vivo in the range (450-650 nm). In this study, the following three oral mucosal tissues were considered: masticatory mucosa, lining mucosa, and specialized mucosa. Spectral features based on spectral intensity ratios were determined at five specific wavelengths (512, 540, 558, 575, and 620 nm). Total hemoglobin based on spectral ratios for the three anatomical regions have also been evaluated. The three studied groups representing different anatomical regions in the oral cavity were compared using analysis of variance and post hoc least significant difference tests. Statistical analysis showed a significant difference in the mean of diffuse spectral ratios between the groups (P oral sites in terms of total hemoglobin content. Diffuse reflectance spectroscopy might be used for creating a DRS databank of normal oral mucosal tissue with specific spectral ratios featuring the total hemoglobin concentrations. That would further enhance the discrimination of oral tissue for examining the histological nature of oral mucosa and diagnosis of early precancerous changes in the oral cavity based on non-invasive monitoring of neovascularization. PMID:25987341

  19. A pyrene-benzthiazolium conjugate portraying aggregation induced emission, a ratiometric detection and live cell visualization of HSO3(.).

    Science.gov (United States)

    Diwan, Uzra; Kumar, Virendra; Mishra, Rakesh K; Rana, Nishant Kumar; Koch, Biplob; Singh, Manish Kumar; Upadhyay, K K

    2016-07-27

    The present study deals with the photophysical property of a pyrene-benzthiazolium conjugate R1, as a strong intramolecular charge transfer (ICT) probe exhibiting long wavelength emission in the red region. Unlike traditional planar polyaromatic hydrocarbons whose aggregation generally quenches the light emission, the pyrene based R1 was found to display aggregation-induced emission (AIE) property along with simultaneous increase in its quantum yield upon increasing the water content of the medium. The R1 exhibits high specificity towards HSO3(-)/SO3(2-) by interrupting its own ICT producing there upon a large ratiometric blue shift of ∼220 nm in its emission spectrum. The lowest detection limit for the above measurement was found to be 8.90 × 10(-8) M. The fluorescent detection of HSO3(-) was also demonstrated excellently by test paper strip and silica coated TLC plate incorporating R1. The live cell imaging of HSO3(─) through R1 in HeLa cells was studied using fluorescence microscopic studies. The particle size and morphological features of R1 and R1-HSO3(-) aggregates in aqueous solution were characterized by DLS along with SEM analysis. PMID:27251947

  20. Construction of near-infrared photonic crystal glucose-sensing materials for ratiometric sensing of glucose in tears.

    Science.gov (United States)

    Hu, Yumei; Jiang, Xiaomei; Zhang, Laiying; Fan, Jiao; Wu, Weitai

    2013-10-15

    Noninvasive monitoring of glucose in tears is highly desirable in tight glucose control. The polymerized crystalline colloidal array (PCCA) that can be incorporated into contact lens represents one of the most promising materials for noninvasive monitoring of glucose in tears. However, low sensitivity and slow time response of the PCCA reported in previous arts has limited its clinical utility. This paper presents a new PCCA, denoted as NIR-PCCA, comprising a CCA of glucose-responsive sub-micrometered poly(styrene-co-acrylamide-co-3-acrylamidophenylboronic acid) microgels embedded within a slightly positive charged hydrogel matrix of poly(acrylamide-co-2-(dimethylamino)ethyl acrylate). This newly designed NIR-PCCA can reflect near-infrared (NIR) light, whose intensity (at 1722 nm) would decrease evidently with increasing glucose concentration over the physiologically relevant range in tears. The lowest glucose concentration reliably detectable was as low as ca. 6.1 μg/dL. The characteristic response time τ(sensing) was 22.1±0.2s when adding glucose to 7.5 mg/dL, and the higher the glucose concentration is, the faster the time response. Such a rationally designed NIR-PCCA is well suited for ratiometric NIR sensing of tear glucose under physiological conditions, thereby likely to bring this promising glucose-sensing material to the forefront of analytical devices for diabetes. PMID:23651573

  1. Two-dimensional Oxygen Distribution in a Surface Sediment Layer Measured Using an RGB Color Ratiometric Oxygen Planar Optode

    Directory of Open Access Journals (Sweden)

    Jae Seong Lee

    2013-09-01

    Full Text Available We measured two-dimensional (2-D oxygen distribution in the surface sediment layer of intertidal sediment using a simple and inexpensive planar oxygen optode, which is based on a color ratiometric image approach. The recorded emission intensity of red color luminophore light significantly changed with oxygen concentration by O2 quenching of platinum(IIoctaethylporphyrin (PtOEP. The ratios between the intensity of red and green emissions with oxygen concentration variation demonstrated the Stern-Volmer relationship. The 2-D oxygen distribution image showed microtopographic structure, diffusivity boundary layer and burrow in surface sediment layer. The oxygen penetration depth (OPD was about 2 mm and the one-dimensional vertical diffusive oxygen uptake (DOU was 12.6 mmol m−2 d−1 in the undisturbed surface sediment layer. However, those were enhanced near burrow by benthic fauna, and the OPD was two times deeper and DOU was increased by 34%. The simple and inexpensive oxygen planar optode has great application potential in the study of oxygen dynamics with high spatiotemporal resolution, in benthic boundary layers.

  2. Zinc(II)-selective ratiometric fluorescent sensors based on inhibition of excited-state intramolecular proton transfer.

    Science.gov (United States)

    Henary, Maged M; Wu, Yonggang; Fahrni, Christoph J

    2004-06-21

    To develop a zinc(II)-selective emission ratiometric probe suitable for biological applications, we explored the cation-induced inhibition of excited-state intramolecular proton transfer (ESIPT) with a series of 2-(2'-benzenesulfonamidophenyl)benzimidazole derivatives. In the absence of Zn(II) at neutral pH, the fluorophores undergo ESIPT to yield a highly Stokes' shifted emission from the proton-transfer tautomer. Coordination of Zn(II) inhibits the ESIPT process and yields a significant hypsochromic shift of the fluorescence emission maximum. Whereas the paramagnetic metal cations Cu(II), Fe(II), Ni(II), Co(II), and Mn(II) result in fluorescence quenching, the emission response is not altered by millimolar concentrations of Ca(II) or Mg(II), rendering the sensors selective for Zn(II) among all biologically important metal cations. Due to the modular architecture of the fluorophore, the Zn(II) binding affinity can be readily tuned by implementing simple structural modifications. The synthesized probes are suitable to gauge free Zn(II) concentrations in the micromolar to picomolar range under physiological conditions.

  3. Mapping of healthy oral mucosal tissue using diffuse reflectance spectroscopy: ratiometric-based total hemoglobin comparative study.

    Science.gov (United States)

    Hafez, Razan; Hamadah, Omar; Bachir, Wesam

    2015-11-01

    The objective of this study is to clinically evaluate the diffuse reflectance spectroscopy (DRS) ratiometric method for differentiation of normal oral mucosal tissues with different histological natures and vascularizations in the oral cavity. Twenty-one healthy patients aged 20-44 years were diagnosed as healthy and probed with a portable DRS system. Diffuse reflectance spectra were recorded in vivo in the range (450-650 nm). In this study, the following three oral mucosal tissues were considered: masticatory mucosa, lining mucosa, and specialized mucosa. Spectral features based on spectral intensity ratios were determined at five specific wavelengths (512, 540, 558, 575, and 620 nm). Total hemoglobin based on spectral ratios for the three anatomical regions have also been evaluated. The three studied groups representing different anatomical regions in the oral cavity were compared using analysis of variance and post hoc least significant difference tests. Statistical analysis showed a significant difference in the mean of diffuse spectral ratios between the groups (P spectroscopy might be used for creating a DRS databank of normal oral mucosal tissue with specific spectral ratios featuring the total hemoglobin concentrations. That would further enhance the discrimination of oral tissue for examining the histological nature of oral mucosa and diagnosis of early precancerous changes in the oral cavity based on non-invasive monitoring of neovascularization.

  4. Fluorescence Ratiometric Assay Strategy for Chemical Transmitter of Living Cells Using H2O2-Sensitive Conjugated Polymers.

    Science.gov (United States)

    Wang, Yunxia; Li, Shengliang; Feng, Liheng; Nie, Chenyao; Liu, Libing; Lv, Fengting; Wang, Shu

    2015-11-01

    A new water-soluble conjugated poly(fluorene-co-phenylene) derivative (PFP-FB) modified with boronate-protected fluorescein (peroxyfluor-1) via PEG linker has been designed and synthesized. In the presence of H2O2, the peroxyfluor-1 group can transform into green fluorescent fluorescein by deprotecting the boronate protecting groups. In this case, upon selective excitation of PFP-FB backbone at 380 nm, efficient fluorescence resonance energy transfer (FRET) from PFP-FB backbone to fluorescein occurs, and accordingly, the fluorescence color of PFP-FB changes from blue to green. Furthermore, the emission color of PFP-FB and the FRET ratio change in a concentration-dependent manner. By taking advantage of PFP-FB, ratiometric detection of choline and acetylcholine (ACh) through cascade enzymatic reactions and further dynamic monitoring of the choline consumption process of cancer cells have been successfully realized. Thus, this new polymer probe promotes the development of enzymatic biosensors and provides a simpler and more effective way for detecting the chemical transmitter of living cells.

  5. Reaction-Driven Self-Assembled Micellar Nanoprobes for Ratiometric Fluorescence Detection of CS2 with High Selectivity and Sensitivity.

    Science.gov (United States)

    Lu, Wei; Xiao, Peng; Liu, Zhenzhong; Gu, Jincui; Zhang, Jiawei; Huang, Youju; Huang, Qing; Chen, Tao

    2016-08-10

    The detection of highly toxic CS2, which is known as a notorious occupational hazard in various industrial processes, is important from both environmental and public safety perspectives. We describe here a robust type of chemical-reaction-based supramolecular fluorescent nanoprobes for ratiometric determination of CS2 with high selectivity and sensitivity in water medium. The micellar nanoprobes self-assemble from amphiphilic pyrene-modified hyperbranched polyethylenimine (Py-HPEI) polymers with intense pyrene excimer emission. Selective sensing is based on a CS2-specific reaction with hydrophilic amino groups to produce hydrophobic dithiocarbamate moieties, which can strongly quench the pyrene excimer emission via a known photoinduced electron transfer (PET) mechanism. Therefore, the developed micellar nanoprobes are free of the H2S interference problem often encountered in the widely used colorimetric assays and proved to show high selectivity over many potentially competing chemical species. Importantly, the developed approach is capable of CS2 sensing even in complex tap and river water samples. In addition, in view of the modular design principle of these powerful micellar nanoprobes, the sensing strategy used here is expected to be applicable to the development of various sensory systems for other environmentally important guest species. PMID:27419849

  6. Ratiometric measurement of hydrogen sulfide and cysteine/homocysteine ratios using a dual-fluorophore fragmentation strategy.

    Science.gov (United States)

    Hammers, Matthew D; Pluth, Michael D

    2014-07-15

    Hydrogen sulfide (H2S) is an integral signaling molecule in biology with complex generation, translocation, and metabolism processes that are intertwined with cellular thiols. Differentiating the complex interplay between H2S and biological thiols, however, remains challenging due to the difficulty of monitoring H2S and thiol levels simultaneously in complex redox environments. As a step toward unraveling the complexities of H2S and thiols in sulfur redox homeostasis, we present a dual-fluorophore fragmentation strategy that allows for the ratiometric determination of relative H2S and cysteine (Cys) or homocysteine (Hcy) concentrations, two important metabolites in H2S biosynthesis. The key design principle is based on a nitrobenzofurazan-coumarin (NBD-Coum) construct, which fragments into spectroscopically differentiable products upon nucleophilic aromatic substitution with either H2S or Cys/Hcy. Measurement of the ratio of fluorescence intensities from coumarin and the NBD-Cys or NBD-Hcy adducts generates a sigmoidal response with a dynamic range of 3 orders of magnitude. The developed scaffold displays a rapid response (selective for sulfhydryl-containing nucleophiles over other reactive sulfur, oxygen, and nitrogen species, including alcohol- and amine-functionalized amino acids, polyatomic anionic sulfur species, NO, and HNO. Additionally, NBD-Coum is demonstrated to differentiate and report on different oxidative stress stimuli in simulated sulfur pools containing H2S, Cys, and cystine.

  7. Facile and high spatial resolution ratio-metric luminescence thermal mapping in microfluidics by near infrared excited upconversion nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yu; Li, Shunbo; Wen, Weijia, E-mail: phwen@ust.hk [Department of Physics, KAUST-HKUST Joint Micro/Nanofluidic Laboratory, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon (Hong Kong); Cao, Wenbin [Nano Science and Technology Program, Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon (Hong Kong)

    2016-02-01

    A local area temperature monitor is important for precise control of chemical and biological processes in microfluidics. In this work, we developed a facile method to realize micron spatial resolution of temperature mapping in a microfluidic channel quickly and cost effectively. Based on the temperature dependent fluorescence emission of NaYF{sub 4}:Yb{sup 3+}, Er{sup 3+} upconversion nanoparticles (UCNPs) under near-infrared irradiation, ratio-metric imaging of UCNPs doped polydimethylsiloxane can map detailed temperature distribution in the channel. Unlike some reported strategies that utilize temperature sensitive organic dye (such as Rhodamine) to achieve thermal sensing, our method is highly chemically inert and physically stable without any performance degradation in long term operation. Moreover, this method can be easily scaled up or down, since the spatial and temperature resolution is determined by an optical imaging system. Our method supplied a simple and efficient solution for temperature mapping on a heterogeneous surface where usage of an infrared thermal camera was limited.

  8. Carbon-Dot and Quantum-Dot-Coated Dual-Emission Core-Satellite Silica Nanoparticles for Ratiometric Intracellular Cu(2+) Imaging.

    Science.gov (United States)

    Zou, Chenchen; Foda, Mohamed Frahat; Tan, Xuecai; Shao, Kang; Wu, Long; Lu, Zhicheng; Bahlol, Hagar Shendy; Han, Heyou

    2016-07-19

    Copper (Cu(2+)) is physiologically essential, but excessive Cu(2+) may cause potential risk to plants and animals due to the bioaccumulative properties. Hence, sensitive recognition is crucial to avoid overintake of Cu(2+), and visual recognition is more favored for practical application. In this work, a dual-emission ratiometric fluorescent nanoprobe was developed possessing the required intensity ratio, which can facilitate the sensitive identification of Cu(2+) by the naked eye. The probe hybridizes two fluorescence nanodots (quantum dots (QDs) and carbon dots (CDs)). Although both of them can be viable fluorescence probes for metal ion detection, rarely research has coupled this two different kinds of fluorescence material in one nanosensor to fabricate a selectively ratiometric fluorescence probe for intracellular imaging. The red emitting CdTe/CdS QDs were capped around the silica microsphere to serve as the response signal label, and the blue-emitting CDs, which is insensitive to the analyte, were covalently attached to the QDs surface to act as the reference signal. This core-satellite hybrid sphere not only improves the stability and brightness of QDs significantly but also decreases the cytotoxicity toward HeLa cells tremendously. Moreover, the Cu(2+) could quench the QDs emission effectively but have no ability for reduction of the CDs emission. Accordingly, a simple, efficient, and precise method for tracing Cu(2+) was proposed. The increase of Cu(2+) concentration in the series of 0-3 × 10(-6) M was in accordance with linearly decrease of the F650/F425 ratio. As for practical application, this nanosensor was utilized to the ratiometric fluorescence imaging of copper ions in HeLa cells. PMID:27347813

  9. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Mengjuan [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Chengquan [School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013 (China); Qian, Jing, E-mail: qianj@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kun, E-mail: wangkun@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China)

    2015-08-12

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg{sup 2+}. The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg{sup 2+}, the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg{sup 2+} in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg{sup 2+} contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg{sup 2+} quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg{sup 2+} detection. • The Hg{sup 2+} content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg{sup 2+} detection. • The ratiometric probe is of good simplicity, low toxicity, and

  10. Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

    Energy Technology Data Exchange (ETDEWEB)

    Noor, M. Omair; Hrovat, David [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Moazami-Goudarzi, Maryam [Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Espie, George S. [Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada); Krull, Ulrich J., E-mail: ulrich.krull@utoronto.ca [Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6 (Canada)

    2015-07-23

    Highlights: • Solid-phase QD-FRET transduction of isothermal tHDA amplicons on paper substrates. • Ratiometric QD-FRET transduction improves assay precision and lowers the detection limit. • Zeptomole detection limit by an iPad camera after isothermal amplification. • Tunable assay sensitivity by immobilizing different amounts of QD–probe bioconjugates. - Abstract: Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric

  11. Novel Pyrene-armed Calix[4]arenes through Triazole Connec-tion: Ratiometric Fluorescent Chemosensor for Zn2+ and Promising Structure for Integrated Logic Gates

    Institute of Scientific and Technical Information of China (English)

    ZHU Lin-Na; GONG Shao-Long; GONG Shu-Ling; YANG Chu-Luo; QIN Jin-Gui

    2008-01-01

    Two novel pyrene-armed calix[4]arenes by triazole connection were synthesized using "click" chemistry. Com-pound 1 with two pyrene subunits appended to the lower rims of the calix[4]arene shows ratiometric fluorescence response toward Zn2+, and selective fluorescence quenching toward heavy metal ions such as Cu2+, Hg2+ and pb2+; while compound 2 with one pyrene subunit exhibits significant fluorescence quenching toward Cu2+ and moderate quenching behaviour toward Hg2+. By utilizing the different fluorescence behavior of 1 toward Zn2+and Cu2+, inhi-bition (INH) and not or (NOR) logic gates were established.

  12. Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides.

    Science.gov (United States)

    Tang, Tingting; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-01-01

    Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core-shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO-aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO-aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO-aptamer duplex changed in the detection process of CE-LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE-LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer-specific small molecule targets by simply changing the types of aptamer sequences.

  13. Preparation of europium-quantum dots and europium-fluorescein composite nanoparticles available for ratiometric luminescent detection of metal ions

    Science.gov (United States)

    Dong, Haitao; Liu, Yan; Wang, Dandan; Zhang, Wenzhu; Ye, Zhiqiang; Wang, Guilan; Yuan, Jingli

    2010-10-01

    The silica-encapsulated luminescent lanthanide nanoparticles have been developed for the selective tagging of a wide range of important targets in recent years, however, they are mainly limited to europium and terbium compounds. In this work, two types of europium-containing dual-luminophore silica nanoparticles, silica-encapsulated CdTe quantum dots (CdTe QDs)-BHHCT-Eu3 + complex nanoparticles and BHHCT-Eu3 + surface-bound silica-encapsulated fluorescein isothiocyanate (FITC) nanoparticles (BHHCT: 4, 4'-bis(1'', 1'', 1'', 2'', 2'', 3'', 3''-heptafluoro-4'', 6''-hexanedion-6''-yl)chlorosulfo-o-terphenyl), were successfully prepared using a water-in-oil (W/O) reverse microemulsion method. The results of transmission electron microscopy and luminescence spectroscopy characterizations indicate that the two types of nanoparticles are all monodisperse, spherical and uniform in size (~50 nm in diameter), and have well-resolved and stable dual luminescence emission properties. The CdTe QDs-BHHCT-Eu3 + nanoparticles can be excited at 365 nm to give dual-emission peaks at 535 and 610 nm, and the FITC-BHHCT-Eu3 + nanoparticles can be excited at 335 nm to give dual-emission peaks at 515 and 610 nm. The luminescence response investigations of the nanoparticles to different metal ions indicate that the new nanoparticles can be used as ratiometric luminescent sensing probes for the selective detection of Cu2 + and Fe2 + ions, respectively. The performance of the nanoparticle probe for metal ion detection was investigated.

  14. rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

    Science.gov (United States)

    Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

    2016-04-01

    Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry.

  15. Visual and fluorescent detection of acetamiprid based on the inner filter effect of gold nanoparticles on ratiometric fluorescence quantum dots

    International Nuclear Information System (INIS)

    Highlights: • The RF-QDs were fabricated by two different QDs using layer-by-layer assembly methods. • The PL intensity of RF-QDs could be quenched by AuNPs based on inner-filter effect. • Acetamiprid can adsorb on AuNPs led to the PL intensity of RF-QDs recover properly. • AuNPs serve a dual function as fluorescence quencher and colorimetric reporter in the sensor. - Abstract: In this work, we develop a simple and rapid sensing method for the visual and fluorescent detection of acetamiprid (AC) based on the inner-filter effect (IFE) of gold nanoparticles (AuNPs) on ratiometric fluorescent quantum dots (RF-QDs). The RF-QDs based dual-emission nanosensor was fabricated by assembling green emissive QDs (QDs539 nm, λem = 539 nm) on the surface of red emissive QDs (QDs661 nm, λem = 661 nm)-doped silica microspheres. The photoluminescence (PL) intensity of RF-QDs could be quenched by AuNPs based on IFE. Acetamiprid can adsorb on the surface of AuNPs due to its cyano group that has good affinity with gold, which could induce the aggregation of AuNPs accompanying color change from red to blue. Thus, the IFE of AuNPs on RF-QDs was weakened and the PL intensity of RF-QDs was recovered accordingly. Under the optimized conditions, the PL intensity of the RF-QDs/AuNPs system was proportional to the concentration of AC in the range of 0.025–5.0 μg mL−1, with a detection limit of 16.8 μg L−1. The established method had been used for AC detection in environmental and agricultural samples with satisfactory results

  16. On-Resonance Fluorescence, Resonance Rayleigh Scattering, and Ratiometric Resonance Synchronous Spectroscopy of Molecular- and Quantum Dot-Fluorophores.

    Science.gov (United States)

    Siriwardana, Kumudu; Nettles, Charles B; Vithanage, Buddhini C N; Zhou, Yadong; Zou, Shengli; Zhang, Dongmao

    2016-09-20

    Existing studies on molecular fluorescence have almost exclusively been focused on Stokes-shifted fluorescence spectroscopy (SSF) in which the emitted photon is detected at the wavelengths longer than that for the excitation photons. Information on fluorophore on-resonance fluorescence (ORF) and resonance Rayleigh scattering (RRS) is limited and often problematic due to the complex interplay of the fluorophore photon absorption, ORF emission, RRS, and solvent Rayleigh scattering. Reported herein is a relatively large-scale systematic study on fluorophore ORF and RRS using the conventional UV-vis extinction and SSF measurements in combination with the recently reported ratiometric resonance synchronous spectroscopic (R2S2, pronounced as "R-Two-S-Two") method. A series of fundamental parameters including fluorophore ORF cross sections and quantum yields have been quantified for the first time for a total of 12 molecular and 6 semiconductor quantum dot (QD) fluorophores. All fluorophore spectra comprise a well-defined Gaussian peak with a full width at half-maximum ranging from 4 to 30 nm. However, the RRS features of fluorophores differ drastically. The effect of fluorophore aggregation on its RRS, UV-vis, R2S2, and SSF spectra was also discussed. This work highlights the critical importance of the combined UV-vis extinction, SSF, and R2S2 spectroscopic measurements for material characterizations. The method and insights described in this work can be directly used for improving the reliability of RRS spectroscopic methods in chemical analysis. In addition, it should pave the way for developing novel R2S2-based analytical applications.

  17. Preparation of europium-quantum dots and europium-fluorescein composite nanoparticles available for ratiometric luminescent detection of metal ions

    Energy Technology Data Exchange (ETDEWEB)

    Dong Haitao; Liu Yan; Wang Dandan; Zhang Wenzhu; Ye Zhiqiang; Wang Guilan; Yuan Jingli, E-mail: zhiqiangye2001@yahoo.com.cn, E-mail: jingliyuan@yahoo.com.cn [State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116012 (China)

    2010-10-01

    The silica-encapsulated luminescent lanthanide nanoparticles have been developed for the selective tagging of a wide range of important targets in recent years, however, they are mainly limited to europium and terbium compounds. In this work, two types of europium-containing dual-luminophore silica nanoparticles, silica-encapsulated CdTe quantum dots (CdTe QDs)-BHHCT-Eu{sup 3+} complex nanoparticles and BHHCT-Eu{sup 3+} surface-bound silica-encapsulated fluorescein isothiocyanate (FITC) nanoparticles (BHHCT: 4, 4'-bis(1{sup ''}, 1{sup ''}, 1{sup ''}, 2{sup ''}, 2{sup ''}, 3{sup ''}, 3{sup ''}-heptafluoro-4{sup ''}, 6{sup ''}-hexanedion-6{sup ''}-yl)chlorosulfo-o-terphenyl), were successfully prepared using a water-in-oil (W/O) reverse microemulsion method. The results of transmission electron microscopy and luminescence spectroscopy characterizations indicate that the two types of nanoparticles are all monodisperse, spherical and uniform in size ({approx}50 nm in diameter), and have well-resolved and stable dual luminescence emission properties. The CdTe QDs-BHHCT-Eu{sup 3+} nanoparticles can be excited at 365 nm to give dual-emission peaks at 535 and 610 nm, and the FITC-BHHCT-Eu{sup 3+} nanoparticles can be excited at 335 nm to give dual-emission peaks at 515 and 610 nm. The luminescence response investigations of the nanoparticles to different metal ions indicate that the new nanoparticles can be used as ratiometric luminescent sensing probes for the selective detection of Cu{sup 2+} and Fe{sup 2+} ions, respectively. The performance of the nanoparticle probe for metal ion detection was investigated.

  18. Exploring 1,4-dihydroxyanthraquinone as long-range emissive ratiometric fluorescent probe for signaling Zn(2+)/PO4(3-): Ensemble utilization for live cell imaging.

    Science.gov (United States)

    Sinha, Sougata; Gaur, Pankaj; Mukherjee, Trinetra; Mukhopadhyay, Subhrakanti; Ghosh, Subrata

    2015-07-01

    Fluorescent 1,4-dihydroxyanthraquinone 1 was found to demonstrate its ratiometric signaling property upon interaction with divalent zinc (Zn(2+)). While the probe itself exhibited fluorescence emission in the yellow region (λem=544 nm and 567 nm), binding with Zn(2+) induced strong emission in the orange region (λem=600 nm) which was mainly due to a combination of CHEF and ICT mechanism. The probe was found to be highly sensitive toward the detection of zinc and the limit of detection (LOD) was calculated to be 9×10(-7) M. The possibility of using this probe for real-time analysis was strongly supported by the striking stability of fluorescence signal for more than five days with similar fluorescence intensity as observed during instant signaling. The present probe works within physiological pH range and is devoid of any interference caused by the same group elements such as Cd(2+)/Hg(2+). The probe possesses excellent excitation/emission wavelength profile and can penetrate cell membrane to image low concentration of zing inside living system. The in situ formed zinc-probe ensemble was further explored as ratiometric sensing platform for detecting another bio-relevant analyte phosphate anion through a zinc-displacement approach.

  19. Combined surface-enhanced Raman spectroscopy biotags and microfluidic platform for quantitative ratiometric discrimination between noncancerous and cancerous cells in flow

    Science.gov (United States)

    Pallaoro, Alessia; Hoonejani, Mehran R.; Braun, Gary B.; Meinhart, Carl; Moskovits, Martin

    2013-01-01

    Surface-enhanced Raman spectroscopy (SERS) biotags (SBTs) that carry peptides as cell recognition moieties were made from polymer-encapsulated silver nanoparticle dimers, infused with unique Raman reporter molecules. We previously demonstrated their potential use for identification of malignant cells, a central goal in cancer research, through a multiplexed, ratiometric method that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro based on receptor overexpression. Progress has been made toward the application of this quantitative methodology for the identification of cancer cells in a microfluidic flow-focusing device. Beads are used as cell mimics to evaluate the devices. Cells (and beads) are simultaneously incubated with two sets of SBTs while in suspension, then injected into the device for laser interrogation under flow. Each cell event is characterized by a composite Raman spectrum, deconvoluted into its single components to ultimately determine their relative contribution. We have found that using SBTs ratiometrically can provide cell identification in flow, insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, autofluorescence, and turbidity.

  20. Monitoring of the proton electrochemical gradient in reconstituted vesicles: quantitative measurements of both transmembrane potential and intravesicular pH by ratiometric fluorescent probes.

    Science.gov (United States)

    Holoubek, Ales; Vecer, Jaroslav; Sigler, Karel

    2007-03-01

    Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi response can be calibrated by the K(+)/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Deltapsi and DeltapH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.

  1. A ratiometric rhodamine–naphthalimide pH selective probe built on the basis of a PAMAM light-harvesting architecture

    Energy Technology Data Exchange (ETDEWEB)

    Alamry, Khalid A. [Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Georgiev, Nikolai I. [Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Street, 1756 Sofia (Bulgaria); El-Daly, Samy Abdullah [Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, P.O. Box 80203 (Saudi Arabia); Taib, Layla A. [Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Bojinov, Vladimir B., E-mail: vlbojin@uctm.edu [Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Street, 1756 Sofia (Bulgaria)

    2015-02-15

    PAMAM light harvesting antenna of second generation was synthesized and investigated. Novel compound was configured as a wavelength-shifting bichromophoric molecule where the system surface is labeled with yellow-green emitting 4-(N,N-dimethylamino)ethylamino-1,8-naphthalimide “donor” units capable of absorbing light and efficiently transferring the energy to a focal Rhodamine 6G “acceptor”. Furthermore, the 1,8-naphthalimide periphery of the system was designed on the “fluorophore-spacer-receptor” format, capable of acting as a molecular fluorescence photoinduced electron transfer based probe. Due to the both effects, photoinduced electron transfer in the periphery of the system and pH dependent rhodamine core absorption, novel antenna is able to act as a selective ratiometric pH fluorescence probe in aqueous medium. Thus, the distinguishing features of light-harvesting systems (fluorescence resonance energy transfer) were successfully combined with the properties of classical ring-opening sensor systems, which may be beneficial for monitoring pH variations in complex samples. - Highlights: • PAMAM antenna decorated with Rhodamine 6G and 1,8-naphthalimides is synthesized. • Periphery of the antenna is designed as a PET based fluorescence probe. • System manifests excellent selective response to protons in aqueous medium. • Core emission of the systems is enhanced more than 10 times as a function of pH. • Bichromophoric system acts as a selective ratiometric probe in complex samples.

  2. A regenerative ratiometric electrochemical biosensor for selective detecting Hg²⁺ based on Y-shaped/hairpin DNA transformation.

    Science.gov (United States)

    Jia, Jing; Chen, Hong Guo; Feng, Ji; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing

    2016-02-18

    Inspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions (Hg(2+)) based on Y-shaped DNA (Y-DNA) was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene (Fc) signal and a weak methylene blue (MB) signal were observed. Upon addition of Hg(2+), the Y-DNA structure was transformed to hairpin structure based on the formation of T-Hg(2+)-T complex. During the transformation, the redox MB gets close to and the redox Fc gets far away from the electrode surface, respectively. This special design allows a reliable Hg(2+) detection with a detection range from 1 nM to 5 μM and a low detection limit down to 0.094 nM. Furthermore, this biosensor exhibits good selectivity and repeatability, and can be easily regenerated by using L-cysteine. This study offers a simple and effective method for designing ratiometric biosensors for detecting other ions and biomolecules.

  3. Efficient On-Off Ratiometric Fluorescence Probe for Cyanide Ion Based on Perturbation of the Interaction between Gold Nanoclusters and a Copper(II)-Phthalocyanine Complex.

    Science.gov (United States)

    Shojaeifard, Zahra; Hemmateenejad, Bahram; Shamsipur, Mojtaba

    2016-06-22

    A new ratiometric fluorescent sensor was developed for the sensitive and selective detection of cyanide ion (CN(-)) in aqueous media. The ratiometric sensing system is based on CN(-) modulated recovery of copper(II) phthalocyanine (Cu(PcTs)) fluorescence signal at the expense of diminished fluorescence intensity of gold nanoclusters (AuNCs). Preliminary experiments revealed that the AuNCs and Cu(PcTs) possess a turn-off effect on each other, the interaction of which being verified through studying their interactions by principle component analysis (PCA) and multivariate cure resolution-alternating least-squares (MCR-ALS) methods. In the presence of CN(-) anion, the AuNCs and Cu(PcTs) interaction was perturbed, so that the fluorescence of Cu (PcTs), already quenched by AuNCs, was found to be efficiently recovered, while the fluorescence intensity of AuNCs was quenched via the formation of a stable [Au(CN)2](-) species. The ratiometric variation of AuNCs and Cu(PcTs) fluorescence intensities leads to designing a highly sensitive probe for CN(-) ion detection. Under the optimal conditions, CN(-) anion was detected without needing any etching time, over the concentration range of 100 nM-220 μM, with a detection limit of 75 nM, which is much lower than the allowable level of CN(-) in water permitted by the World Health Organization (WHO). Moreover, the detection of CN(-) was developed based on the CN(-) effects on the blue and red florescent colors of Cu(PcTs) and AuNCs, respectively. The designed probe displays a continuous color change from red to blue by addition of CN(-), which can be clearly observed by the naked eye in the range of 7-350 μM, under UV lamp. The prepared AuNCs/Cu(PcTs) probe was successfully utilized for the selective and sensitive determination of CN(-) anion in two different types of natural water (Rodbal dam and rainwater) and also in blood serum as a biological sample. PMID:27211049

  4. Ratiometric fluorescent ion detection in water with high sensitivity via aggregation-mediated fluorescence resonance energy transfer using a conjugated polyelectrolyte as an optical platform.

    Science.gov (United States)

    Le, Van Sang; Kim, Boram; Lee, Wonho; Jeong, Ji-Eun; Yang, Renqiang; Woo, Han Young

    2013-05-14

    A cationic conjugated polyelectrolyte was designed and synthesized based on poly(fluorene-co-phenylene) containing 5 mol% benzothiadiazole (BT) as a low energy trap and 15-crown-5 as a recognizing group for potassium ions. A potassium ion can form a sandwich-type 2:1 Lewis acid-based complex with 15-crown-5, to cause the intermolecular aggregation of polymers. This facilitates inter-chain fluorescence resonance energy transfer (FRET) to a low-energy BT segment, resulting in fluorescent signal amplification, even at dilute analyte concentrations. Highly sensitive and selective detection of K(+) ions was demonstrated in water. The linear response of ratiometric fluorescent signal as a function of [K(+) ] allows K(+) quantification in a range of nanomolar concentrations with a detection limit of ≈0.7 × 10(-9) M. PMID:23417971

  5. Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore.

    Science.gov (United States)

    Chib, Rahul; Mummert, Mark; Bora, Ilkay; Laursen, Bo W; Shah, Sunil; Pendry, Robert; Gryczynski, Ignacy; Borejdo, Julian; Gryczynski, Zygmunt; Fudala, Rafal

    2016-05-01

    In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase. PMID:26993308

  6. A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.

    Science.gov (United States)

    Kim, Sung Bae; Ozawa, Takeaki; Umezawa, Yoshio

    2016-01-01

    We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147-149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: •Cytokine activities are determined within 2 h after stimulation.•Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.•Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity. PMID:27489781

  7. Neuronal acid-induced [Zn²⁺]i elevations calibrated using the low-affinity ratiometric probe FuraZin-1.

    Science.gov (United States)

    Kiedrowski, Lech

    2015-11-01

    The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn(2+) ] ([Zn(2+) ]i ) elevations, a low affinity (Kd = 39 μM at pH 6.1) ratiometric Zn(2+) probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn(2+) ]i elevations reaching 2 μM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn(2+) ]i increased further to 5.6 μM; analogous acid- and NEM-induced [Zn(2+) ]i elevations could also be detected but not calibrated, using the high affinity Zn(2+) probe FluoZin-3. The data indicate that NEM causes Zn(2+) release from ligands that chelate Zn(2+) at pH 6.1. ATP could also chelate Zn(2+) at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn(2+) ]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn(2+) ]i increases. These data suggest that the total amount of Zn(2+) accumulated in intracellular ATP-dependent stores (Zn(2+) -ATP complexes and organelles that accumulate Zn(2+) in an ATP-dependent manner) is negligible compared to the amount of Zn(2+) accumulated in the acid-sensitive intracellular ligands. In vitro, upon acidification, Zn(2+) -cysteine complexes release Zn(2+) and ATP chelates the released Zn(2+) . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid-induced [Zn(2+) ]i elevations. These [Zn(2+) ]i elevations were calibrated using a low affinity ratiometric probe FuraZin-1; they reached 2 µM levels and increased to 5 µM when a thiol-reactive agent, N-ethylmaleimide, compromised Zn(2+) binding by cysteines. PMID:26263185

  8. 9-Aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one (Seoul-Fluor) as a smart platform for colorful ratiometric fluorescent pH sensors.

    Science.gov (United States)

    Kim, Eunha; Lee, Sanghee; Park, Seung Bum

    2011-07-21

    In this communication, we report that 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one (Seoul-Fluor) can serve as a potential platform for colorful ratiometric fluorescent pH sensors by simple incorporation of pH responsive elements on Seoul-Fluor. Seoul-Fluor-based fluorescent pH sensors allow the emission- and pH-tuning ability upon protonation by varying their pK(a) values and electronic characteristics of substituents by a rational design.

  9. Computational analysis and ratiometric comparison approaches aimed to assist column selection in hydrophilic interaction liquid chromatography-tandem mass spectrometry targeted metabolomics.

    Science.gov (United States)

    Sampsonidis, Ioannis; Witting, Michael; Koch, Wendelin; Virgiliou, Christina; Gika, Helen G; Schmitt-Kopplin, Philippe; Theodoridis, Georgios A

    2015-08-01

    In the present work two different approaches, a semi-quantitative and a Derringer function approach, were developed to assist column selection for method development in targeted metabolomics. These approaches were applied in the performance assessment of three HILIC columns with different chemistries (an amide, a diol and a zwitterionic phase). This was the first step for the development of a HILIC UPLC-MS/MS method that should be capable to analyze a large number of polar metabolites. Two gradient elution profiles and two mobile phase pH values were tested for the analysis of multi-analyte mixtures. Acquired chromatographic data were firstly treated by a ratiometric, "semi-quantitative" approach which quantifies various overall analysis parameters (e.g. the percent of detected compounds, retentivity and resolved critical pairs). These parameters were used to assess chromatographic performance in a rather conventional/traditional and cumbersome/labor-intensive way. Secondly, a comprehensive and automated comparison of the three columns was performed by monitoring several well-known chromatographic parameters (peak width, resolution, tailing factor, etc.) using a lab-built programming script which calculates overall desirability utilizing Derringer functions. Derringer functions exhibit the advantage that column performance is ultimately expressed in an objective single and quantitative value which can be easily interpreted. In summary, results show that each column exhibits unique strengths in metabolic profiling of polar compounds. The applied methodology proved useful for the selection of the most effective chromatographic system during method development for LC-MS/MS targeted metabolomics, while it could further assist in the selection of chromatographic conditions for the development of multi-analyte methods.

  10. Design of NIR Chromenylium-Cyanine Fluorophore Library for "Switch-ON" and Ratiometric Detection of Bio-Active Species In Vivo.

    Science.gov (United States)

    Wei, Yanfen; Cheng, Dan; Ren, Tianbing; Li, Yinhui; Zeng, Zebing; Yuan, Lin

    2016-02-01

    The real-time monitoring of key biospecies in the living systems has received thrusting attention during the past decades. Specifically, fluorescent detection based on near-infrared (NIR) fluorescent probes is highly favorable for live cells, live tissues, and even animal imaging, owing to the substantial merits of the NIR window, such as minimal phototoxicity, deep penetration into tissues, and low autofluorescence background. Nevertheless, developing potent NIR fluorescent probes still poses serious challenges to the chemists because traditional NIR fluorophores are less tunable than visible-wavelength fluorophores. To address this issue, here we report a set of novel NIR hybrid fluorophores, namely, the hybrid chromenylium-cyanine fluorophore (CC-Fluor), in which both the fluorescence intensity and the emission wavelength can be easily adjusted by the conformational changes and substitution groups. Compared to known NIR fluorophores, the new CC-Fluors are substantially advantageous for NIR probe development: (1) CC-Fluors display tunable and moderate Stokes shifts and quantum yields; (2) the fluorophores are stable at physiological conditions after long-term incubation; (3) the absorption maxima of CC-Fluors coincide with the common laser spectral lines in mainstream in vivo imaging systems; (4) most importantly, CC-Fluors can be easily modified to prepare NIR probes targeting various biospecies. To fully demonstrate the practical utility of CC-Fluors, we report two innovative NIR probes, a ratiometric pH probe and a turn-on Hg(2+) probe, both are successfully employed in live animal imaging. Hence, the detailed studies allow us to confirm that CC-Fluors can work as an excellent platform for developing NIR probes for the detection of species in living systems. PMID:26730493

  11. Design of a dual-signaling sensing system for fluorescent ratiometric detection of Al3+ ion based on the inner-filter effect.

    Science.gov (United States)

    Wang, Yongxiang; Xiong, Limin; Geng, Fenghua; Zhang, Fuqiang; Xu, Maotian

    2011-11-21

    A dual-signal sensing system based on the inner-filter effect (IFE) was demonstrated, in which the combination of two signaling mechanisms allows metal binding to turn on two fluorescence emission bands, independently. A proof-of-concept fluorescent ratiometric assay for Al(3+) in pure aqueous solution is presented. The proposed assay is based on the Al(3+)-induced color and fluorescence changes of Alizarin red S (ARS) and IFE between ARS and meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP). In the absence of Al(3+), the absorption spectrum of the ARS in 0.2 M HAc-NaAc buffer (pH 5.5) has a strong peak at 420 nm, significantly overlapping with the excitation of TMPyP. ARS is expected to be capable of functioning as a powerful absorber to tune the emission of TMPyP on account of the spectral overlap. Binding of Al(3+) with ARS forms a fluorometric ARS/Al(3+) complex and shifts the maximum absorbance from 420 nm to 480 nm, which overlaps negligibly with the excitation of TMPyP and turns on the proper emission spectrum for TMPyP. Under the optimum conditions, The fluorescence intensity ratio, F(585)/F(651), responds to Al(3+) over a dynamic range of 0.1-1.5 μM, with a limit of detection of 40 nM, where F(585) and F(651) are the fluorescence intensity at 585 nm and 651 nm in the absence or presence of Al(3+), respectively. Further application in Al(3+)-spiked water samples suggested a recovery between 95 and 108%. The fluorescence response is highly selective for Al(3+) over other metal ions with the addition of thiourea as the masking agent.

  12. Development of dual-emission ratiometric probe-based on fluorescent silica nanoparticle and CdTe quantum dots for determination of glucose in beverages and human body fluids.

    Science.gov (United States)

    Zhai, Hong; Feng, Ting; Dong, Lingyu; Wang, Liyun; Wang, Xiangfeng; Liu, Hailing; Liu, Yuan; Chen, Luan; Xie, MengXia

    2016-08-01

    A novel dual emission ratiometric fluorescence probe for determination of glucose has been developed. The reference dye fluorescence isothiocyanate (FITC) has been encapsulated in the silica nanoparticles and then the red emission CdTe QDs were grafted on the surface of the silica particles to obtain the fluorescence probe. With glucose and dopamine as substrates, the glucose level was proportional to the fluorescence ratio change of above probe caused by dopamine oxidation, which was produced via bienzyme catalysis (glucose oxidase and horseradish peroxidase). The established approach was sensitive and selective, and has been applied to determine the glucose in beverage, urine and serum samples. The average recoveries of the glucose at various spiking levels ranged from 95.5% to 108.9% with relative standard deviations from 1.5% to 4.3%. The results provided a clue to develop sensors for rapid determination of the target analytes from complex matrices. PMID:26988523

  13. Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal

    Directory of Open Access Journals (Sweden)

    John J. Willoughby

    2011-10-01

    Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.

  14. Genetically encoded sensors enable real-time observation of metabolite production

    OpenAIRE

    Rogers, Jameson K.; Church, George M.

    2016-01-01

    Advances in biotechnology are enabling engineers to harness natural processes for the production of valuable new chemicals and materials. Cells engineered for chemical production act as renewable factories and redefine what is possible in industries as diverse as manufacturing, pharmaceuticals, and energy. Despite this potential, long and uncertain timelines for biobased product development hinder progress. Engineering cells for chemical production is challenging because the complexity of bio...

  15. Statistical sulcal shape comparisons: application to the detection of genetic encoding of the central sulcus shape

    DEFF Research Database (Denmark)

    Le Goualher, G; Argenti, A.M.; Duyme, M;

    2000-01-01

    encoding. When applied to real data, this study highlighted genetic constraints on the shape of the central sulcus. We found from 10 pairs of monozygotic twins that the intrapair modal distance of the central sulcus was significantly smaller than the interpair modal distance, for both the left central...... sulcus (Z = -2.66; P sulcus (Z = -2.26; P sulcus shape were confirmed by applying the same experiment to 10 pairs of normal young individuals (Z = -1.39; Z = -0.63, i.e., values not significant at the P

  16. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  17. Abscisic acid dynamics in roots detected with genetically encoded FRET sensors.

    Science.gov (United States)

    Jones, Alexander M; Danielson, Jonas Ah; Manojkumar, Shruti N; Lanquar, Viviane; Grossmann, Guido; Frommer, Wolf B

    2014-04-15

    Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2-800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI: http://dx.doi.org/10.7554/eLife.01741.001.

  18. Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids.

    Science.gov (United States)

    Lim, Sung In; Kwon, Inchan

    2016-10-01

    The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering. PMID:26036278

  19. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors

    Science.gov (United States)

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin

    2015-08-01

    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

  20. Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

    OpenAIRE

    Ma, Limei; Haga-Yamanaka, Sachiko; Yu, Qingfeng Elden; Qiu, Qiang; Kim, Sangseong; Yu, C. Ron

    2011-01-01

    The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the det...

  1. Using genetically encoded fluorescent reporters to image lipid signalling in living plants

    NARCIS (Netherlands)

    J.E.M. Vermeer; T. Munnik

    2013-01-01

    The discovery of the green fluorescent protein has revolutionized cell biology as it allowed researchers to visualize dynamic processes in living cells. The fusion of fluorescent protein variants with lipid binding domains that bind to specific phospholipids have been very instrumental in investigat

  2. Use of genetically encoded, light-gated ion translocators to control tumorigenesis

    Science.gov (United States)

    Chernet, Brook T.; Adams, Dany S.; Lobikin, Maria; Levin, Michael

    2016-01-01

    It has long been known that the resting potential of tumor cells is depolarized relative to their normal counterparts. More recent work has provided evidence that resting potential is not just a readout of cell state: it regulates cell behavior as well. Thus, the ability to control resting potential in vivo would provide a powerful new tool for the study and treatment of tumors, a tool capable of revealing living-state physiological information impossible to obtain using molecular tools applied to isolated cell components. Here we describe the first use of optogenetics to manipulate ion-flux mediated regulation of membrane potential specifically to prevent and cause regression of oncogene-induced tumors. Injection of mutant-KRAS mRNA induces tumor-like structures with many documented similarities to tumors, in Xenopus tadpoles. We show that expression and activation of either ChR2D156A, a blue-light activated cation channel, or Arch, a green-light activated proton pump, both of which hyperpolarize cells, significantly lowers the incidence of KRAS tumor formation. Excitingly, we also demonstrate that activation of co-expressed light-activated ion translocators after tumor formation significantly increases the frequency with which the tumors regress in a process called normalization. These data demonstrate an optogenetic approach to dissect the biophysics of cancer. Moreover, they provide proof-of-principle for a novel class of interventions, directed at regulating cell state by targeting physiological regulators that can over-ride the presence of mutations. PMID:26988909

  3. Imaging bacterial protein expression using genetically encoded sensors composed of RNA

    OpenAIRE

    Song, Wenjiao; Strack, Rita L.; Jaffrey, Samie R.

    2013-01-01

    We show that the difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins, and exhibit fluorescence increases that enable protein expression to be imaged in living cells. These sensors provide a general strategy to image protein expression in single bacteria in real-time.

  4. Herpesvirus-Mediated Delivery of a Genetically Encoded Fluorescent Ca2+ Sensor to Canine Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    János Prorok

    2009-01-01

    Full Text Available We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca2+ sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (Ito, kinetics of the intracellular Ca2+ transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the Ito current was significantly changed and no major shifts occurred in parameters of [Ca2+]i transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

  5. Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

    Science.gov (United States)

    Kim, Sung Bae; Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

    2016-02-17

    Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects. PMID:26322739

  6. Photophysical Properties of a Red-Shift Cu(II) Ratiometric Fluorescent Chemosensor%红移型Cu(II)离子比率荧光探针的光物理性质

    Institute of Scientific and Technical Information of China (English)

    崔俐丽; 周丹红; 李苗苗

    2013-01-01

      应用密度泛函理论(DFT)及含时密度泛函理论(TDDFT)方法研究了N-丁基-4,5-二[2-(苯胺基)乙胺基]-l,8萘酰亚胺红移型铜离子比率荧光探针的光物理性质。通过探针分子与金属离子结合前后的几何构型优化,结合自然键轨道分析,揭示了探针分子对铜离子的识别作用。通过激发态计算阐明了光诱导分子内电荷转移(ICT)机理。研究结果表明,由于Cu(II)离子络合导致萘胺脱氢,带负电荷的胺基N原子与萘环形成C=N双键,延长了共轭体系;N的非键电子向Cu(II)离子的空d轨道转移一个电子,抑制了Cu(II)离子的顺磁效应所导致的荧光淬灭,受光激发后,共轭N与萘环之间发生n→π*电子转移导致ICT效应和荧光红移。%Density functional theory (DFT) and time-dependent DFT (TDDFT) were used to study the photophysical properties of N-butyl-4,5-di[2-(phenylamino)ethylamino]-1,8-naphthalimide, a ratiometric fluorescent sensor for Cu(II). The geometric structures of the compounds at the ground state were optimized by DFT. Combined with natural bond orbital (NBO) analysis, the binding characteristics of the chemosensor molecule coordinated with a Cu(II) ion were identified. The excitation states of the compounds were investigated and the internal charge transfer (ICT) mechanism was elucidated by theoretical calculations. The results indicated that the coordinated Cu(II) ion induced the dehydrogenation of naphthylamine. The negatively-charged amino N atom then formed a C=N double bond with the naphthalene ring, extending the conjugation of the system. The nonbonding electron of N was transferred to the unoccupied d orbital of Cu(II), preventing fluorescence quenching by paramagnetic Cu(II). It was proposed that in the excited state, n→π*electron transfer from the amino N to the naphthalene ring led to internal charge transfer and resulted in the red shift of fluorescence.

  7. Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme.

    Science.gov (United States)

    Xiong, Hai; Reynolds, Noah M; Fan, Chenguang; Englert, Markus; Hoyer, Denton; Miller, Scott J; Söll, Dieter

    2016-03-14

    Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications. PMID:26914285

  8. Benzimidazole based ratiometric and colourimetric chemosensor for Ni(II)

    Science.gov (United States)

    Sarkar, Deblina; Pramanik, Ajoy Kumar; Mondal, Tapan Kumar

    2016-01-01

    A highly sensitive and selective benzimidazole based colourimetric chemosensor (HL) for the efficient detection of Ni2 + has been reported. The synthesized chemosensor HL is highly efficient in detecting Ni2 + over other metal ions that commonly coexist with Ni2 + in physiological and environmental samples. HL also shows distinct color change from orange yellow to blue visible under the naked eye due to specific binding with Ni2 +. This color change corresponds to a large red shift of the UV-Vis spectrum from 403 nm to 600 nm with a distinct isosbestic point at around 500 nm. The cation sensing property of the receptor HL has been examined by UV-Vis spectroscopy. Electronic structure of the HL-Ni2 + complex and sensing mechanism has been interpreted by DFT and TDDFT calculations.

  9. Ratiometric fluorescence imaging of free Zn2+ in brain

    Science.gov (United States)

    Thompson, Richard B.; Suh, Sang W.; Frederickson, Christopher J.

    2001-05-01

    Recently, the function of zinc in the axonal boutons of hippocampal neurons has come under increased scrutiny as evidence has emerged of a putative role for this metal ion in neural damage following insults such as ischemia, blunt force trauma, and seizure. Indeed, the nonpathological role of free zinc in the brain remains cryptic after more than 40 years. We have used a biosensing approach to determine free zinc ion concentrations by fluorescence lifetime, intensity, intensity ratio, or anisotropy changes caused by binding of zinc to variants of a protein, apocarbonic anhydrase II (apo-CA). This approach permits real time measurement of zinc down to picomolar levels, with no perceptible interference from other divalent metal ions abundant in serum and tissue, such as calcium and magnesium. Recently, we used apo-CA together with a fluorescent ligand whose binding is metal-dependent to obtain the first fluorescence micrographs of zinc release from a rat hippocampus model in response to electrical stimulus. In our view, elucidation of the zinc fluxes in neural tissue ultimately requires quantitation, as in the case of calcium. Recent results will be shown.

  10. A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.

    Directory of Open Access Journals (Sweden)

    Gerard N M van der Krogt

    Full Text Available We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.

  11. Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

    Science.gov (United States)

    VanBrunt, Michael P; Shanebeck, Kurt; Caldwell, Zachary; Johnson, Jeffrey; Thompson, Pamela; Martin, Thomas; Dong, Huifang; Li, Gary; Xu, Hengyu; D'Hooge, Francois; Masterson, Luke; Bariola, Pauline; Tiberghien, Arnaud; Ezeadi, Ebele; Williams, David G; Hartley, John A; Howard, Philip W; Grabstein, Kenneth H; Bowen, Michael A; Marelli, Marcello

    2015-11-18

    Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties. PMID:26332743

  12. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

    Directory of Open Access Journals (Sweden)

    Kurt F Ahrens

    2012-03-01

    Full Text Available A fluorescent voltage sensor protein Flare was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular visual crescent in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo.

  13. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

    Science.gov (United States)

    Ahrens, Kurt F.; Heider, Barbara; Lee, Hanson; Isacoff, Ehud Y.; Siegel, Ralph M.

    2012-01-01

    A fluorescent voltage sensor protein “Flare” was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular region of visual cortex in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo. PMID:22461770

  14. Heat transfer analysis of unsteady graphene oxide nanofluid flow using a fuzzy identifier evolved by genetically encoded mutable smart bee algorithm

    Directory of Open Access Journals (Sweden)

    Mohammadreza Azimi

    2015-03-01

    Full Text Available In the current research, the unsteady two dimensional Graphene Oxide water based nanofluid heat transfer between two moving parallel plates is analyzed using an intelligent black-box identifier. The developed intelligent tool is known as evolvable evolutionary fuzzy inference system (EE-FIS which is based on the integration of low-level fuzzy programming and hyper-level evolutionary computing concepts. Here, the authors propose the use of a modified evolutionary algorithm (EA which is called hybrid genetic mutable smart bee algorithm (HGMSBA. The proposed HGMSBA is used to evolve both antecedent and consequent parts of fuzzy rule base. Besides, it tries to prune the rule base of fuzzy inference system (FIS to decrease its computational complexity and increase its interpretability. By considering the prediction error of the fuzzy identifier as the objective function of HGMSBA, an automatic soft interpolation machine is developed which can intuitively increase the robustness and accuracy of the final model. Here, HGMSBA-FIS is used to provide a nonlinear map between inputs, i.e. nanoparticles solid volume fraction (ϕ, Eckert number (Ec and a moving parameter which describes the movements of plates (S, and output, i.e. Nusselt number (Nu. Prior to proceeding with the modeling process, a comprehensive numerical comparative study is performed to investigate the potentials of the proposed model for nonlinear system identification. After demonstrating the efficacy of HGMSBA for training the FIS, the system is applied to the considered problem. Based on the obtained results, it can be inferred that the developed HGMSBA-FIS black-box identifier can be used as a very authentic tool with respect to accuracy and robustness. Besides, as the proposed black-box is not a physics-based identifier, it frees experts from the cumbersome mathematical formulations, and can be used for advanced real-time applications such as model-based control. The simulations indicate that the gradient of Nu has a direct nonlinear relation with the values of ϕ and Ec. It is also observed that an increase in the value of S decreases the value of Nu.

  15. Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors.

    Science.gov (United States)

    Tarrago, Lionel; Péterfi, Zalán; Lee, Byung Cheon; Michel, Thomas; Gladyshev, Vadim N

    2015-05-01

    Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli.

  16. Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy.

    Directory of Open Access Journals (Sweden)

    Alex Costa

    Full Text Available Selective Plane Illumination Microscopy (SPIM is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET. Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+ signal percolation, predicted in previous studies, has been directly visualized.

  17. An engineered palette of metal ion quenchable fluorescent proteins.

    Directory of Open Access Journals (Sweden)

    Xiaozhen Yu

    Full Text Available Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+. Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.

  18. Linear approaches to intramolecular Forster resonance energy transfer probe measurements for quantitative modeling.

    Directory of Open Access Journals (Sweden)

    Marc R Birtwistle

    Full Text Available Numerous unimolecular, genetically-encoded Förster Resonance Energy Transfer (FRET probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1 fluorescence lifetime imaging (FLIM or (2 ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R(alt is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R(alt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.

  19. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Pan [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Xi, Zhaoyong; Wang, Hu [School of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026 (China); Shi, Chaowei [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Xiong, Ying, E-mail: yxiong73@ustc.edu.cn [School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Tian, Changlin, E-mail: cltian@ustc.edu.cn [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China)

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  20. Ratio-metric sensor to detect riboflavin via fluorescence resonance energy transfer with ultrahigh sensitivity

    Science.gov (United States)

    Wang, Jilong; Su, Siheng; Wei, Junhua; Bahgi, Roya; Hope-Weeks, Louisa; Qiu, Jingjing; Wang, Shiren

    2015-08-01

    In this paper, a novel fluorescence resonance energy transfer (FRET) ration-metric fluorescent probe based on heteroatom N, S doped carbon dots (N, S-CDs) was developed to determine riboflavin in aqueous solutions. The ratio of two emission intensities at different wavelengths is applied to determine the concentration of riboflavin (RF). This method is more effective in reducing the background interference and fluctuation of diverse conditions. Therefore, this probe obtains high sensitivity with a low limit of detection (LOD) of 1.9 nM (0.7 ng/ml) which is in the highest level of all riboflavin detection approaches and higher than single wavelength intensity detection (1.9 μM). In addition, this sensor has a high selectivity of detecting riboflavin in deionized water (pH=7) with other biochemical like amino acids. Moreover, riboflavin in aqueous solution is very sensitive to sunlight and can be degraded to lumiflavin, which is toxic. Because the N, S doped carbon dots cannot serve as an energy donor for N, S doped carbon dots and lumiflavin system, this system makes it easy to determine whether the riboflavin is degraded or not, which is first to be reported. This platform may provide possibilities to build a new and facile fluorescence resonance energy transfer based sensor to detect analytes and metamorphous analytes in aqueous solution.

  1. Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

    OpenAIRE

    Schlafer, Sebastian; Garcia, Javier E.; Greve, Matilde; Merete K Raarup; Nyvad, Bente; Dige, Irene

    2014-01-01

    pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in rea...

  2. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Science.gov (United States)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  3. Ratiometric Organic Fibers for Localized and Reversible Ion Sensing with Micrometer‐Scale Spatial Resolution

    Science.gov (United States)

    Moffa, Maria; Rinaldi, Rosaria

    2015-01-01

    A fundamental issue in biomedical and environmental sciences is the development of sensitive and robust sensors able to probe the analyte of interest, under physiological and pathological conditions or in environmental samples, and with very high spatial resolution. In this work, novel hybrid organic fibers that can effectively report the analyte concentration within the local microenvironment are reported. The nanostructured and flexible wires are prepared by embedding fluorescent pH sensors based on seminaphtho‐rhodafluor‐1‐dextran conjugate. By adjusting capsule/polymer ratio and spinning conditions, the diameter of the fibers and the alignment of the reporting capsules are both tuned. The hybrid wires display excellent stability, high sensitivity, as well as reversible response, and their operation relies on effective diffusional kinetic coupling of the sensing regions and the embedding polymer matrix. These devices are believed to be a powerful new sensing platform for clinical diagnostics, bioassays and environmental monitoring. PMID:26539625

  4. Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

    Science.gov (United States)

    2016-01-01

    Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time. PMID:27547982

  5. Characterization of the ER-Targeted Low Affinity Ca(2+) Probe D4ER.

    Science.gov (United States)

    Greotti, Elisa; Wong, Andrea; Pozzan, Tullio; Pendin, Diana; Pizzo, Paola

    2016-01-01

    Calcium ion (Ca(2+)) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca(2+) store and the free Ca(2+) concentration ([Ca(2+)]) within its lumen ([Ca(2+)]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca(2+) sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca(2+) probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca(2+), low dynamic range, and an affinity for the cation that is too high for the levels of [Ca(2+)] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca(2+) affinity and dynamic range for monitoring [Ca(2+)] variations within the ER. As an example, resting [Ca(2+)]ER have been evaluated in a known paradigm of altered ER Ca(2+) homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca(2+) affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca(2+) content in cells expressing mutated PS2, compared to controls. PMID:27598166

  6. Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

    Directory of Open Access Journals (Sweden)

    Elisa Greotti

    2016-09-01

    Full Text Available Calcium ion (Ca2+ is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+] within its lumen ([Ca2+]ER can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2. The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.

  7. Tunable Carbon-Dot-Based Dual-Emission Fluorescent Nanohybrids for Ratiometric Optical Thermometry in Living Cells.

    Science.gov (United States)

    Wang, Chuanxi; Lin, Huihui; Xu, Zhenzhu; Huang, Yijun; Humphrey, Mark G; Zhang, Chi

    2016-03-01

    The use of carbon-dot-based dual-emission fluorescent nanohybrids (DEFNs) as versatile nanothermometry devices for spatially resolved temperature measurements in living cells is demonstrated. The carbon dots (CDs) are prepared in the organic phase and display tunable photoluminescence (PL) across a wide visible range by adjusting the excitation wavelengths and extend of N-doping. DEFNs are formed in a straightforward fashion from CDs (emitting blue PL) and gold nanoclusters (AuNCs, emitting red PL). The DEFNs display ideal single-excitation, dual-emission with two well-resolved, intensity-comparable fluorescence peaks, and function in optical thermometry with high reliability and accuracy by exploiting the temperature sensitivity of their fluorescence intensity ratio (blue/red). Furthermore, the DEFNs have been introduced into cells, exhibiting good biocompatibility, and have facilitated physiological temperature measurements in the range of 25-45 °C; the DEFNs can therefore function as "non-contact" tools for the accurate measurement of temperature and its gradient inside a living cell. PMID:26909643

  8. Synthesis and Application of Ratiometric and "Turn-On" Fluorescent pH Sensors: An Advanced Organic Undergraduate Laboratory

    Science.gov (United States)

    Hutt, Johnathon T.; Aron, Zachary D.

    2014-01-01

    An upper-division organic chemistry laboratory experiment exploring fluorescent sensing over two laboratory periods and part of a third is described. Two functionally distinct pH-responsive sensors are prepared through a dehydrative three-component coupling reaction. During the abbreviated (<1 h) first laboratory period, students set up…

  9. Internal charge transfer based ratiometric interaction of anionic surfactant with calf thymus DNA bound cationic surfactant: Study I

    Science.gov (United States)

    Mukherjee, Abhijit; Chaudhuri, Tandrima; Moulik, Satya Priya; Banerjee, Manas

    2016-01-01

    Cetyl trimethyl ammonium bromide (CTAB) binds calf thymus (ct-) DNA like anionic biopolymers electrostatically and established equilibrium both in the ground as well as in excited state in aqueous medium at pH 7. Anionic sodium dodecyl sulfate (SDS) does not show even hydrophobic interaction with ct-DNA at low concentration. On contrary, SDS can establish well defined equilibrium with DNA bound CTAB in ground state where the same CTAB-DNA isosbestic point reappears. First report of internal charge transfer (ICT) based binding of CTAB with ct-DNA as well as ICT based interaction of anionic SDS with DNA bound CTAB that shows dynamic quenching contribution also. The reappearance of anodic peak and slight increase in cathodic peak current with increasing concentration (at lower range) of anionic SDS, possibly reflect the release of CTAB from DNA bound CTAB by SDS.

  10. Highly sensitive and selective ratiometric fluorescent copper sensors: Different binding affinities modulated by three separate side chains of naphthalimide

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A series of compounds 1-11 with different side chains of naphthalimide as fluorescent copper sensors were designed and synthesized. Compounds 1, 9, 10 and 11 presented a high selectivity to Cu2+ in a neutral aqueous environment. Here 1, 9 and 10 showed selectivity and affinity to Cu2+ with an association constant of about ~106. It gave somewhat response to Ag+, Co2+, Ni2+ and Fe2+ while 1 detected copper. 9 and 10 displayed better selectivity by changing their hydrophobic side chains to the hydrophilic ones on imide moieties. 11, with one flexible side chain, showed high selectivity and an association constant (Ka = 2.2 × 108), which were much higher than those of 1, 9 and 10. These results indicated that the selectivity and affinity could be improved by changing side chains of naphthalimide. That might provide a novel strategy or method for the development of fluorescent sensors.

  11. Highly sensitive and selective ratiometric fluorescent copper sensors: Different binding affinities modulated by three separate side chains of naphthalimide

    Institute of Scientific and Technical Information of China (English)

    XU YuFang; LU Feng; XU ZhaoChao; CHENG TanYu; QIAN XuHong

    2009-01-01

    A series of compounds 1 --11 with different side chains of naphthalimide as fluorescent copper sensors were designed and synthesized. Compounds 1, 9, 10 and 11 presented a high selectivity to Cu2+ in a neutral aqueous environment. Here 1, 9 and 10 showed selectivity and affinity to Cu2+ with an association constant of about ~106. It gave somewhat response to Ag+, Co2+, Ni2+ and Fe2+ while 1 detected copper. 9 and 10 displayed better selectivity by changing their hydrophobic side chains to the hydrophilic ones on imide moieties. 11, with one flexible side chain, showed high selectivity and an association constant (Ka = 2.2 × 108), which were much higher than those of 1, 9 and 10. These results indicated that the selectivity and affinity could be improved by changing side chains of naphthalimide. That might provide a novel strategy or method for the development of fluorescent sensors.

  12. Stable DNA Nanomachine Based on Duplex-Triplex Transition for Ratiometric Imaging Instantaneous pH Changes in Living Cells.

    Science.gov (United States)

    Yang, Mengqi; Zhang, Xiaoling; Liu, Haipeng; Kang, Huaizhi; Zhu, Zhi; Yang, Wen; Tan, Weihong

    2015-06-16

    DNA nanomachines are becoming useful tools for molecular recognition, imaging, and diagnostics and have drawn gradual attention. Unfortunately, the present application of most DNA nanomachines is limited in vitro, so expanding their application in organism has become a primary focus. Hence, a novel DNA nanomachine named t-switch, based on the DNA duplex-triplex transition, is developed for monitoring the intracellular pH gradient. Our strategy is based on the DNA triplex structure containing C(+)-G-C triplets and pH-dependent Förster resonance energy transfer (FRET). Our results indicate that the t-switch is an efficient reporter of pH from pH 5.3 to 6.0 with a fast response of a few seconds. Also the uptake of the t-switch is speedy. In order to protect the t-switch from enzymatic degradation, PEI is used for modification of our DNA nanomachine. At the same time, the dynamic range could be extended to pH 4.6-7.8. The successful application of this pH-depended DNA nanomachine and motoring spatiotemporal pH changes associated with endocytosis is strong evidence of the possibility of self-assembly DNA nanomachine for imaging, targeted therapies, and controllable drug delivery. PMID:26016566

  13. Smart protein biogate as a mediator to regulate competitive host-guest interaction for sensitive ratiometric electrochemical assay of prion

    Science.gov (United States)

    Yu, Peng; Zhang, Xiaohua; Zhou, Jiawan; Xiong, Erhu; Li, Xiaoyu; Chen, Jinhua

    2015-11-01

    A novel competitive host-guest strategy regulated by protein biogate was developed for sensitive and selective analysis of prion protein. The methylene blue (MB)-tagged prion aptamer (MB-Apt) was introduced to the multiwalled carbon nanotubes-β-cyclodextrins (MWCNTs-β-CD) composites-modified glassy carbon (GC) electrode through the host-guest interaction between β-CD and MB. In the absence of prion, MB-Apt could be displaced by ferrocenecarboxylic acid (FCA) due to its stronger binding affinity to β-CD, resulting in a large oxidation peak of FCA. However, in the presence of prion, the specific prion-aptamer interaction drove the formation of protein biogate to seal the cavity of β-CD, which hindered the guest displacement of MB by FCA and resulted in the oxidation peak current of MB (IMB) increased and that of FCA (IFCA) decreased. The developed aptasensor showed good response towards the target (prion protein) with a low detection limit of 160 fM. By changing the specific aptamers, this strategy could be easily extended to detect other proteins, showing promising potential for extensive applications in bioanalysis.

  14. Ratiometric Optical Temperature Sensor Using Two Fluorescent Dyes Dissolved in an Ionic Liquid Encapsulated by Parylene Film

    OpenAIRE

    Isao Shimoyama; Nguyen Binh-Khiem; Tetsuo Kan; Hironori Aoki; Kiyoshi Matsumoto

    2013-01-01

    A temperature sensor that uses temperature-sensitive fluorescent dyes is developed. The droplet sensor has a diameter of 40 µm and uses 1 g/L of Rhodamine B (RhB) and 0.5 g/L of Rhodamine 110 (Rh110), which are fluorescent dyes that are dissolved in an ionic liquid (1-ethyl-3-methylimidazolium ethyl sulfate) to function as temperature indicators. This ionic liquid is encapsulated using vacuum Parylene film deposition (which is known as the Parylene-on-liquid-deposition (PoLD) method). The dro...

  15. In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Wine

    Full Text Available To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (~50 individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ~0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with 'CFTR-related' conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

  16. A simple and inexpensive high resolution color ratiometric planar optode imaging approach: application to oxygen and pH sensing

    DEFF Research Database (Denmark)

    Larsen, M.; Borisov, S. M.; Grunwald, B.;

    2011-01-01

    ) salt derivate for O-2 and pH measurements, respectively. The brightness of both indicators is dramatically enhanced by making use of energy transfer from a donor molecule (Macrolex yellow coumarin). Furthermore, the emission from the donor serves as an internal reference for the O-2 sensor...

  17. A fluorescent coumarin-thiophene hybrid as a ratiometric chemosensor for anions: Synthesis, photophysics, anion sensing and orbital interactions

    Science.gov (United States)

    Yanar, Ufuk; Babür, Banu; Pekyılmaz, Damla; Yahaya, Issah; Aydıner, Burcu; Dede, Yavuz; Seferoğlu, Zeynel

    2016-03-01

    A colorimetric and fluorimetric fluorescent chemosensor (CT-2), having a coumarin ring as a signaling unit and an acetamido thiophene ring as an H-donor receptor, has been synthesized from amino derivative (CT-1) of CT-2 for the purpose of recognition of anions in DMSO. The absorption and emission maxima were both determined for the fluorescent dye in different solvents. Both hypsochromic shift at the absorption maximum, and quenching of fluorescence after interactions between the anions and the receptoric part, were observed. This phenomenon was explained using orbital interactions based on quantum chemical calculations. The selectivity and sensitivity of CT-2 for F-, Cl-, Br-, I-, AcO-, CN-, H2PO4-, HSO4- and ClO4- anions were determined with spectrophotometric, fluorimetric and 1H NMR titration techniques and it was found that CT-2 be utilized for the detection of CN-, F- and AcO- in the presence of other ions as competitors. Color and fluorescence changes visible to the naked eye and under UV (365 nm) were observed upon addition of CN-, F- and AcO- to the solution of chemosensor (CT-2) in DMSO. The sensor showed no colorimetric and fluorimetric response for the anions such as Cl-, Br-, I-, H2PO4-, HSO4-, and ClO4-. However, 1H NMR titration shows that the chemosensor was more sensitive to CN-, than F- and AcO- at the stochiometric ratio of 1:2.5 respectively. Additionally, the compounds CT-1 and CT-2 showed good thermal stability for practical applications.

  18. Internal charge transfer based ratiometric interaction of anionic surfactant with calf thymus DNA bound cationic surfactant: Study I.

    Science.gov (United States)

    Mukherjee, Abhijit; Chaudhuri, Tandrima; Moulik, Satya Priya; Banerjee, Manas

    2016-01-01

    Cetyl trimethyl ammonium bromide (CTAB) binds calf thymus (ct-) DNA like anionic biopolymers electrostatically and established equilibrium both in the ground as well as in excited state in aqueous medium at pH 7. Anionic sodium dodecyl sulfate (SDS) does not show even hydrophobic interaction with ct-DNA at low concentration. On contrary, SDS can establish well defined equilibrium with DNA bound CTAB in ground state where the same CTAB-DNA isosbestic point reappears. First report of internal charge transfer (ICT) based binding of CTAB with ct-DNA as well as ICT based interaction of anionic SDS with DNA bound CTAB that shows dynamic quenching contribution also. The reappearance of anodic peak and slight increase in cathodic peak current with increasing concentration (at lower range) of anionic SDS, possibly reflect the release of CTAB from DNA bound CTAB by SDS.

  19. Ratiometric, filter-free optical sensor based on a complementary metal oxide semiconductor buried double junction photodiode.

    Science.gov (United States)

    Yung, Ka Yi; Zhan, Zhiyong; Titus, Albert H; Baker, Gary A; Bright, Frank V

    2015-07-16

    We report a complementary metal oxide semiconductor integrated circuit (CMOS IC) with a buried double junction (BDJ) photodiode that (i) provides a real-time output signal that is related to the intensity ratio at two emission wavelengths and (ii) simultaneously eliminates the need for an optical filter to block Rayleigh scatter. We demonstrate the BDJ platform performance for gaseous NH3 and aqueous pH detection. We also compare the BDJ performance to parallel results obtained by using a slew scanned fluorimeter (SSF). The BDJ results are functionally equivalent to the SSF results without the need for any wavelength filtering or monochromators and the BDJ platform is not prone to errors associated with source intensity fluctuations or sensor signal drift.

  20. Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

    Directory of Open Access Journals (Sweden)

    Richard W Daniels

    Full Text Available Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A and Thosea asigna virus (T2A worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

  1. Transient light-induced intracellular oxidation revealed by redox biosensor

    International Nuclear Information System (INIS)

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition

  2. Transient light-induced intracellular oxidation revealed by redox biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Kolossov, Vladimir L., E-mail: viadimer@illinois.edu [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Beaudoin, Jessica N. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Hanafin, William P. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); DiLiberto, Stephen J. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Kenis, Paul J.A. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801 (United States); Rex Gaskins, H. [Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801 (United States); Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61801 (United States); Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  3. A single and rapid calcium wave at egg activation in Drosophila

    Directory of Open Access Journals (Sweden)

    Anna H. York-Andersen

    2015-03-01

    Full Text Available Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca2+ concentration in mammals, ascidians and polychaete worms and a single Ca2+ peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca2+ levels occur. Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.

  4. Transient increase in neuronal chloride concentration by neuroactive amino acids released from glioma cells

    Directory of Open Access Journals (Sweden)

    Cristina eBertollini

    2012-11-01

    Full Text Available Neuronal chloride concentration ([Cl-]i is known to be dynamically modulated and alterations in Cl- homeostasis may occur in the brain at physiological and pathological conditions, being also likely involved in glioma-related seizures. However, the mechanism leading to changes in neuronal [Cl-]i during glioma invasion are still unclear. To characterize the potential effect of glioma released soluble factors on neuronal [Cl-]i, we used genetically encoded CFP/YFP-based ratiometric Cl-Sensor transiently expressed in cultured hippocampal neurons. Exposition of neurons to glioma conditioned medium (GCM caused rapid and transient elevation of [Cl-]i, resulting in the increase of fluorescence ratio, which was strongly reduced by blockers of ionotropic glutamate receptors APV and NBQX. Furthermore, in HEK cells expressing GluR1-AMPA receptors, GCM activated ionic current with efficacy similar to those caused by glutamate, supporting the notion that GCM contains glutamate or glutamatergic agonists, which cause neuronal depolarization, activation of NMDA and AMPA/KA receptors leading to elevation of [Cl-]i. Chromatographic analysis of the GCM showed that it contained several aminoacids, including glutamate, whose release from glioma cells did not occur via the most common glial mechanisms of transport, or in response to hypoosmotic stress. GCM also contained glycine, whose action contrasted the glutamate effect. Indeed, strychnine application significantly increased GCM-induced depolarization and [Cl-]i rise. GCM-evoked [Cl-]i elevation was not inhibited by antagonists of Cl- transporters and significantly reduced in the presence of anion channels blocker NPPB, suggesting that Cl-selective channels are a major route for GCM-induced Cl- influx. Altogether, these data show that glioma released aminoacids may dynamically alter Cl- equilibrium in surrounding neurons, deeply interfering with their inhibitory balance, likely leading to physiological and

  5. Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.

    Directory of Open Access Journals (Sweden)

    Franck P Martial

    Full Text Available Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded

  6. A water-soluble ESIPT fluorescent probe with high quantum yield and red emission for ratiometric detection of inorganic and organic palladium.

    Science.gov (United States)

    Gao, Tang; Xu, Pengfei; Liu, Meihui; Bi, Anyao; Hu, Pengzhi; Ye, Bin; Wang, Wei; Zeng, Wenbin

    2015-05-01

    A novel fluorescent probe with a high quantum yield (0.41), large Stokes shifts (255 nm), and red emission (635 nm) was designed to detect all typical oxidation states of palladium species (0, +2, +4) by palladium-mediated terminal propargyl ethers cleavage reaction in water solution without any organic media. The probe showed a high selectivity and excellent sensitivity for palladium species, with a detection as low as 57 nM (6.2 μg L(-1)). PMID:25757156

  7. Ratiometric measurements of adiponectin by mass spectrometry in bottlenose dolphins (Tursiops truncatus with iron overload reveal an association with insulin resistance and glucagon

    Directory of Open Access Journals (Sweden)

    Benjamin A Neely

    2013-09-01

    Full Text Available High molecular weight (HMW adiponectin levels are reduced in humans with type 2 diabetes and insulin resistance. Similar to humans with insulin resistance, managed bottlenose dolphins (Tursiops truncatus diagnosed with hemochromatosis (iron overload have higher levels of 2 h post-prandial plasma insulin than healthy controls. A parallel reaction monitoring assay for dolphin serum adiponectin was developed based on tryptic peptides identified by mass spectrometry. Using identified post-translational modifications, a differential measurement was constructed. Total and unmodified adiponectin levels were measured in sera from dolphins with (n=4 and without (n=5 iron overload. This measurement yielded total adiponectin levels as well as site specific percent unmodified adiponectin that may inversely correlate with HMW adiponectin. Differences in insulin levels between iron overload cases and controls were observed 2 h post-prandial, but not during the fasting state. Thus, post-prandial as well as fasting serum adiponectin levels were measured to determine whether adiponectin and insulin would follow similar patterns. There was no difference in total adiponectin or percent unmodified adiponectin from case or control fasting animals. There was no difference in post-prandial total adiponectin levels between case and control dolphins (mean ± S.D. at 763 ± 298 and 727 ± 291 pmol/ml, respectively (p = 0.91; however, percent unmodified adiponectin was significantly higher in post-prandial cases compared controls (30.0 ± 6.3 versus 17.0 ± 6.6%, respectively; p = 0.016. Interestingly, both total and percent unmodified adiponectin were correlated with glucagon levels in controls (r = 0.999, p < 0.001, but not in cases, which is possibly a reflection of insulin resistance. Although total adiponectin levels were not significantly different, the elevated percent unmodified adiponectin follows a trend similar to HMW adiponectin reported for humans with metabolic disorders.

  8. A colorimetric and ratiometric turn-on BODIPY-based fluorescent probe for double-channel detection of Cu{sup 2+} and Hg{sup 2+}

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jinlong; Ma, Xiaowei; Liu, Bin; Cai, Libo; Li, Qi; Zhang, Yeqin [College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036 (China); Jiang, Kezhi [Key Laboratory of Organosilicon Chemistry and Material Technology of Ministry of Education, Hangzhou Normal University, Hangzhou 310036 (China); Yin, Shouchun, E-mail: yinsc@ustc.edu [College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036 (China)

    2013-09-15

    A colorimetric and turn-on fluorescent chemosensor (RS1) for the detection of Hg{sup 2+} and Cu{sup 2+} based on BODIPY has been synthesized. RS1 selectively binds with Hg{sup 2+} and Cu{sup 2+} ions in CH{sub 3}CN among various metal ions. RS1 shows a significant red-shift in absorption spectra from 522 nm to 581 nm for Hg{sup 2+} and 600 nm for Cu{sup 2+}, which induces naked-eye color changes from pink to purple and blue. Upon excitation at 520 nm, RS1 upon interaction with Hg{sup 2+} ions shows the increase of the ratio of fluorescent intensities (I{sub 589}/I{sub 576}) from 0.84 to 1.87, while RS1 with Cu{sup 2+} exhibits a huge increase of I{sub 610}/I{sub 576} from 0.72 to 7.87. The binding stoichiometries of RS1 with Hg{sup 2+} or Cu{sup 2+} have been determined to be 1:1 by Job's plot and ESI. The binding of RS1 with Hg{sup 2+} is chemically reversible, while the sensing processing of RS1 in response to Cu{sup 2+} ions is irreversible. Highlights: • A new colorimetric and turn-on fluorescent chemosensor (RS1) was synthesized. • RS1 exhibits different fluorescence enhancement in the presence of Hg{sup 2+} and Cu{sup 2+} upon excitation at 520 nm in CH{sub 3}CN. • The remarkable and different naked-eye visible color changes also provide RS1 as a colorimetric senor for Hg{sup 2+} and Cu{sup 2+}.

  9. Ratiometric Measurements of Adiponectin by Mass Spectrometry in Bottlenose Dolphins (Tursiops truncatus) with Iron Overload Reveal an Association with Insulin Resistance and Glucagon

    OpenAIRE

    Neely, Benjamin A.; Kevin P Carlin; Arthur, John M.; McFee, Wayne E.; Janech, Michael G.

    2013-01-01

    High molecular weight (HMW) adiponectin levels are reduced in humans with type 2 diabetes and insulin resistance. Similar to humans with insulin resistance, managed bottlenose dolphins (Tursiops truncatus) diagnosed with hemochromatosis (iron overload) have higher levels of 2 h post-prandial plasma insulin than healthy controls. A parallel reaction monitoring assay for dolphin serum adiponectin was developed based on tryptic peptides identified by mass spectrometry. Using identified post-tran...

  10. Highly Luminescent Microporous Organic Polymer with Lewis Acidic Boron Sites on the Pore Surface: Ratiometric Sensing and Capture of F(-) Ions.

    Science.gov (United States)

    Suresh, Venkata M; Bandyopadhyay, Arkamita; Roy, Syamantak; Pati, Swapan K; Maji, Tapas Kumar

    2015-07-20

    Reversible and selective capture/detection of F(-) ions in water is of the utmost importance, as excess intake leads to adverse effects on human health. Highly robust Lewis acidic luminescent porous organic materials have potential for efficient sequestration and detection of F(-) ions. Herein, the rational design and synthesis of a boron-based, Lewis acidic microporous organic polymer (BMOP) derived from tris(4-bromo-2,3,5,6-tetramethylphenyl)boron nodes and diethynylbiphenyl linkers with a pore size of 1.08 nm for selective turn-on sensing and capture of F(-) ion are reported. The presence of a vacant pπ orbital on the boron center of BMOP results in intramolecular charge transfer (ICT) from the linker to boron. BMOP shows selective turn-on blue emission for F(-) ions in aqueous mixtures with a detection limit of 2.6 μM. Strong B-F interactions facilitate rapid sequestration of F(-) by BMOP. The ICT emission of BMOP can be reversibly regenerated by addition of an excess of water, and the polymer can be reused several times. PMID:26074403

  11. Towards Behavior Control for Evolutionary Robot Based on RL with ENN

    Directory of Open Access Journals (Sweden)

    Jingan Yang

    2013-03-01

    In comparison with the conventional behavior network and the adaptive behavior method, our algorithm simplified the genetic encoding complexity, improved the convergence rate  and the network performance.

  12. Fast kinetics of calcium signaling and sensor design.

    Science.gov (United States)

    Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J

    2015-08-01

    Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change.

  13. Achieving sustainable cultivation of potatoes

    Science.gov (United States)

    Every phase of the production cycle impacts the sustainability of potato. Potato physiology determines how genetically encoded developmental attributes interact with local environmental conditions as modified through agricultural practice to produce a perishable crop. In this chapter we highlight ho...

  14. Sensing Cardiac Electrical Activity With a Cardiac Myocyte--Targeted Optogenetic Voltage Indicator

    NARCIS (Netherlands)

    Chang Liao, Mei-Ling; de Boer, Teun P; Mutoh, Hiroki; Raad, Nour; Richter, Claudia; Wagner, Eva; Downie, Bryan R; Unsöld, Bernhard; Arooj, Iqra; Streckfuss-Bömeke, Katrin; Döker, Stephan; Luther, Stefan; Guan, Kaomei; Wagner, Stefan; Lehnart, Stephan E; Maier, Lars S; Stühmer, Walter; Wettwer, Erich; van Veen, Toon; Morlock, Michael M; Knöpfel, Thomas; Zimmermann, Wolfram-Hubertus

    2015-01-01

    RATIONALE: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac

  15. 系列混合碳源条件下颗粒化EBPR系统茵群结构变化规律研究%A study of microbial diversity of granule-based enhanced biological phosphorus removal systems cultivated with ratiometric propionate and acetate as mixed carbon sources

    Institute of Scientific and Technical Information of China (English)

    蒋涛; 孙培德; 金均

    2012-01-01

    A series of mixed carbon sources with different ratios of propionate and acetate was applied in granule-based enhanced biological phosphorus removal (EBPR) sludge in SBR reactor. Microbial diversity change during the granular process and functional bacteria competition under different carbon sources were studied. Significant microbial diversity change in EBPR system was exhibited during granulation. Uncultured bacteria previously dominated in the system disappeared rapidly, while uncultured rhodocyclaceae bacterium and portions of candidatus competibacter phosphatis, denitrifying bacterium, acinetobacter and uncultured alpha proteobacterium were gradually washed out. Uncultured chlorobi bacterium and uncultured alpha proteobacterium were the primary phosphorus removal bacteria in developed granular EBPR system. The change of bacteria population ofcandidatus competibacterphosphatis and uncultured chlorobi bacterium was evidenced as a result of microbial diversity under different ratios of mixed carbon sources. The population of candidatus competibacter phosphatis increased monotonically with acetate concentration, decreaseing the system phosphorus removal efficiency. Meanwhile, the population of uncultured chlorobi bacterium had a positive correlation with propionate concentration, which maintained good phosphorus removal efficiency of the EBPR system.%在SBR反应器中接种富含聚磷菌的活性污泥,采用一系列不同丙酸/乙酸比例混合的碳源进行EBPR系统污泥的颗粒化培养,并考察了颗粒化进程中的系统菌群结构变化,以及不同混合碳源条件对系统功能菌种竞争的影响.结果表明,污泥颗粒化过程对EBPR系统菌群结构产生了较大的筛选作用.原本在系统中占优势的一类Uncultured bacterium被迅速淘汰;Uncultured Rhodocyclaceae bacterium、部分Candidatus Competibacter phosphatis、部分Denitrifying bacterium、Acinetobacter及部分Uncultured alpha proteobacterium分别逐渐被淘汰.在各个成熟的颗粒化EBPR系统中,除磷微生物主要为Uncultured Chlorobi bacterium与Uncultured alpha proteobacterium.不同混合碳源条件培养的颗粒化EBPR系统菌群结构差异主要表现为Candidatus Competibacter phosphatis(聚糖菌)与Uncultured Chlorobi bacterium(聚磷菌)菌群数量的不同.混合碳源中乙酸比例的提高可造成颗粒化EBPR系统中Candidatus Competibacter phosphatis的增长,使系统的除磷效率下降.而碳源中丙酸比例相对较高的条件有利于Uncultured Chlorobi bacterium增长,从而有助于颗粒化EBPR系统维持较好的除磷效率.

  16. Ru( dpp)3(ClO4)2掺杂的聚丙烯腈纳米颗粒的制备及其在比率荧光pH检测中的应用%Synthesis of Ru (dpp) 3 ( ClO4 ) 2 doped polyacrylonitrile nanoparticles and its applications in ratiometric pH sensing

    Institute of Scientific and Technical Information of China (English)

    叶廷秀; 王旭东; 陈小霞; 张英雪; 屈艳勤; 陈曦

    2011-01-01

    In this paper Ru (dpp ) 3 ( C104) 2 doped polyacrylonitrile nanoparticles ( Ru - PAN ) were prepared using emulsion polymerization method. Morphology characterizations of the nanoparticles were performed using scanning electron microscope, the particle diameter was about 135 ± 15 nm. The in terference of the oxygen, coexisting ions, pH and photo - stability were investigated. A new ratiomet ric fluorescence pH sensing system was established via fluorescein isothiocyanate (FITC) as pH - sen sitive dye and Ru - PAN as a reference.%采用乳液聚合法制备了掺杂有4,7-二苯基-1,10-邻菲咯啉钌(Ru( dpp)3( ClO4)2)的聚丙烯腈纳米颗粒(Ru- PAN).经扫描电子显微镜(SEM)表征,制备的Ru - PAN的尺寸为135±15 nm,呈规则球型,尺寸分布均匀且在水中的分散性较好.实验考察了氧气、共存离子和pH值对其荧光性质的影响以及其荧光稳定性.以异硫氰根荧光素(FITC)为pH荧光指示剂、Ru - PAN为参比信号,初步建立了一种比率荧光pH检测的方法.

  17. A Guide to Fluorescent Protein FRET Pairs

    Science.gov (United States)

    Bajar, Bryce T.; Wang, Emily S.; Zhang, Shu; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. PMID:27649177

  18. A Guide to Fluorescent Protein FRET Pairs

    Directory of Open Access Journals (Sweden)

    Bryce T. Bajar

    2016-09-01

    Full Text Available Förster or fluorescence resonance energy transfer (FRET technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

  19. Calcium Imaging of Pheromone Responses in the Insect Antennal Lobe

    OpenAIRE

    Kim, Susy M.; Wang, Jing W.

    2013-01-01

    Calcium imaging is a powerful technique that permits the visual monitoring of neural responses to pheromones and other odors in large ensembles of neurons. Here, we describe a method that permits the monitoring of Drosophila antennal lobe responses to odors using the genetically encoded calcium monitor GCaMP.

  20. Visualization of Plasticity in Fear-Evoked Calcium Signals in Midbrain Dopamine Neurons

    Science.gov (United States)

    Gore, Bryan B.; Soden, Marta E.; Zweifel, Larry S.

    2014-01-01

    Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium…

  1. A single-stranded architecture for cotranscriptional folding of RNA nanostructures

    DEFF Research Database (Denmark)

    Geary, Cody; Rothemund, Paul; Andersen, Ebbe Sloth

    2014-01-01

    Artificial DNA and RNA structures have been used as scaffolds for a variety of nanoscale devices. In comparison to DNA structures, RNA structures have been limited in size, but they also have advantages: RNA can fold during transcription and thus can be genetically encoded and expressed in cells....

  2. Calcium imaging of vomeronasal organ response using slice preparations from transgenic mice expressing G-CaMP2

    OpenAIRE

    Yu, C. Ron

    2013-01-01

    The vomeronasal organ (VNO) in vertebrate animals detects pheromones and interspecies chemical signals. We describe in this chapter a Ca2+ imaging approach using transgenic mice that express the genetically encoded Ca2+ sensor G-CaMP2 in VNO tissue. This approach allows us to analyze the complex patterns of the vomeronasal neuron response to large numbers of chemosensory stimuli.

  3. Bacterial protein toxins : tools to study mammalian molecular cell biology

    NARCIS (Netherlands)

    Wüthrich, I.W.

    2014-01-01

    Bacterial protein toxins are genetically encoded proteinaceous macromolecules that upon exposure causes perturbation of cellular metabolism in a susceptible host. A bacterial toxin can work at a distance from the site of infection, and has direct and quantifiable actions. Bacterial protein toxins ca

  4. Molecular Neuroanatomy: A Generation of Progress

    OpenAIRE

    Pollock, Jonathan D.; Wu, Da-Yu; Satterlee, John

    2013-01-01

    The neuroscience research landscape has changed dramatically over the past decade. An impressive array of neuroscience tools and technologies have been generated, including brain gene expression atlases, genetically encoded proteins to monitor and manipulate neuronal activity and function, cost effective genome sequencing, new technologies enabling genome manipulation, new imaging methods and new tools for mapping neuronal circuits. However, despite these technological advances, several signi...

  5. Expanding the eukaryotic genetic code

    Science.gov (United States)

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2013-01-22

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  6. Unnatural reactive amino acid genetic code additions

    Science.gov (United States)

    Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.; Anderson, J. Christopher; Schultz, Peter G.

    2014-08-26

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  7. Expanding the eukaryotic genetic code

    Energy Technology Data Exchange (ETDEWEB)

    Chin, Jason W; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G

    2015-02-03

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  8. Unnatural reactive amino acid genetic code additions

    Energy Technology Data Exchange (ETDEWEB)

    Deiters, Alexander; Cropp, Ashton T; Chin, Jason W; Anderson, Christopher J; Schultz, Peter G

    2013-05-21

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  9. Filling a GAP-An Optimized Probe for ER Ca(2+) Imaging In Vivo.

    Science.gov (United States)

    Malli, Roland; Eroglu, Emrah; Waldeck-Weiermair, Markus; Graier, Wolfgang F

    2016-06-23

    In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca(2+) signals within the endoplasmic reticulum (ER) of living animals. PMID:27341431

  10. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    NARCIS (Netherlands)

    Shaner, Nathan C; Campbell, Robert E; Steinbach, Paul A; Giepmans, Ben N G; Palmer, Amy E; Tsien, Roger Y

    2004-01-01

    Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red

  11. Expanding the dynamic measurement range for polymeric nanoparticle pH sensors

    DEFF Research Database (Denmark)

    Sun, Honghao; Almdal, Kristoffer; Andresen, Thomas Lars

    2011-01-01

    Conventional optical nanoparticle pH sensors that are designed for ratiometric measurements in cells have been based on utilizing one sensor fluorophore and one reference fluorophore in each nanoparticle, which results in a relatively narrow dynamic measurement range. This results in substantial...

  12. Poly(L-Lysine)-pyranine-3 coacervate mediated nanoparticle-assembly: fabrication of dynamic pH-responsive containers.

    Science.gov (United States)

    Amali, Arlin Jose; Singh, Shashi; Rangaraj, Nandini; Patra, Digambara; Rana, Rohit Kumar

    2012-01-21

    Counter-ion condensation of Poly(L-Lysine) in the presence of pyranine-3 generates spherical coacervates, which then template the assembly of silica nanoparticles to form microcapsule structures that dynamically control the optical ratiometric sensing of both the change in pH and release of the probe molecule.

  13. Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy

    DEFF Research Database (Denmark)

    Novak, Ivana; Nitschke, Roland; Amstrup, Jan

    2002-01-01

    Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanni...

  14. Luminescence of Y3Al5O12:Eu3+ nanophosphors in blood and organic media

    Science.gov (United States)

    Kolesnikov, I. E.; Povolotskiy, A. V.; Tolstikova, D. V.; Manshina, A. A.; Mikhailov, M. D.

    2015-02-01

    Lanthanide-doped nanoparticles characterized by their large Stokes’ shifts, narrow emission bands, long lifetimes and high photostability are promising candidates for luminescent labels in biological research. That is why investigation of lanthanide-doped nanoparticles’ fluorescence in complex biological media (like blood and Intralipid) is of great importance from the standpoint of applicability in medicine and biology. Nanopowder of yttrium aluminum garnet doped with Eu3+ was synthesized by the Pechini method. X-ray diffraction data demonstrated formation of cubic crystalline phase without any impurities. The synthesized sample was found to be well-defined 20-50 nm nanoparticles agglomerated to clusters no bigger than 200 nm in diameter. The emission spectrum of nanocrystalline powder consists of characteristic narrow lines attributed to 5D0-7FJ transitions. It was found that the long lifetime of 5D0 level (4.13 ms) allows using time-resolved techniques to eliminate fluorescence of the biological background. The possibility of the luminescence signal detection of Y3Al5O12:Eu3+ nanopowder (0.14 mg per 1 ml) against the background of blood auto-luminescence was demonstrated. Y3Al5O12:Eu3+ phosphors luminescence in blood media showed timing stability whereas fluorescein luminescence dropped down by a factor of 3 after 20 min. It was found that the luminescence signal of Y3Al5O12:Eu3+ phosphors could be detected through 5 mm of biological tissue. The possibility of independent registration of nanopowders doped with different REI-ions (Eu3+ and Nd3+) was investigated.

  15. Measuring intracellular redox conditions using GFP-based sensors

    DEFF Research Database (Denmark)

    Björnberg, Olof; Ostergaard, Henrik; Winther, Jakob R

    2006-01-01

    Recent years have seen the development of methods for analyzing the redox conditions in specific compartments in living cells. These methods are based on genetically encoded sensors comprising variants of Green Fluorescent Protein in which vicinal cysteine residues have been introduced at solvent......-exposed positions. Several mutant forms have been identified in which formation of a disulfide bond between these cysteine residues results in changes of their fluorescence properties. The redox sensors have been characterized biochemically and found to behave differently, both spectroscopically and in terms...... of redox properties. As genetically encoded sensors they can be expressed in living cells and used for analysis of intracellular redox conditions; however, which parameters are measured depends on how the sensors interact with various cellular redox components. Results of both biochemical and cell...

  16. SoNar, a Highly Responsive NAD+/NADH Sensor, Allows High-Throughput Metabolic Screening of Anti-tumor Agents.

    Science.gov (United States)

    Zhao, Yuzheng; Hu, Qingxun; Cheng, Feixiong; Su, Ni; Wang, Aoxue; Zou, Yejun; Hu, Hanyang; Chen, Xianjun; Zhou, Hai-Meng; Huang, Xinzhi; Yang, Kai; Zhu, Qian; Wang, Xue; Yi, Jing; Zhu, Linyong; Qian, Xuhong; Chen, Lixin; Tang, Yun; Loscalzo, Joseph; Yang, Yi

    2015-05-01

    The altered metabolism of tumor cells confers a selective advantage for survival and proliferation, and studies have shown that targeting such metabolic shifts may be a useful therapeutic strategy. We developed an intensely fluorescent, rapidly responsive, pH-resistant, genetically encoded sensor of wide dynamic range, denoted SoNar, for tracking cytosolic NAD(+) and NADH redox states in living cells and in vivo. SoNar responds to subtle perturbations of various pathways of energy metabolism in real time, and allowed high-throughput screening for new agents targeting tumor metabolism. Among > 5,500 unique compounds, we identified KP372-1 as a potent NQO1-mediated redox cycling agent that produced extreme oxidative stress, selectively induced cancer cell apoptosis, and effectively decreased tumor growth in vivo. This study demonstrates that genetically encoded sensor-based metabolic screening could serve as a valuable approach for drug discovery.

  17. A simple method for enhancing the bioorthogonality of cyclooctyne reagent.

    Science.gov (United States)

    Tian, He; Sakmar, Thomas P; Huber, Thomas

    2016-04-01

    The cross-reactivity between some cyclooctynes and thiols limits the bioorthogonality of the strain-promoted azide-alkyne cycloaddition reaction. We show that a low concentration of β-mercaptoethanol significantly reduces the undesirable side reaction between bicyclononyne (BCN) and cysteine and while preserving free cysteines. We site-specifically label a genetically-encoded azido group in the visual photoreceptor rhodopsin to demonstrate the utility of the strategy. PMID:27009873

  18. la bioluminescence de l'aequorine en réponse au calcium In vitro et dans le Cortex cerebral

    OpenAIRE

    Tricoire, Ludovic

    2006-01-01

    During my PhD, I investigated in vitro the calcium-dependent bioluminescence of thephotoprotein aequorin and then used its bioluminescence to image neuronal activities in theneocortical network. This genetically encoded calcium sensor can be expressed in specific cell types and its bioluminescence is not toxic and exhibit a high signal/noise ratio.I first search for mutations modifying aequorin bioluminescence, using a randommutagenesis and in vitro evolution approach. I isolated mutants show...

  19. Direct visualization of specifically modified extracellular glycans in living animals.

    Science.gov (United States)

    Attreed, Matthew; Desbois, Muriel; van Kuppevelt, Toin H; Bülow, Hannes E

    2012-05-01

    Modification patterns of heparan sulfate coordinate protein function in metazoans, yet in vivo imaging of such non-genetically encoded structures has been impossible. Here we report a transgenic method in Caenorhabditis elegans that allows direct live imaging of specific heparan sulfate modification patterns. This experimental approach reveals a dynamic and cell-specific heparan sulfate landscape and could in principle be adapted to visualize and analyze any extracellular molecule in vivo. PMID:22466794

  20. Direct visualization of specifically modified extracellular glycans in living animals

    OpenAIRE

    Attreed, Matthew; Desbois, Muriel; van Kuppevelt, Toin H.; Bülow, Hannes E.

    2012-01-01

    Modification patterns of the extracellular glycan heparan sulfate coordinate protein function in metazoans, yet in vivo imaging of such non-genetically encoded structures has been impossible. Here we report a transgenic method in Caenorhabditis elegans that allows direct live imaging of specific heparan sulfate modification patterns. This experimental approach reveals a dynamic and cell-specific heparan sulfate landscape and could in principle be adapted to visualize and analyze any extracell...

  1. Synthetic Physiology: Strategies for Adapting Tools from Nature for Genetically-Targeted Control of Fast Biological Processes

    OpenAIRE

    Chow, Brian Y.; Chuong, Amy S.; Klapoetke, Nathan C; Boyden, Edward S.

    2011-01-01

    The life and operation of cells involve many physiological processes that take place over fast timescales of milliseconds to minutes. Genetically-encoded technologies for driving or suppressing specific fast physiological processes in intact cells, perhaps embedded within intact tissues in living organisms, are critical for the ability to understand how these physiological processes contribute to emergent cellular and organismal functions and behaviors. Such “synthetic physiology” tools are o...

  2. Optically Highlighting Basement Membrane Components in C. elegans

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Elliott Hagedorn & David Sherwood ### Abstract Green fluorescent protein (GFP) and other genetically encoded fluorescent proteins provide a means to study gene expression pattern and protein localization in living tissues. Recently discovered GFP-like fluorophores and engineered variants have further expanded the fluorescent protein toolkit for in vivo imaging. Here we describe a technique using transgenic C. elegans that contain laminin or type IV collagen fused to the g...

  3. Skin care during the menopause period: noninvasive procedures of beauty studies

    OpenAIRE

    Herman, Joanna; Rost-Roszkowska, Magdalena; Skotnicka-Graca, Urszula

    2013-01-01

    Ageing is a resultant of two processes, including genetically encoded changes in an organism and modifications caused by a negative external environment impact. In the histological aspect, the skin ageing, due to endogenous factors and hormonal changes shows: excessive dryness, Malpighian layer thinning, microcirculation disorders, collagenic or elastin fiber degradation and simultaneous glycation, decreased speed of sebum and perspiration secretion. It is said that skin is a functional pictu...

  4. Diversity and Evolution of Coral Fluorescent Proteins

    OpenAIRE

    Alieva, Naila O.; Konzen, Karen A.; Field, Steven F.; Meleshkevitch, Ella A.; Hunt, Marguerite E.; Victor Beltran-Ramirez; Miller, David J.; Jörg Wiedenmann; Anya Salih; Matz, Mikhail V

    2008-01-01

    GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full ...

  5. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

    OpenAIRE

    Kiwamu Takemoto; Tomoki Matsuda; Naoki Sakai; Donald Fu; Masanori Noda; Susumu Uchiyama; Ippei Kotera; Yoshiyuki Arai; Masataka Horiuchi; Kiichi Fukui; Tokiyoshi Ayabe; Fuyuhiko Inagaki; Hiroshi Suzuki; Takeharu Nagai

    2013-01-01

    Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subce...

  6. aequorine bioluminescence response to calcium in vitro and in cerebral cortex

    OpenAIRE

    Tricoire, Ludovic

    2006-01-01

    During my PhD, I investigated in vitro the calcium-dependent bioluminescence of thephotoprotein aequorin and then used its bioluminescence to image neuronal activities in theneocortical network. This genetically encoded calcium sensor can be expressed in specific cell types and its bioluminescence is not toxic and exhibit a high signal/noise ratio.I first search for mutations modifying aequorin bioluminescence, using a randommutagenesis and in vitro evolution approach. I isolated mutants show...

  7. Concurrent Imaging of Synaptic Vesicle Recycling and Calcium Dynamics

    OpenAIRE

    Li, Haiyan; Foss, Sarah M.; Dobryy, Yuriy L.; Park, C. Kevin; Hires, Samuel Andrew; Shaner, Nathan C.; Tsien, Roger Y.; Osborne, Leslie C.; Voglmaier, Susan M.

    2011-01-01

    Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-sh...

  8. Concurrent imaging of synaptic vesicle recycling and calcium dynamics.

    OpenAIRE

    Haiyan eLi; Foss, Sarah M.; Yuriy eDobryy; C. Kevin ePark; Samuel Andrew Hires; Shaner, Nathan C.; Tsien, Roger Y.; Osborne, Leslie C.; Voglmaier, Susan M.

    2011-01-01

    Synaptic transmission involves the calcium-dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-...

  9. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB - E. coli cytoplasm

    OpenAIRE

    Markiv Anatoliy; Beatson Richard; Burchell Joy; Durvasula Ravi V; Kang Angray S

    2011-01-01

    Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains link...

  10. Imaging Light Responses of Foveal Ganglion Cells in the Living Macaque Eye

    OpenAIRE

    Yin, Lu; Masella, Benjamin; Dalkara, Deniz; Zhang, Jie; Flannery, John G.; Schaffer, David V; Williams, David R.; Merigan, William H.

    2014-01-01

    The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-C...

  11. Optogenetics: Molecular and Optical Tools for Controlling Life with Light

    OpenAIRE

    Boyden, Edward Stuart

    2013-01-01

    Optogenetic tools are genetically-encoded reagents that, when targeted to specific brain cells, enable their activity to be controlled by light. These tools are having broad impact on science, and may serve clinical roles as well. 150-word Biography: Ed Boyden is Associate Professor of Biological Engineering and Brain and Cognitive Sciences, at the MIT Media Lab and the MIT McGovern Institute. He leads the Synthetic Neurobiology Group, which develops tools for analyzing and engineering the ci...

  12. Struktur und Reaktionsmechanismus der Pyrrolysinsynthase (PylD)

    KAUST Repository

    Quitterer, Felix

    2013-05-29

    The final step in the biosynthesis of the 22nd genetically encoded amino acid, pyrrolysine, is catalyzed by PylD, a structurally and mechanistically unique dehydrogenase. This catalyzed reaction includes an induced-fit mechanism achieved by major structural rearrangements of the N-terminal helix upon substrate binding. Different steps of the reaction trajectory are visualized by complex structures of PylD with substrate and product.

  13. Culture Prefigures Cognition in Pan/Homo Bonobos

    Directory of Open Access Journals (Sweden)

    Sue SAVAGE-RUMBAUGH

    2010-01-01

    Full Text Available This article questions traditional approaches to the study of primate cognition. Because of a widespread assumption that cognition in non-human primates is genetically encoded, these approaches neglect how profoundly apes’ cultural rearing experiences affect test results. We describe how three advanced cognitive abilities – imitation, theory of mind and language – emerged in bonobos maturing in a Pan/Homo culture.

  14. Optical highlighter molecules in neurobiology.

    Science.gov (United States)

    Datta, Sandeep Robert; Patterson, George H

    2012-02-01

    The development of advanced optical methods has played a key role in propelling progress in neurobiology. Genetically-encoded fluorescent molecules found in nature have enabled labeling of individual neurons to study their physiology and anatomy. Here we discuss the recent use of both native and synthetic optical highlighter proteins to address key problems in neurobiology, including questions relevant to synaptic function, neuroanatomy, and the organization of neural circuits.

  15. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.

    Science.gov (United States)

    Liss, Viktoria; Barlag, Britta; Nietschke, Monika; Hensel, Michael

    2015-12-08

    Research in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches.

  16. A Fiber Optic Ammonia Sensor Using a Universal pH Indicator

    Science.gov (United States)

    Rodríguez, Adolfo J.; Zamarreño, Carlos R.; Matías, Ignacio R.; Arregui, Francisco. J.; Domínguez Cruz, Rene F.; May-Arrioja, Daniel. A.

    2014-01-01

    A universal pH indicator is used to fabricate a fiber optic ammonia sensor. The advantage of this pH indicator is that it exhibits sensitivity to ammonia over a broad wavelength range. This provides a differential response, with a valley around 500 nm and a peak around 650 nm, which allows us to perform ratiometric measurements. The ratiometric measurements provide not only an enhanced signal, but can also eliminate any external disturbance due to humidity or temperature fluctuations. In addition, the indicator is embedded in a hydrophobic and gas permeable polyurethane film named Tecoflex®. The film provides additional advantages to the sensor, such as operation in dry environments, efficient transport of the element to be measured to the sensitive area of the sensor, and prevent leakage or detachment of the indicator. The combination of the universal pH indicator and Tecoflex® film provides a reliable and robust fiber optic ammonia sensor. PMID:24583969

  17. Easily Accessible and Highly Selective "Turn-on" Fluorescent Sensor for Imaging Cadmium in Living Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; ZHANG Ying-mu; LI Yao-xian; ZHAO Qing

    2013-01-01

    A new schiff base of phenothiazine derivative was designed for ratiometric sensing of Cd2+ selectively.Upon the addition of Cd2+ to the solution of phenothiazine derivative,the fluorescence intensity of it was enhanced in a linear fashion and the maximum fluorescence peak exhibited a blue shift from 575 nm to 525 nm.This ratiometric fluorescent sensor displays a very high sensitivity(detection limits were 0.34 and 1.0 μmol/L of Cd2+ using the visual fluorescence changes and UV-Vis changes,respectively),a rapid response time(<10 s) and high selectivity for Cd2+ over other transition metal ions.Moreover,the living cells image experiments also demonstrate the value of the sensor in fluorescent visualization of Cd2+ in the environmental and biological systems.

  18. Fluorescent Ensemble Based on Bispyrene Fluorophore and Surfactant Assemblies: Sensing and Discriminating Proteins in Aqueous Solution.

    Science.gov (United States)

    Fan, Junmei; Ding, Liping; Bo, Yu; Fang, Yu

    2015-10-14

    A particular bispyrene fluorophore (1) with two pyrene moieties covalently linked via a hydrophilic spacer was synthesized. Fluorescence measurements reveal that the fluorescence emission of 1 could be well modulated by a cationic surfactant, dodecyltrimethylammonium bromide (DTAB). Protein sensing studies illustrate that the selected ensemble based on 1/DTAB assemblies exhibits ratiometric responses to nonmetalloproteins and turn-off responses to metalloproteins, which can be used to differentiate the two types of proteins. Moreover, negatively charged nonmetalloproteins can be discriminated from the positively charged ones according to the difference in ratiometric responses. Fluorescence sensing studies with control bispyrenes indicate that the polarity of the spacer connecting two pyrene moieties plays an important role in locating bispyrene fluorophore in DTAB assemblies, which further influences its sensing behaviors to noncovalent interacting proteins. This study sheds light on the influence of the probe structure on the sensing performance of a fluorescent ensemble based on probe and surfactant assemblies.

  19. Intravital FRET: Probing Cellular and Tissue Function in Vivo.

    Science.gov (United States)

    Radbruch, Helena; Bremer, Daniel; Mothes, Ronja; Günther, Robert; Rinnenthal, Jan Leo; Pohlan, Julian; Ulbricht, Carolin; Hauser, Anja E; Niesner, Raluca

    2015-01-01

    The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo-ratiometrically and time-resolved by fluorescence lifetime imaging-and show their concrete application in the context of neuroinflammation in adult mice. PMID:26006244

  20. Intravital FRET: Probing Cellular and Tissue Function in Vivo

    OpenAIRE

    Helena Radbruch; Daniel Bremer; Ronja Mothes; Robert Günther; Jan Leo Rinnenthal; Julian Pohlan; Carolin Ulbricht; Hauser, Anja E.; Raluca Niesner

    2015-01-01

    The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context o...

  1. A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

    Science.gov (United States)

    Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

    2013-06-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting.

  2. Temperature Induced Instabilities in Macro-bend Fiber Based Wavelength Measurement Systems

    OpenAIRE

    Rajan, Ginu; Semenova, Yuliya; Wang, Pengfei; Farrell, Gerald

    2009-01-01

    An investigation of temperature-induced instabilities in a wavelength measurement system based on macro-bend fiber filter used in the ratiometric scheme are presented. Two wavelength measurement systems based on macro-bend fiber, a standard low bend loss single-mode fiber filter based system and a high bend loss fiber filter based system are considered. In the case of a low bend loss fiber filter based system, the oscillatory variation in the ratio response with temperature and the difference...

  3. A plotting optical densitometer for routine radiotherapy dosimetry

    International Nuclear Information System (INIS)

    In this note the authors describe a modified densitometer for evaluation of film blackening during electron dosimetry in radiotherapy quality assurance work. The instability and drift of previous plotting densitometers have been eliminated. Using a cold light source and a ratiometric measurement technique has removed the major sources of error inherent in these units. Using a minimum of components and a high quality integrated circuit log amplifier has improved accuracy and linearily. (author)

  4. Nanosensors for biotechnological and medical research; Nanosensoren fuer die biotechnologische und medizinische Forschung

    Energy Technology Data Exchange (ETDEWEB)

    Mohr, Gerhard J.; Trupp, Sabine [Fraunhofer-Institut fuer Zuverlaessigkeit und Mikrointegration (IZM-M), Regensburg (Germany). Arbeitsgruppe Sensormaterialien; Schulz, Anja [Dublin City University (Ireland). National Center for Sensor Research, Optical Sensors Laboratory; Doussineau, Tristan [Friedrich-Schiller-Universitaet Jena (Germany). Institut fuer Physikalische Chemie

    2010-07-01

    Nanosensors are introduced that enable a reliable and continuous monitoring of pH. The pH-nanosensors are obtained by covalent immobilisation of indicator dyes to different types of polymer particles. Additional reference dyes allow for a ratiometric and quantitative detection of signal changes. Possible applications are in biological and medical analytics, e.g. monitoring the cellular pH of cancer cells to evaluate drug effects, and also in micro-reactors and sensor-arrays. (orig.)

  5. Green fluorescent protein: A perspective

    OpenAIRE

    Remington, S James

    2011-01-01

    A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994–2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationship...

  6. High-content screening identifies a role for Na+ channels in insulin production

    OpenAIRE

    Szabat, Marta; Modi, Honey; Ramracheya, Reshma; Girbinger, Vroni; Chan, Forson; Lee, Jason T. C.; Piske, Micah; Kamal, Sepehr; Carol Yang, Yu Hsuan; Welling, Andrea; Rorsman, Patrik; Johnson, James D.

    2015-01-01

    Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, dri...

  7. Laser Induced Breakdown Spectroscopy for Classification of High Energy Materials using Elemental Intensity Ratios

    OpenAIRE

    Sreedhar, S.; Manoj Kumar Gundawar; Venugopal Rao, S.

    2014-01-01

    A simple, yet efficient, methodology is proposed to classify three high energy materials (HEMs) with diverse composition using nanosecond laser induced breakdown spectroscopic data. We have calculated O/N, N/H, and O/H elemental peaks ratios using a ratiometric method. The present work describes a novel way to construct 1D, 2D, and 3D classification model using the above mentioned ratios. Multivariate statistical methods are followed for construction of the classification models. A detailed p...

  8. Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

    OpenAIRE

    Ewins, B. A.; Vassiliadou, M.; Minihane, A. M.; Rimbach, G. H.; Weinberg, P.D.

    2006-01-01

    Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non-standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemica...

  9. Role of Synthetic and Dimensional Synthetic Organic Chemistry in Block Copolymer Micelle Nanosensor Engineering

    OpenAIRE

    Ek, Pramod Kumar; Andresen, Thomas Lars; Almdal, Kristoffer

    2012-01-01

    This thesis investigated the role of amphiphilic triblock copolymer micelle nanomaterials in nanosensors, with emphasis on the synthesis of micelle particle sensors. The thesis is focused on the role of synthetic and dimensional synthetic organic chemistry in amphiphilic triblock core-shellcorona micelle based ratiometric fluorescence pH nanosensor fabrications. Two synthetic strategies such as post micelle modification and mixed micellisation (co-micellisation) were employed for pH nanosenso...

  10. An All-fiber Temperature Sensor Based on a Macro-bend Singlemode Fiber Loop

    OpenAIRE

    Rajan, Ginu; Semenova, Yuliya; Farrell, Gerald

    2008-01-01

    An all-fibre temperature sensor is proposed based on a macro-bend singlemode fibre loop using a ratiometric power measurement scheme. The sensor has a linear characteristic with temperature at a fixed wavelength and bend radius. A direct linear relationship between the bend loss of the singlemode fibre and temperature is reported for the first time. By measuring the change in bend loss of the system a change in temperature can be measured assuming the system is calibrated. The proposed sensor...

  11. Experimental Analysis and Demonstration of a Low Cost Fibre Optic Temperature Sensor System for Engineering Applications

    OpenAIRE

    Rajan, Ginu; Semenova, Yuliya; Mathew, Jinesh; Farrell, Gerald

    2010-01-01

    An epoxy packaged surface mountable fibre temperature sensor for engineering applications is presented in this paper. The temperature sensor is based on a macro-bend single-mode fibre loop employed in a ratiometric power measurement scheme and has a linear characteristic with temperature at a fixed wavelength and bend radius. The sensor head consists of a single turn of a bare bend sensitive single-mode fibre with an applied absorption coating. The temperature of the sensor head is varied up ...

  12. Photoacoustic Imaging: Semiconducting Oligomer Nanoparticles as an Activatable Photoacoustic Probe with Amplified Brightness for In Vivo Imaging of pH (Adv. Mater. 19/2016).

    Science.gov (United States)

    Miao, Qingqing; Lyu, Yan; Ding, Dan; Pu, Kanyi

    2016-05-01

    Despite the great potential of photoacoustic imaging in the life sciences, the development of smart activatable photoacoustic probes remains elusive. On page 3662, K. Pu and co-workers report a facile nanoengineering approach based on semiconducting oligomer nano-particles to develop ratiometric photoacoustic probes with amplified brightness and enhanced sensing capability for accurate photoacoustic mapping of pH in the tumors of living mice.

  13. Spermine detection via metal-mediated ethynylarene ‘turn-on’ fluorescence signaling

    OpenAIRE

    Fletcher, James T.; Bruck, Brent S.

    2015-01-01

    A dicarboxylated ethynylarene was shown to behave as a fluorescent chemosensor for millimolar concentrations of polyamines when mixed with Cd(II), Pb(II) or Zn(II) ions at micromolar concentrations. A bathochromic shift and intensification of fluorescence emission was observed with increasing amounts of metal ion in the presence of aqueous polyamines buffered at pH = 7.6. Such perturbations manifested as ‘turn-on’ signals from a ratiometric comparison of emission intensities at 390 nm versus ...

  14. A graphitic carbon nitride based fluorescence resonance energy transfer detection of riboflavin.

    Science.gov (United States)

    Han, Jing; Zou, Hong Yan; Gao, Ming Xuan; Huang, Cheng Zhi

    2016-01-01

    Fluorescence resonance energy transfer (FRET), which occurs between two luminescent chromophores, can greatly improve the selectivity and sensitivity of a fluorescent assay when a ratiometric signaling with the fluorescence enhancement of the acceptor at the expense of the donor is adopted. In this study, a fluorescence ratiometric detection (FRD) of riboflavin (RF) has been made based on FRET, as the strong overlap occurred between the emission spectrum of graphitic carbon nitride (g-C3N4) and absorption spectrum of RF, in which g-C3N4 acts as the energy donor and RF as the energy acceptor. With increasing concentration of RF, the fluorescence intensity of g-C3N4 emission at 444 nm decreased and the fluorescence peak at 523 nm for RF increased regularly, making the fluorescence intensity ratio of 523 nm to 444 nm linearly dependent on the concentration of RF in the range from 0.4 μM to 10 μM, giving a limit of the detection of 170 nM. This method can be used to quantify RF in complex systems such as milk and drink, showing that the novel FRET-based fluorescence ratiometric detection can enable an attractive assay platform for analytes of interest.

  15. A graphitic carbon nitride based fluorescence resonance energy transfer detection of riboflavin.

    Science.gov (United States)

    Han, Jing; Zou, Hong Yan; Gao, Ming Xuan; Huang, Cheng Zhi

    2016-01-01

    Fluorescence resonance energy transfer (FRET), which occurs between two luminescent chromophores, can greatly improve the selectivity and sensitivity of a fluorescent assay when a ratiometric signaling with the fluorescence enhancement of the acceptor at the expense of the donor is adopted. In this study, a fluorescence ratiometric detection (FRD) of riboflavin (RF) has been made based on FRET, as the strong overlap occurred between the emission spectrum of graphitic carbon nitride (g-C3N4) and absorption spectrum of RF, in which g-C3N4 acts as the energy donor and RF as the energy acceptor. With increasing concentration of RF, the fluorescence intensity of g-C3N4 emission at 444 nm decreased and the fluorescence peak at 523 nm for RF increased regularly, making the fluorescence intensity ratio of 523 nm to 444 nm linearly dependent on the concentration of RF in the range from 0.4 μM to 10 μM, giving a limit of the detection of 170 nM. This method can be used to quantify RF in complex systems such as milk and drink, showing that the novel FRET-based fluorescence ratiometric detection can enable an attractive assay platform for analytes of interest. PMID:26653450

  16. RNA fluorescence with light-up aptamers

    Science.gov (United States)

    Ouellet, Jonathan

    2016-06-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way.

  17. At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code.

    Science.gov (United States)

    Xiao, Han; Schultz, Peter G

    2016-01-01

    The ability to site-specifically incorporate noncanonical amino acids (ncAAs) with novel structures into proteins in living cells affords a powerful tool to investigate and manipulate protein structure and function. More than 200 ncAAs with diverse biological, chemical, and physical properties have been genetically encoded in response to nonsense or frameshift codons in both prokaryotic and eukaryotic organisms with high fidelity and efficiency. In this review, recent advances in the technology and its application to problems in protein biochemistry, cellular biology, and medicine are highlighted. PMID:27413101

  18. Development of biosensors and their application in metabolic engineering

    DEFF Research Database (Denmark)

    Zhang, Jie; Jensen, Michael Krogh; Keasling, Jay

    2015-01-01

    for the desired phenotypes. However, methods available for microbial genome diversification far exceed our ability to screen and select for those variants with optimal performance. Genetically encoded biosensors have shown the potential to address this gap, given their ability to respond to small molecule binding...... and ease of implementation with high-throughput analysis. Here we describe recent progress in biosensor development and their applications in a metabolic engineering context. We also highlight examples of how biosensors can be integrated with synthetic circuits to exert feedback regulation...

  19. RNA Fluorescence with Light-Up Aptamers

    Science.gov (United States)

    Ouellet, Jonathan

    2016-01-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way. PMID:27446908

  20. Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells

    OpenAIRE

    Jichuan Zhang; Jingyi Fei; Leslie, Benjamin J.; Kyu Young Han; Kuhlman, Thomas E; Taekjip Ha

    2015-01-01

    Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs po...

  1. Arduino Due based tool to facilitate in vivo two-photon excitation microscopy.

    Science.gov (United States)

    Artoni, Pietro; Landi, Silvia; Sato, Sebastian Sulis; Luin, Stefano; Ratto, Gian Michele

    2016-04-01

    Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra. This tool is portable, fast, affordable and precise. It allowed studying the impact of scattering and of blood absorption on two-photon excitation light. In this way, we determined the wavelength-dependent deformation of excitation spectra occurring in deep tissues in vivo. PMID:27446677

  2. Stimulus responsive elastin biopolymers: applications in medicine and biotechnology

    Science.gov (United States)

    Chilkoti, Ashutosh; Christensen, Trine; MacKay, J Andrew

    2013-01-01

    Elastin-like polypeptides (ELPs) are artificial polypeptides, derived from Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide repeats found in human tropoelastin, that reversibly coacervate above a critical temperature. Genetically encodable ELPs are monodisperse, stimuli responsive, and biocompatible, properties that make them attractive for drug delivery and tissue engineering. The potential of ELPs to self-assemble into nanostructures in response to environmental triggers is another interesting feature of these polypeptides that promises to lead to a host of new applications. PMID:17055770

  3. Peptide-based Biopolymers in Biomedicine and Biotechnology

    Science.gov (United States)

    Chow, Dominic; Nunalee, Michelle L.; Lim, Dong Woo; Simnick, Andrew J.; Chilkoti, Ashutosh

    2008-01-01

    Peptides are emerging as a new class of biomaterials due to their unique chemical, physical, and biological properties. The development of peptide-based biomaterials is driven by the convergence of protein engineering and macromolecular self-assembly. This review covers the basic principles, applications, and prospects of peptide-based biomaterials. We focus on both chemically synthesized and genetically encoded peptides, including poly-amino acids, elastin-like polypeptides, silk-like polymers and other biopolymers based on repetitive peptide motifs. Applications of these engineered biomolecules in protein purification, controlled drug delivery, tissue engineering, and biosurface engineering are discussed. PMID:19122836

  4. Fluorogen-based reporters for fluorescence imaging: a review

    Science.gov (United States)

    Jullien, Ludovic; Gautier, Arnaud

    2015-12-01

    Fluorescence bioimaging has recently jumped into a new area of spatiotemporal resolution and sensitivity thanks to synergistic advances in both optical physics and probe/biosensor design. This review focuses on the recent development of genetically encodable fluorescent reporters that bind endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activate their fluorescence. We highlight the innovative engineering and design that gave rise to these new natural and synthetic fluorescent reporters, and describe some of the emerging applications in imaging and biosensing.

  5. Visualizing voltage dynamics in zebrafish heart.

    Science.gov (United States)

    Tsutsui, Hidekazu; Higashijima, Shin-ichi; Miyawaki, Atsushi; Okamura, Yasushi

    2010-06-15

    The zebrafish heart provides a useful vertebrate cardiovascular model with outstanding advantages, including genetic manipulatability, optical accessibility and rapid development. In addition, an emerging topic in cardiotoxicity assay and drug discovery is its use in phenotype-based chemical screening. Here, we report a technique that allows non-invasive voltage mapping in beating heart using a genetically encoded probe for transmembrane potential. Application of the anti-allergy compound astemizole resulted in aberrant propagation of excitation, which accounted for a lack of ventricular contraction. This optical method will provide new opportunities in broad areas of physiological, developmental and pharmacological cardiovascular research. PMID:20421282

  6. X-ray irradiation activates K+ channels via H2O2 signaling

    OpenAIRE

    Gibhardt, Christine S.; Bastian Roth; Indra Schroeder; Sebastian Fuck; Patrick Becker; Burkhard Jakob; Claudia Fournier; Anna Moroni; Gerhard Thiel

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1...

  7. Arduino Due based tool to facilitate in vivo two-photon excitation microscopy.

    Science.gov (United States)

    Artoni, Pietro; Landi, Silvia; Sato, Sebastian Sulis; Luin, Stefano; Ratto, Gian Michele

    2016-04-01

    Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra. This tool is portable, fast, affordable and precise. It allowed studying the impact of scattering and of blood absorption on two-photon excitation light. In this way, we determined the wavelength-dependent deformation of excitation spectra occurring in deep tissues in vivo.

  8. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J;

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases....

  9. Structure and Reaction Mechanism of Pyrrolysine Synthase (PylD)

    KAUST Repository

    Quitterer, Felix

    2013-05-29

    The final step in the biosynthesis of the 22nd genetically encoded amino acid, pyrrolysine, is catalyzed by PylD, a structurally and mechanistically unique dehydrogenase. This catalyzed reaction includes an induced-fit mechanism achieved by major structural rearrangements of the N-terminal helix upon substrate binding. Different steps of the reaction trajectory are visualized by complex structures of PylD with substrate and product. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release

    OpenAIRE

    Vishnu, Neelanjan; Jadoon Khan, Muhammad; Karsten, Felix; Groschner, Lukas N.; Waldeck-Weiermair, Markus; Rost, Rene; Hallström, Seth; Imamura, Hiromi; Graier, Wolfgang F; Malli, Roland

    2014-01-01

    Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca2+ and redox insensitive, which makes it possible to monitor Ca2+-coupled ER ATP dynamics specifically. The approach uncovers a c...

  11. SLAP: Small Labeling Pair for Single-Molecule Super-Resolution Imaging.

    Science.gov (United States)

    Wieneke, Ralph; Raulf, Anika; Kollmannsperger, Alina; Heilemann, Mike; Tampé, Robert

    2015-08-24

    Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).

  12. ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc

    OpenAIRE

    Wang, DA; Hosteen, Olijahwon; Fierke, Carol A.

    2012-01-01

    In E. coli, ZitB and ZntA are important metal exporters that enhance cell viability under high environmental zinc. To understand their functions in maintaining zinc homeostasis, we applied a novel genetically-encoded fluorescent zinc sensor to monitor the intracellular free zinc changes in wild type, ΔzitB and ΔzntA E. coli cells upon sudden exposure to toxic levels of zinc (“zinc shock”). The intracellular readily exchangeable zinc concentration (or “free” zinc) increases transiently from pi...

  13. The evolution of secondary organization in immune system gene libraries

    Energy Technology Data Exchange (ETDEWEB)

    Hightower, R.; Forrest, S. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science; Perelson, A.S. [Los Alamos National Lab., NM (United States)

    1993-02-01

    A binary model of the immune system is used to study the effects of evolution on the genetic encoding for antibody molecules. We report experiments which show that the evolution of immune system genes, simulated by the genetic algorithm, can induce a high degree of genetic organization even though that organization is not explicitly required by the fitness function. This secondary organization is related to the true fitness of an individual, in contrast to the sampled fitness which is the explicit fitness measure used to drive the process of evolution.

  14. Optogenetics for gene expression in mammalian cells.

    Science.gov (United States)

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  15. Optogenetic control of transcription in zebrafish.

    Directory of Open Access Journals (Sweden)

    Hongtao Liu

    Full Text Available Light inducible protein-protein interactions are powerful tools to manipulate biological processes. Genetically encoded light-gated proteins for controlling precise cellular behavior are a new and promising technology, called optogenetics. Here we exploited the blue light-induced transcription system in yeast and zebrafish, based on the blue light dependent interaction between two plant proteins, blue light photoreceptor Cryptochrome 2 (CRY2 and the bHLH transcription factor CIB1 (CRY-interacting bHLH 1. We demonstrate the utility of this system by inducing rapid transcription suppression and activation in zebrafish.

  16. A novel fluorescent turn-on probe for bisulfite based on NBD chromophore

    Indian Academy of Sciences (India)

    Puhui Xie; Guangqin Gao; Wenjie Zhang; Guoyu Yang; Qiu Jin

    2015-07-01

    A novel fluorescent turn-on probe (compound 1) for bisulfite based on 7-nitrobenz-2-oxa-1,3-diazole (NBD) chromophore has been developed. Its sensing behavior toward various anions was investigated by absorption and fluorescence techniques. This probe shows a selective, turn-on fluorescent response and ratiometric colorimetric response toward bisulfite in aqueous acetonitrile solutions. The possible recognition mechanism of probe 1 toward bisulfite was illustrated by MS spectra analysis and DFT calculations Probe 1 was used to determine bisulfite in real-life samples with good recoveries.

  17. Fluorescence lifetime-based biosensing of zinc: Origin of the broad dynamic range.

    Science.gov (United States)

    Thompson, R B; Patchan, M W

    1995-06-01

    Fluorescence lifetime-based chemical sensors have recently been described for applications in medicine, environmental monitoring, and bioprocess control. These sensors transduce the level of the analyte as a change in the apparent fluorescence lifetime of an indicator phase. We have previously developed a wavelength-ratiometric fluorescence biosensor for zinc based on binding of zinc and dansylamide to apo-carbonic anhydrase which exhibited high sensitivity and selectivity. We demonstrate that the apo-carbonic anhydrase/dansylamide indicator system is very well suited for lifetime-based sensing, with a subnanomolar detection limit and greater than 1000-fold dynamic range. The theoretical basis for the wide dynamic range is discussed.

  18. Cytoplasmic pH Response to Acid Stress in Individual Cells of Escherichia coli and Bacillus subtilis Observed by Fluorescence Ratio Imaging Microscopy

    OpenAIRE

    Martinez, Keith A.; Ryan D Kitko; Mershon, J. Patrick; Adcox, Haley E.; Malek, Kotiba A.; Berkmen, Melanie B.; Slonczewski, Joan L.

    2012-01-01

    The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switch...

  19. Study on the Interferation of the Concentration of Free Calcium in Peripheral Human Lymphocyte by Low Dose Penicillin, Using Fura-2 as Fluorescent Probe

    Institute of Scientific and Technical Information of China (English)

    Hai Yan WANG; Dan Dan WANG; Chun Gui ZHAO; Ye Hong ZHOU; Shao Min SHUANG; Chuan DONG

    2006-01-01

    The effect of penicillin on the human peripheral lymphocytes was studied by steady fluorescent technique and ratiometric fluorescence dye, Fura-2. The change of the free calcium concentration in cytosol was examined under different conditions. A characterization of Fura-2-Ca interaction in an isotonic saline solution showed that Ca2+ formed a 1:1 Fura-2-Ca complex with the apparent dissociation constant 1.81×10-7 mol/L. The mechanism, by which penicillin induced the decrease of [Ca2+]i, was discussed in detail. The low dose of penicillin might modify the lymphocytes' immunology response by interfering the increase in the intracellular free calcium concentration.

  20. Azadioxatriangulenium

    DEFF Research Database (Denmark)

    Sørensen, Thomas Just; Thyrhaug, Erling; Szabelski, Mariusz;

    2013-01-01

    %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes.......Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable...

  1. Fluorescence Resonance Energy Transfer (FRET) sensor

    CERN Document Server

    Hussain, Syed Arshad; Chakraborty, Sekhar; Saha, Jaba; Roy, Arpan Datta; Chakraborty, Santanu; Debnath, Pintu; Bhattacharjee, D

    2014-01-01

    The applications of Fluorescence resonance energy transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. FRET can be used as spectroscopic ruler in various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting, and metallic nanomaterials etc. Based on the mechanism of FRET a variety of novel chemical sensors and Biosensors have been developed. This review highlights the recent applications of sensitive and selective ratiometric FRET based sensors.

  2. A simple optode based imaging technique to measure O2 distribution and dynamics in tap water biofilms

    DEFF Research Database (Denmark)

    Staal, Marc Jaap; Prest, E.; Vrouwenvelder, H.;

    2011-01-01

    ratiometric method and compare the results with O2 distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O2 distributions with a high signal......-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O2 concentration...

  3. Intravital FRET: Probing Cellular and Tissue Function in Vivo

    Directory of Open Access Journals (Sweden)

    Helena Radbruch

    2015-05-01

    Full Text Available The development of intravital Förster Resonance Energy Transfer (FRET is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.

  4. The future of fluorescence sensor arrays.

    Science.gov (United States)

    Demchenko, Alexander P

    2005-09-01

    The rapid progress in sensor and biosensor array technologies needs a general strategy in the design of fluorescence reporters. Such reporters should provide a high density of sensor elements, allow analysis of targets of different affinities, and be internally calibrated, reproducible and have a rapid readout. Several criteria are introduced here for the comparative evaluation of fluorescence-sensing techniques. It is shown that only the two-band wavelength ratiometric sensing with a single reporter dye exhibiting rapid reversible excited-state reaction can satisfy all these criteria and is a prospective candidate for further development. PMID:15967523

  5. Monitoring of extracellular pH in young dental biofilms grown in vivo in the presence and absence of sucrose

    OpenAIRE

    Dige, Irene; Baelum, Vibeke; Nyvad, Bente; Schlafer, Sebastian

    2016-01-01

    Background and objective: pH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply.Design: Dental biofilms (48 h) from 10 individuals were collected on glass slabs mounted on intra-oral appliances. During growth, appliances were immersed extra-orally in either physiological saline or 4% s...

  6. Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents

    Directory of Open Access Journals (Sweden)

    Jennifer H Hou

    2014-09-01

    Full Text Available The cardiac action potential (AP and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf. We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 – 102 hours post fertilization (hpf, the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

  7. Development of a Cell-Based Fluorescence Resonance Energy Transfer Reporter for Bacillus anthracis Lethal Factor Protease

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, R H; Steenblock, E R; Camarero, J A

    2007-03-22

    We report the construction of a cell-based fluorescent reporter for anthrax lethal factor (LF) protease activity using the principle of fluorescence resonance energy transfer (FRET). This was accomplished by engineering an Escherichia coli cell line to express a genetically encoded FRET reporter and LF protease. Both proteins were encoded in two different expression plasmids under the control of different tightly controlled inducible promoters. The FRET-based reporter was designed to contain a LF recognition sequence flanked by the FRET pair formed by CyPet and YPet fluorescent proteins. The length of the linker between both fluorescent proteins was optimized using a flexible peptide linker containing several Gly-Gly-Ser repeats. Our results indicate that this FRET-based LF reporter was readily expressed in E. coli cells showing high levels of FRET in vivo in the absence of LF. The FRET signal, however, decreased 5 times after inducing LF expression in the same cell. These results suggest that this cell-based LF FRET reporter may be used to screen genetically encoded libraries in vivo against LF.

  8. Intelligent Design of Nano-Scale Molecular Imaging Agents

    Directory of Open Access Journals (Sweden)

    Takeaki Ozawa

    2012-12-01

    Full Text Available Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs, biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on–off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents.

  9. Studying the mechanism of neurostimulation by infrared laser light using GCaMP6s and Rhodamine B imaging

    Science.gov (United States)

    Moreau, David; Lefort, Claire; Bardet, Sylvia M.; O'Connor, Rodney P.

    2016-03-01

    Infrared laser light radiation can be used to depolarize neurons and to stimulate neural activity. The absorption of infrared radiation and heating of biological tissue is thought to be the underlying mechanism of this phenomenon whereby local temperature increases in the plasma membrane of cells either directly influence membrane properties or act via temperature sensitive ion channels. Action potentials are typically measured electrically in neurons with microelectrodes, but they can also be observed using fluorescence microscopy techniques that use synthetic or genetically encoded calcium indicators. In this work, we studied the impact of infrared laser light on neuronal calcium signals to address the mechanism of these thermal effects. Cultured primary mouse hippocampal neurons expressing the genetically encoded calcium indicator GCaMP6s were used in combination with the temperature sensitive fluorophore Rhodamine B to measure calcium signals and temperature changes at the cellular level. Here we present our all-optical strategy for studying the influence of infrared laser light on neuronal activity.

  10. Fiber-optic control and thermometry of single-cell thermosensation logic

    Science.gov (United States)

    Fedotov, I. V.; Safronov, N. A.; Ermakova, Yu. G.; Matlashov, M. E.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Belousov, V. V.; Zheltikov, A. M.

    2015-11-01

    Thermal activation of transient receptor potential (TRP) cation channels is one of the most striking examples of temperature-controlled processes in cell biology. As the evidence indicating the fundamental role of such processes in thermosensation builds at a fast pace, adequately accurate tools that would allow heat receptor logic behind thermosensation to be examined on a single-cell level are in great demand. Here, we demonstrate a specifically designed fiber-optic probe that enables thermal activation with simultaneous online thermometry of individual cells expressing genetically encoded TRP channels. This probe integrates a fiber-optic tract for the delivery of laser light with a two-wire microwave transmission line. A diamond microcrystal fixed on the fiber tip is heated by laser radiation transmitted through the fiber, providing a local heating of a cell culture, enabling a well-controlled TRP-assisted thermal activation of cells. Online local temperature measurements are performed by using the temperature-dependent frequency shift of optically detected magnetic resonance, induced by coupling the microwave field, delivered by the microwave transmission line, to nitrogen—vacancy centers in the diamond microcrystal. Activation of TRP channels is verified by using genetically encoded fluorescence indicators, visualizing an increase in the calcium flow through activated TRP channels.

  11. Engineering an NADPH/NADP+ Redox Biosensor in Yeast

    DEFF Research Database (Denmark)

    Zhang, Jie; Sonnenschein, Nikolaus; Pihl, Thomas Peter Boye;

    2016-01-01

    Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biote......Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science...... and biotechnology. Still, there is a need for bioprospecting and engineering of more biosensors to enable real-time monitoring of specific cellular states and controlling downstream actuation. In this study, we report the engineering and application of a transcription factor-based NADPH/NADP+ redox biosensor...... in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP+ ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon...

  12. Optogenetic control of ROS production

    Directory of Open Access Journals (Sweden)

    Andrew P. Wojtovich

    2014-01-01

    Full Text Available Reactive Oxygen Species (ROS are known to cause oxidative damage to DNA, proteins and lipids. In addition, recent evidence suggests that ROS can also initiate signaling cascades that respond to stress and modify specific redox-sensitive moieties as a regulatory mechanism. This suggests that ROS are physiologically-relevant signaling molecules. However, these sensor/effector molecules are not uniformly distributed throughout the cell. Moreover, localized ROS damage may elicit site-specific compensatory measures. Thus, the impact of ROS can be likened to that of calcium, a ubiquitous second messenger, leading to the prediction that their effects are exquisitely dependent upon their location, quantity and even the timing of generation. Despite this prediction, ROS signaling is most commonly intuited through the global administration of chemicals that produce ROS or by ROS quenching through global application of antioxidants. Optogenetics, which uses light to control the activity of genetically-encoded effector proteins, provides a means of circumventing this limitation. Photo-inducible genetically-encoded ROS-generating proteins (RGPs were originally employed for their phototoxic effects and cell ablation. However, reducing irradiance and/or fluence can achieve sub-lethal levels of ROS that may mediate subtle signaling effects. Hence, transgenic expression of RGPs as fusions to native proteins gives researchers a new tool to exert spatial and temporal control over ROS production. This review will focus on the new frontier defined by the experimental use of RGPs to study ROS signaling.

  13. Albumin-NIR dye self-assembled nanoparticles for photoacoustic pH imaging and pH-responsive photothermal therapy effective for large tumors.

    Science.gov (United States)

    Chen, Qian; Liu, Xiaodong; Zeng, Jianfeng; Cheng, Zhenping; Liu, Zhuang

    2016-08-01

    Real-time in vivo pH imaging in the tumor, as well as designing therapies responsive to the acidic tumor microenvironment to achieve optimized therapeutic outcomes have been of great interests in the field of nanomedicine. Herein, a pH-responsive near-infrared (NIR) croconine (Croc) dye is able to induce the self-assembly of human serum albumin (HSA) to form HSA-Croc nanoparticles useful not only for real-time ratiometric photoacoustic pH imaging of the tumor, but also for pH responsive photothermal therapy with unexpected great performance against tumors with relatively large sizes. Such HSA-Croc nanoparticles upon intravenous injection exhibit efficient tumor homing. As the decrease of pH, the absorption of Croc at 810 nm would increase while that at 680 nm would decrease, allowing real-time pH sensing in the tumor by double-wavelength ratiometric photoacoustic imaging, which reveals the largely decreased pH inside the cores of large tumors. Moreover, utilizing HSA-Croc as a pH-responsive photothermal agent, effective photothermal ablation of large tumors is realized, likely owing to the more evenly distributed intratumoral heating compared to that achieved by conventional pH-insensitive photothermal agents, which are effective mostly for tumors with small sizes. PMID:27177219

  14. An evolutionary optimized nonlinear function to improve the linearity of transducer characteristics

    Science.gov (United States)

    Abudhahir, A.; Baskar, S.

    2008-04-01

    This paper proposes a nonlinear optimal-function-based algorithm which can be utilized to replace electronic circuitry traditionally employed to linearize the characteristics of commonly used temperature transducers such as resistance temperature detectors, thermistors and thermocouples. The function exploits ratiometric-logarithmic operation for linearization. The optimal parameters of the function are determined using a covariance matrix adopted evolutionary strategy (CMAES) algorithm. Transducers' input-output data are derived from the Yokogawa handy calibrator model CA 150 and subjected to the proposed algorithm to evaluate the performance of the method. The performance measures such as full-scale error and mean square error are considered to compare the performance of the proposed technique with other methods reported for transducers. The present linearization algorithm was implemented using LabVIEW 7.1 Professional Development System in a personal computer that provides the facility to interface with the National Instruments data acquisition module NI DAQCard PCI-6221. Experimental results reveal that the proposed evolutionary optimized nonlinear function based software linearizer does its job efficiently in a better way than that of the conventional hardware and software methods. Also, the results obtained using the CMAES algorithm are compared with the results of a real-coded genetic algorithm. The comparison shows that the CMAES algorithm is more consistent in determining the best solution for the proposed ratiometric-logarithmic function with reasonable computation time.

  15. Dual-emissive Polymer Dots for Rapid Detection of Fluoride in Pure Water and Biological Systems with Improved Reliability and Accuracy

    Science.gov (United States)

    Zhao, Qiang; Zhang, Chuanqi; Liu, Shujuan; Liu, Yahong; Zhang, Kenneth Yin; Zhou, Xiaobo; Jiang, Jiayang; Xu, Wenjuan; Yang, Tianshe; Huang, Wei

    2015-11-01

    It is of paramount importance to develop new probes that can selectively, sensitively, accurately and rapidly detect fluoride in aqueous media and biological systems, because F- is found to be closely related to many health and environmental concerns. Herein, a dual-emissive conjugated polyelectrolyte P1 containing phosphorescent iridium(III) complex was designed and synthesized, which can form ultrasmall polymer dots (Pdots) in aqueous media. The F--responsive tert-butyldiphenylsilyl moiety was introduced into iridium(III) complex as the signaling unit for sensing F- with the quenched phosphorescence. Thus, the dual-emissive Pdots can rapidly and accurately detect F- in aqueous media and live cells as a ratiometric probe by measuring the change in the ratio of the F--sensitive red phosphorescence from iridium(III) complex to the F--insensitive blue fluorescence from polyfluorene. Moreover, the interaction of Pdots with F- also changes its emission lifetime, and the lifetime-based detection of F- in live cells has been realized through photoluminescence lifetime imaging microscopy for the first time. Both the ratiometric luminescence and lifetime imaging have been demonstrated to be resistant to external influences, such as the probe’s concentration and excitation power. This study provides a new perspective for the design of promising Pdots-based probes for biological applications.

  16. Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes

    Science.gov (United States)

    Niko, Yosuke; Didier, Pascal; Mely, Yves; Konishi, Gen-Ichi; Klymchenko, Andrey S.

    2016-01-01

    Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

  17. Polypeptide micelles with dual pH activatable dyes for sensing cells and cancer imaging.

    Science.gov (United States)

    Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao

    2014-05-21

    pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.

  18. Research of the relationship of intracellular acidification and apoptosis in Hela cells based on pH nanosensors

    Institute of Scientific and Technical Information of China (English)

    HE XiaoXiao; WANG Yan; WANG KeMin; PENG JiaoFeng; LIU Fang; TAN WeiHong

    2007-01-01

    In this paper, the relationship of intracellular acidification and apoptosis in Hela cells induced by vincristine sulfate has been studied by use of the ratiometric pH nanosensors that have been developed by our group, employing fluorescein isothiocyanate (FITC) doped as the pH-sensitive dye and Tris(2,2'-bipyidyl) dichlororuthenium(Ⅱ) hexahydrate (RuBpy) doped as reference dye. The pH change of the Hela cells induced by vincristine sulfate has been monitored in vivo, in situ and real time by use of the ratiometric pH nanosensors. The experimental results show that the pH of the apoptotic Hela cells induced by vincristine sulfate has been acidified from 7.11 to 6.51, and the percentage of intracellular acidification is correlated with the induced concentration and incubation time of the vincristine sulfate. The further study of the percentage of intracellular acidification and the percentage of apoptosis of Hela cells at the same time reveals that apoptosis of Hela cells induced by vincristine sulfate is preceded by intracellular acidification. These results would provide theoretical foundation for the therapy of cancer through interfering the pH of cells by use of vincristine sulfate or other anti-cancer drugs.

  19. Research of the relationship of intracellular acidification and apoptosis in Hela cells based on pH nanosensors

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this paper,the relationship of intracellular acidification and apoptosis in Hela cells induced by vin-cristine sulfate has been studied by use of the ratiometric pH nanosensors that have been developed by our group,employing fluorescein isothiocyanate(FITC) doped as the pH-sensitive dye and Tris(2,2’-bipyidyl) dichlororuthenium(II) hexahydrate(RuBpy) doped as reference dye. The pH change of the Hela cells induced by vincristine sulfate has been monitored in vivo,in situ and real time by use of the ratiometric pH nanosensors. The experimental results show that the pH of the apoptotic Hela cells induced by vincristine sulfate has been acidified from 7.11 to 6.51,and the percentage of intra-cellular acidification is correlated with the induced concentration and incubation time of the vincristine sulfate. The further study of the percentage of intracellular acidification and the percentage of apop-tosis of Hela cells at the same time reveals that apoptosis of Hela cells induced by vincristine sulfate is preceded by intracellular acidification. These results would provide theoretical foundation for the therapy of cancer through interfering the pH of cells by use of vincristine sulfate or other anti-cancer drugs.

  20. Multi-parametric imaging of tumor spheroids with ultra-bright and tunable nanoparticle O2 probes

    Science.gov (United States)

    Dmitriev, Ruslan I.; Borisov, Sergey M.; Jenkins, James; Papkovsky, Dmitri B.

    2015-03-01

    Multi-modal probes allow for flexible choice of imaging equipment when performing quenched-phosphorescence O2 measurements: one- or two-photon, PLIM or intensity-based ratiometric read-outs. Spectral and temporal (e.g. FLIMPLIM) discrimination can be used to image O2 together with pH, Ca2+, mitochondrial membrane potential, cell death markers or cell/organelle specific markers. However, the main challenge of existing nanoparticle probes is their limited diffusion across thick (> 20-50 μm) 3D cell models such as tumor spheroids. Here, we present new class of polymeric nanoparticle probes having tunable size, charge, cell-penetrating ability, and reporter dyes. Being spectrally similar to the recently described MM2, PA2 and other O2 probes, they are 5-10 times brighter, demonstrate improved ratiometric response and their surface chemistry can be easily modified. With cultures of 2D and 3D cell models (fibroblasts, PC12 aggregates, HCT116 human colon cancer spheroids) we found cell-specific staining by these probes. However, the efficient staining of model of interest can be tuned by changing number of positive and negative surface groups at nanoparticle, to allow most efficient loading. We also demonstrate how real-time monitoring of oxygenation can be used to select optimal spheroid production with low variability in size and high cell viability.

  1. 0.5 V and 0.43 pJ/bit Capacitive Sensor Interface for Passive Wireless Sensor Systems.

    Science.gov (United States)

    Beriain, Andoni; Gutierrez, Iñigo; Solar, Hector; Berenguer, Roc

    2015-01-01

    This paper presents an ultra low-power and low-voltage pulse-width modulation based ratiometric capacitive sensor interface. The interface was designed and fabricated in a standard 90 nm CMOS 1P9M technology. The measurements show an effective resolution of 10 bits using 0.5 V of supply voltage. The active occupied area is only 0.0045 mm2 and the Figure of Merit (FOM), which takes into account the energy required per conversion bit, is 0.43 pJ/bit. Furthermore, the results show low sensitivity to PVT variations due to the proposed ratiometric architecture. In addition, the sensor interface was connected to a commercial pressure transducer and the measurements of the resulting complete pressure sensor show a FOM of 0.226 pJ/bit with an effective linear resolution of 7.64 bits. The results validate the use of the proposed interface as part of a pressure sensor, and its low-power and low-voltage characteristics make it suitable for wireless sensor networks and low power consumer electronics. PMID:26343681

  2. Molecular recognition of 4′-Nitrobenzo-15-crown-5 by bis(benzimidazolium)propane borontetrafluoride in acetonitrile

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhuri, Tandrima, E-mail: tanchem_bu@yahoo.co.in [Department of Chemistry, Dr. Bhupendranath Dutta Smriti Mahavidyalaya, Burdwan 713407 (India); Karmakar, Animesh [Department of Chemistry, Dr. Bhupendranath Dutta Smriti Mahavidyalaya, Burdwan 713407 (India); Ghosh, Sabari; Mukhopadhyay, Chhanda [Department of Chemistry, University of Calcutta, 92 APC Road, Kolkata 700009 (India); Pal, Sunanda [Department of Chemistry, Dr. Bhupendranath Dutta Smriti Mahavidyalaya, Burdwan 713407 (India); Banerjee, Manas [Department of Chemistry, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal (India)

    2015-05-15

    ICT based ratiometric sensing due to H-bonding interaction among three different crown ethers (C): Dibenzo-24-crown-8 (DB24C8 or C1), Benzo-15-crown-5 (B15C5 or C2) and 4′-Nitrobenzo-15-crown-5 (4′–NB15C5 or C3) along with the axle bis(benzimidazolium)propane borontetrafluoride (BBIM-propane) (3a–3d) have been studied. The association were initially ascertained from isosbestic formation and later corroborated by iso-emissive formation where C3 fails to establish iso-emissive. Stoichiometry of adducts were 1:1 both in the ground as well as in excited state. The threading or external association was finally distinguished by Monte Carlo simulation and frontier molecular orbital interaction. - Highlights: • The first report of ICT–based wavelength ratiometric interaction of crown–axle system. • Photophysical recognition of 4′-Nitrobenzo-15-crown-5 (4′-NB15C5 or C3) in acetonitrile. • Evidence of charge transfer interaction along with H-bond formation even in excited state is reported. • Monte Carlo simulation and FMO interaction justified the experimental findings.

  3. Enhanced fluorescence cyanide detection at physiologically lethal levels: reduced ICT-based signal transduction.

    Science.gov (United States)

    Badugu, Ramachandram; Lakowicz, Joseph R; Geddes, Chris D

    2005-03-16

    Three water-soluble fluorescent probes have been specifically designed to determine free cyanide concentrations up to physiologically lethal levels, >20 microM. The probes have been designed in such a way as to afford many notable sensing features, which render them unique with regard to signal transduction, photophysical characteristics, and their application to physiological cyanide determination and safeguard. The probes are readily able to reversibly bind free aqueous cyanide with dissociation constants around 4 microM3. Subsequent cyanide binding modulates the intramolecular charge transfer within the probes, a change in the electronic properties within the probes, resulting in enhanced fluorescence optical signals as a function of increased solution cyanide concentration. The ground-state chelation with cyanide produces wavelength shifts, which also enable the probes to sense cyanide in both an excitation and emission ratiometric manner, in addition to enhanced fluorescence signaling. This has enabled a generic cyanide sensing platform to be realized that is not dependent on fluorescent probe concentration, probe photodegradation, or fluctuations in the intensity of any employed excitation sources, ideal for remote cyanide sensing applications. Further, the >600 nm fluorescence emission of the probes potentially allows for enhanced fluorescence ratiometric cyanide sensing in the optical window of tissues and blood, facilitating their use for the transdermal monitoring of cyanide for mammalian safeguard or postmortem in fire victims, both areas of active research. PMID:15755185

  4. 0.5 V and 0.43 pJ/bit Capacitive Sensor Interface for Passive Wireless Sensor Systems

    Directory of Open Access Journals (Sweden)

    Andoni Beriain

    2015-08-01

    Full Text Available This paper presents an ultra low-power and low-voltage pulse-width modulation based ratiometric capacitive sensor interface. The interface was designed and fabricated in a standard 90 nm CMOS 1P9M technology. The measurements show an effective resolution of 10 bits using 0.5 V of supply voltage. The active occupied area is only 0.0045 mm2 and the Figure of Merit (FOM, which takes into account the energy required per conversion bit, is 0.43 pJ/bit. Furthermore, the results show low sensitivity to PVT variations due to the proposed ratiometric architecture. In addition, the sensor interface was connected to a commercial pressure transducer and the measurements of the resulting complete pressure sensor show a FOM of 0.226 pJ/bit with an effective linear resolution of 7.64 bits. The results validate the use of the proposed interface as part of a pressure sensor, and its low-power and low-voltage characteristics make it suitable for wireless sensor networks and low power consumer electronics.

  5. In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach.

    Science.gov (United States)

    Pineda Rodó, Albert; Váchová, Libuše; Palková, Zdena

    2012-01-01

    Many essential cellular processes are affected by transmembrane H(+) gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.

  6. Luminescent Gold Nanoparticles with Size-Independent Emission.

    Science.gov (United States)

    Liu, Jinbin; Duchesne, Paul N; Yu, Mengxiao; Jiang, Xingya; Ning, Xuhui; Vinluan, Rodrigo D; Zhang, Peng; Zheng, Jie

    2016-07-25

    Size-independent emission has been widely observed for ultrasmall thiolated gold nanoparticles (AuNPs) but our understanding of the photoluminescence mechanisms of noble metals on the nanoscale has remained limited. Herein, we report how the emission wavelength of a AuNP and the local binding geometry of a thiolate ligand (glutathione) on the AuNP are correlated, as these AuNPs emit at different wavelengths in spite of their identical size (ca. 2.5 nm). By using circular dichroism, X-ray absorption, and fluorescence spectroscopy, we found that a high Au-S coordination number (CN) and a high surface coverage resulted in strong Au(I) -ligand charge transfer, a chiral conformation, and 600 nm emission, whereas a low Au-S CN and a low surface coverage led to weak charge transfer, an achiral conformation, and 810 nm emission. These two size-independent emissions can be integrated into one single 2.5 nm AuNP by fine-tuning of the surface coverage; a ratiometric pH response was then observed owing to strong energy transfer between two emission centers, opening up new possibilities for the design of ultrasmall ratiometric pH nanoindicators. PMID:27348584

  7. Nanoparticle/Polymer assembled microcapsules with pH sensing property.

    Science.gov (United States)

    Zhang, Pan; Song, Xiaoxue; Tong, Weijun; Gao, Changyou

    2014-10-01

    The dual-labeled microcapsules via nanoparticle/polymer assembly based on polyamine-salt aggregates can be fabricated for the ratiometric intracellular pH sensing. After deposition of SiO2 nanoparticles on the poly(allylamine hydrochloride)/multivalent anionic salt aggregates followed by silicic acid treatment, the generated microcapsules are stable in a wide pH range (3.0 ∼ 8.0). pH sensitive dye and pH insensitive dye are simultaneously labeled on the capsules, which enable the ratiometric pH sensing. Due to the rough and positively charged surface, the microcapsules can be internalized by several kinds of cells naturally. Real-time measurement of intracellular pH in several living cells shows that the capsules are all located in acidic organelles after being taken up. Furthermore, the negatively charged DNA and dyes can be easily encapsulated into the capsules via charge interaction. The microcapsules with combination of localized pH sensing and drug loading abilities have many advantages, such as following the real-time transportation and processing of the carriers in cells. PMID:25081194

  8. In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach.

    Directory of Open Access Journals (Sweden)

    Albert Pineda Rodó

    Full Text Available Many essential cellular processes are affected by transmembrane H(+ gradients and intracellular pH (pHi. The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.

  9. A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time.

    Science.gov (United States)

    Seo, Daeha; Southard, Kaden M; Kim, Ji-Wook; Lee, Hyun Jung; Farlow, Justin; Lee, Jung-Uk; Litt, David B; Haas, Thomas; Alivisatos, A Paul; Cheon, Jinwoo; Gartner, Zev J; Jun, Young-Wook

    2016-06-01

    Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.

  10. Initial Photophysical Characterization of the Proteorhodopsin Optical Proton Sensor (PROPS

    Directory of Open Access Journals (Sweden)

    Jay eNadeau

    2015-09-01

    Full Text Available Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS, which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanoscale time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested.

  11. Lysosome-associated miniSOG as a photosensitizer for mammalian cells.

    Science.gov (United States)

    Ryumina, Alina P; Serebrovskaya, Ekaterina O; Staroverov, Dmitry B; Zlobovskaya, Olga A; Shcheglov, Alexander S; Lukyanov, Sergey A; Lukyanov, Konstantin A

    2016-01-01

    Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis. PMID:27528074

  12. Understanding Synaptogenesis and Functional Connectome in C. elegans by Imaging Technology

    Science.gov (United States)

    Hong, Jung-Hwa; Park, Mikyoung

    2016-01-01

    Formation of functional synapses is a fundamental process for establishing neural circuits and ultimately for expressing complex behavior. Extensive research has interrogated how such functional synapses are formed and how synapse formation contributes to the generation of neural circuitry and behavior. The nervous system of Caenorhabditis elegans, due to its relatively simple structure, the transparent body, and tractable genetic system, has been adapted as an excellent model to investigate synapses and the functional connectome. Advances in imaging technology together with the improvement of genetically encoded molecular tools enabled us to visualize synapses and neural circuits of the animal model, which provide insights into our understanding of molecules and their signaling pathways that mediate synapse formation and neuronal network modulation. Here, we review synaptogenesis in active zones and the mapping of local connectome in C. elegans nervous system whose understandings have been extended by the advances in imaging technology along with the genetic molecular tools. PMID:27445787

  13. Live-cell imaging of mammalian RNAs with Spinach2

    Science.gov (United States)

    Strack, Rita L.; Jaffrey, Samie R.

    2015-01-01

    The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of “RNA mimics of GFP” or Spinach technology for tagging mammalian RNAs, and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag. PMID:25605384

  14. Geometrical assembly of ultrastable protein templates for nanomaterials

    Science.gov (United States)

    Glover, Dominic J.; Giger, Lars; Kim, Steve S.; Naik, Rajesh R.; Clark, Douglas S.

    2016-06-01

    The fabrication of nanoscale devices requires architectural templates on which to position functional molecules in complex arrangements. Protein scaffolds are particularly promising templates for nanomaterials due to inherent molecular recognition and self-assembly capabilities combined with genetically encoded functionalities. However, difficulties in engineering protein quaternary structure into stable and well-ordered shapes have hampered progress. Here we report the development of an ultrastable biomolecular construction kit for the assembly of filamentous proteins into geometrically defined templates of controllable size and symmetry. The strategy combines redesign of protein-protein interaction specificity with the creation of tunable connector proteins that govern the assembly and projection angles of the filaments. The functionality of these nanoarchitectures is illustrated by incorporation of nanoparticles at specific locations and orientations to create hybrid materials such as conductive nanowires. These new structural components facilitate the manufacturing of nanomaterials with diverse shapes and functional properties over a wide range of processing conditions.

  15. Analog Module Placement Design Using Genetic Algorithm

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    This paper presents a novel genetic algorithm for analog module placement based on ageneralization of the two-dimensional bin packing problem. The genetic encoding and operators assure that allproblem constraints are always satisfied. Thus the potential problems of adding penalty terms to the costfunction are eliminated so that the search configuration space is drastically decreased. The dedicated costfunction is based on the special requirements of analog integrated circuits. A fractional factorial experimentwas conducted using an orthogonal array to study the algorithm parameters. A meta GA was applied todetermine the optimal parameter values. The algorithm was tested with several local benchmark circuits. Theexperimental results show that the algorithm has better performance than the simulated annealing approachwith satisfactory results comparable to manual placement. This study demonstrates the effectiveness of thegenetic algorithm in the analog module placement problem. The algorithm has been successfully used in alayout synthesis tool.

  16. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    Science.gov (United States)

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  17. Density-dependent adaptive resistance allows swimming bacteria to colonize an antibiotic gradient.

    Science.gov (United States)

    Hol, Felix J H; Hubert, Bert; Dekker, Cees; Keymer, Juan E

    2016-01-01

    During antibiotic treatment, antibiotic concentration gradients develop. Little is know regarding the effects of antibiotic gradients on populations of nonresistant bacteria. Using a microfluidic device, we show that high-density motile Escherichia coli populations composed of nonresistant bacteria can, unexpectedly, colonize environments where a lethal concentration of the antibiotic kanamycin is present. Colonizing bacteria establish an adaptively resistant population, which remains viable for over 24 h while exposed to the antibiotic. Quantitative analysis of multiple colonization events shows that collectively swimming bacteria need to exceed a critical population density in order to successfully colonize the antibiotic landscape. After colonization, bacteria are not dormant but show both growth and swimming motility under antibiotic stress. Our results highlight the importance of motility and population density in facilitating adaptive resistance, and indicate that adaptive resistance may be a first step to the emergence of genetically encoded resistance in landscapes of antibiotic gradients.

  18. Biophysical Properties of Optogenetic Tools and Their Application for Vision Restoration Approaches.

    Science.gov (United States)

    Klapper, Simon D; Swiersy, Anka; Bamberg, Ernst; Busskamp, Volker

    2016-01-01

    Optogenetics is the use of genetically encoded light-activated proteins to manipulate cells in a minimally invasive way using light. The most prominent example is channelrhodopsin-2 (ChR2), which allows the activation of electrically excitable cells via light-dependent depolarization. The combination of ChR2 with hyperpolarizing-light-driven ion pumps such as the Cl(-) pump halorhodopsin (NpHR) enables multimodal remote control of neuronal cells in culture, tissue, and living animals. Very soon, it became obvious that this method offers a chance of gene therapy for many diseases affecting vision. Here, we will give a brief introduction to retinal function and retinal diseases; optogenetic vision restoration strategies will be highlighted. We will discuss the functional and structural properties of rhodopsin-based optogenetic tools and analyze the potential for the application of vision restoration. PMID:27642278

  19. Human SepSecS or SLA/LP: selenocysteine formation and autoimmune hepatitis.

    Science.gov (United States)

    Palioura, Sotiria; Herkel, Johannes; Simonović, Miljan; Lohse, Ansgar W; Söll, Dieter

    2010-07-01

    Selenocysteine, the 21st genetically encoded amino acid, is the major form of the antioxidant trace element selenium in the human body. In eukaryotes and archaea its synthesis proceeds through a phosphorylated intermediate in a tRNA-dependent fashion. The final step of selenocysteine formation is catalyzed by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) that converts phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). The human SepSecS protein is also known as soluble liver antigen/liver pancreas (SLA/LP), which represents one of the antigens of autoimmune hepatitis. Here we review the discovery of human SepSecS and the current understanding of the immunogenicity of SLA/LP in autoimmune hepatitis.

  20. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    Science.gov (United States)

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. PMID:23242824

  1. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy

    Directory of Open Access Journals (Sweden)

    Segall Jeffrey E

    2005-05-01

    Full Text Available Abstract Background The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. Results Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1 the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2 direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. Conclusion Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

  2. Remote control of myosin and kinesin motors using light-activated gearshifting.

    Science.gov (United States)

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C; Schindler, Tony D; Nogales, Eva; Bryant, Zev

    2014-09-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

  3. VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis.

    Science.gov (United States)

    Nicholson-Fish, Jessica C; Kokotos, Alexandros C; Gillingwater, Thomas H; Smillie, Karen J; Cousin, Michael A

    2015-12-01

    The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.

  4. Imaging light responses of foveal ganglion cells in the living macaque eye.

    Science.gov (United States)

    Yin, Lu; Masella, Benjamin; Dalkara, Deniz; Zhang, Jie; Flannery, John G; Schaffer, David V; Williams, David R; Merigan, William H

    2014-05-01

    The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses. PMID:24806684

  5. Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

    Directory of Open Access Journals (Sweden)

    Juan A. González-Vera

    2015-11-01

    Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

  6. The old 3-oxoadipate pathway revisited: new insights in the catabolism of aromatics in the saprophytic fungus Aspergillus nidulans.

    Science.gov (United States)

    Martins, Tiago M; Hartmann, Diego O; Planchon, Sébastien; Martins, Isabel; Renaut, Jenny; Silva Pereira, Cristina

    2015-01-01

    Aspergilli play major roles in the natural turnover of elements, especially through the decomposition of plant litter, but the end catabolism of lignin aromatic hydrocarbons remains largely unresolved. The 3-oxoadipate pathway of their degradation combines the catechol and the protocatechuate branches, each using a set of specific genes. However, annotation for most of these genes is lacking or attributed to poorly- or un-characterised families. Aspergillus nidulans can utilise as sole carbon/energy source either benzoate or salicylate (upstream aromatic metabolites of the protocatechuate and the catechol branches, respectively). Using this cultivation strategy and combined analyses of comparative proteomics, gene mining, gene expression and characterisation of particular gene-replacement mutants, we precisely assigned most of the steps of the 3-oxoadipate pathway to specific genes in this fungus. Our findings disclose the genetically encoded potential of saprophytic Ascomycota fungi to utilise this pathway and provide means to untie associated regulatory networks, which are vital to heightening their ecological significance.

  7. The nature of chemical innovation: new enzymes by evolution.

    Science.gov (United States)

    Arnold, Frances H

    2015-11-01

    I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N-H insertion, C-H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists' favorite transformations with the exquisite selectivity and tunability of enzymes.

  8. Rapid adaptation to climate change.

    Science.gov (United States)

    Hancock, Angela M

    2016-08-01

    In recent years, amid growing concerns that changing climate is affecting species distributions and ecosystems, predicting responses to rapid environmental change has become a major goal. In this issue, Franks and colleagues take a first step towards this objective (Franks et al. 2016). They examine genomewide signatures of selection in populations of Brassica rapa after a severe multiyear drought. Together with other authors, Franks had previously shown that flowering time was reduced after this particular drought and that the reduction was genetically encoded. Now, the authors have sequenced previously stored samples to compare allele frequencies before and after the drought and identify the loci with the most extreme shifts in frequencies. The loci they identify largely differ between populations, suggesting that different genetic variants may be responsible for reduction in flowering time in the two populations. PMID:27463237

  9. Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast.

    Science.gov (United States)

    Jagadish, Krishnappa; Gould, Andrew; Borra, Radhika; Majumder, Subhabrata; Mushtaq, Zahid; Shekhtman, Alexander; Camarero, Julio A

    2015-07-13

    We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

  10. From jellyfish to biosensors: the use of fluorescent proteins in plants.

    Science.gov (United States)

    Voss, Ute; Larrieu, Antoine; Wells, Darren M

    2013-01-01

    The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors. PMID:24166435

  11. Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1 and electroporation: methods and optogenetic applications

    Directory of Open Access Journals (Sweden)

    Ming eZou

    2014-05-01

    Full Text Available The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1 or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications.

  12. Epigenetic mechanisms in diurnal cycles of metabolism and neurodevelopment.

    Science.gov (United States)

    Powell, Weston T; LaSalle, Janine M

    2015-10-15

    The circadian cycle is a genetically encoded clock that drives cellular rhythms of transcription, translation and metabolism. The circadian clock interacts with the diurnal environment that also drives transcription and metabolism during light/dark, sleep/wake, hot/cold and feast/fast daily and seasonal cycles. Epigenetic regulation provides a mechanism for cells to integrate genetic programs with environmental signals in order produce an adaptive and consistent output. Recent studies have revealed that DNA methylation is one epigenetic mechanism that entrains the circadian clock to a diurnal environment. We also review recent circadian findings in the epigenetic neurodevelopmental disorders Prader-Willi, Angelman and Rett syndromes and hypothesize a link between optimal brain development and intact synchrony between circadian and diurnal rhythms. PMID:26105183

  13. Biophysical Properties of Optogenetic Tools and Their Application for Vision Restoration Approaches

    Science.gov (United States)

    Klapper, Simon D.; Swiersy, Anka; Bamberg, Ernst; Busskamp, Volker

    2016-01-01

    Optogenetics is the use of genetically encoded light-activated proteins to manipulate cells in a minimally invasive way using light. The most prominent example is channelrhodopsin-2 (ChR2), which allows the activation of electrically excitable cells via light-dependent depolarization. The combination of ChR2 with hyperpolarizing-light-driven ion pumps such as the Cl− pump halorhodopsin (NpHR) enables multimodal remote control of neuronal cells in culture, tissue, and living animals. Very soon, it became obvious that this method offers a chance of gene therapy for many diseases affecting vision. Here, we will give a brief introduction to retinal function and retinal diseases; optogenetic vision restoration strategies will be highlighted. We will discuss the functional and structural properties of rhodopsin-based optogenetic tools and analyze the potential for the application of vision restoration. PMID:27642278

  14. Large-Scale Fluorescence Calcium-Imaging Methods for Studies of Long-Term Memory in Behaving Mammals.

    Science.gov (United States)

    Jercog, Pablo; Rogerson, Thomas; Schnitzer, Mark J

    2016-01-01

    During long-term memory formation, cellular and molecular processes reshape how individual neurons respond to specific patterns of synaptic input. It remains poorly understood how such changes impact information processing across networks of mammalian neurons. To observe how networks encode, store, and retrieve information, neuroscientists must track the dynamics of large ensembles of individual cells in behaving animals, over timescales commensurate with long-term memory. Fluorescence Ca(2+)-imaging techniques can monitor hundreds of neurons in behaving mice, opening exciting avenues for studies of learning and memory at the network level. Genetically encoded Ca(2+) indicators allow neurons to be targeted by genetic type or connectivity. Chronic animal preparations permit repeated imaging of neural Ca(2+) dynamics over multiple weeks. Together, these capabilities should enable unprecedented analyses of how ensemble neural codes evolve throughout memory processing and provide new insights into how memories are organized in the brain. PMID:27048190

  15. Species specificity in major urinary proteins by parallel evolution.

    Directory of Open Access Journals (Sweden)

    Darren W Logan

    Full Text Available Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1 diversity, to enable the signaling of multiple behaviors, 2 dynamic regulation, to indicate age and dominance, and 3 species-specificity. Recently, the major urinary proteins (Mups have been shown to function themselves as genetically-encoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues.Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species

  16. Reciprocal cholinergic and GABAergic modulation of the small ventrolateral pacemaker neurons of Drosophila's circadian clock neuron network.

    Science.gov (United States)

    Lelito, Katherine R; Shafer, Orie T

    2012-04-01

    The relatively simple clock neuron network of Drosophila is a valuable model system for the neuronal basis of circadian timekeeping. Unfortunately, many key neuronal classes of this network are inaccessible to electrophysiological analysis. We have therefore adopted the use of genetically encoded sensors to address the physiology of the fly's circadian clock network. Using genetically encoded Ca(2+) and cAMP sensors, we have investigated the physiological responses of two specific classes of clock neuron, the large and small ventrolateral neurons (l- and s-LN(v)s), to two neurotransmitters implicated in their modulation: acetylcholine (ACh) and γ-aminobutyric acid (GABA). Live imaging of l-LN(v) cAMP and Ca(2+) dynamics in response to cholinergic agonist and GABA application were well aligned with published electrophysiological data, indicating that our sensors were capable of faithfully reporting acute physiological responses to these transmitters within single adult clock neuron soma. We extended these live imaging methods to s-LN(v)s, critical neuronal pacemakers whose physiological properties in the adult brain are largely unknown. Our s-LN(v) experiments revealed the predicted excitatory responses to bath-applied cholinergic agonists and the predicted inhibitory effects of GABA and established that the antagonism of ACh and GABA extends to their effects on cAMP signaling. These data support recently published but physiologically untested models of s-LN(v) modulation and lead to the prediction that cholinergic and GABAergic inputs to s-LN(v)s will have opposing effects on the phase and/or period of the molecular clock within these critical pacemaker neurons.

  17. Design of lanthanide fingers: compact lanthanide-binding metalloproteins.

    Science.gov (United States)

    am Ende, Christopher W; Meng, Hai Yun; Ye, Mao; Pandey, Anil K; Zondlo, Neal J

    2010-08-16

    Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide-binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a beta beta alpha structural motif. Lanthanide fingers utilize an Asp(2)Glu(2) metal-coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide-binding peptide incorporated the following key elements: 1) residues with high alpha-helix and beta-sheet propensities in the respective secondary structures; 2) an optimized big box alpha-helix N-cap; 3) a Schellman alpha-helix C-cap motif; and 4) an optional D-Pro-Ser type II' beta-turn in the beta-hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25-residue peptide that was a general lanthanide-binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 microM for binding Er(3+). CD spectra of the peptide-lanthanide complexes are similar to those of zinc fingers and other beta beta alpha proteins. Metal binding involves residues from the N-terminal beta-hairpin and the C terminal alpha-helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D-Pro-Ser type II' beta-turn motif could be replaced by Thr-Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF-hand peptide.

  18. Vipericidins: a novel family of cathelicidin-related peptides from the venom gland of South American pit vipers.

    Science.gov (United States)

    Falcao, C B; de La Torre, B G; Pérez-Peinado, C; Barron, A E; Andreu, D; Rádis-Baptista, G

    2014-11-01

    Cathelicidins are phylogenetically ancient, pleiotropic host defense peptides-also called antimicrobial peptides (AMPs)-expressed in numerous life forms for innate immunity. Since even the jawless hagfish expresses cathelicidins, these genetically encoded host defense peptides are at least 400 million years old. More recently, cathelicidins with varying antipathogenic activities and cytotoxicities were discovered in the venoms of poisonous snakes; for these creatures, cathelicidins may also serve as weapons against prey and predators, as well as for innate immunity. We report herein the expression of orthologous cathelicidin genes in the venoms of four different South American pit vipers (Bothrops atrox, Bothrops lutzi, Crotalus durissus terrificus, and Lachesis muta rhombeata)-distant relatives of Asian cobras and kraits, previously shown to express cathelicidins-and an elapid, Pseudonaja textilis. We identified six novel, genetically encoded peptides: four from pit vipers, collectively named vipericidins, and two from the elapid. These new venom-derived cathelicidins exhibited potent killing activity against a number of bacterial strains (S. pyogenes, A. baumannii, E. faecalis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa), mostly with relatively less potent hemolysis, indicating their possible usefulness as lead structures for the development of new anti-infective agents. It is worth noting that these South American snake venom peptides are comparable in cytotoxicity (e.g., hemolysis) to human cathelicidin LL-37, and much lower than other membrane-active peptides such as mastoparan 7 and melittin from bee venom. Overall, the excellent bactericidal profile of vipericidins suggests they are a promising template for the development of broad-spectrum peptide antibiotics. PMID:25100358

  19. Vipericidins: a novel family of cathelicidin-related peptides from the venom gland of South American pit vipers.

    Science.gov (United States)

    Falcao, C B; de La Torre, B G; Pérez-Peinado, C; Barron, A E; Andreu, D; Rádis-Baptista, G

    2014-11-01

    Cathelicidins are phylogenetically ancient, pleiotropic host defense peptides-also called antimicrobial peptides (AMPs)-expressed in numerous life forms for innate immunity. Since even the jawless hagfish expresses cathelicidins, these genetically encoded host defense peptides are at least 400 million years old. More recently, cathelicidins with varying antipathogenic activities and cytotoxicities were discovered in the venoms of poisonous snakes; for these creatures, cathelicidins may also serve as weapons against prey and predators, as well as for innate immunity. We report herein the expression of orthologous cathelicidin genes in the venoms of four different South American pit vipers (Bothrops atrox, Bothrops lutzi, Crotalus durissus terrificus, and Lachesis muta rhombeata)-distant relatives of Asian cobras and kraits, previously shown to express cathelicidins-and an elapid, Pseudonaja textilis. We identified six novel, genetically encoded peptides: four from pit vipers, collectively named vipericidins, and two from the elapid. These new venom-derived cathelicidins exhibited potent killing activity against a number of bacterial strains (S. pyogenes, A. baumannii, E. faecalis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa), mostly with relatively less potent hemolysis, indicating their possible usefulness as lead structures for the development of new anti-infective agents. It is worth noting that these South American snake venom peptides are comparable in cytotoxicity (e.g., hemolysis) to human cathelicidin LL-37, and much lower than other membrane-active peptides such as mastoparan 7 and melittin from bee venom. Overall, the excellent bactericidal profile of vipericidins suggests they are a promising template for the development of broad-spectrum peptide antibiotics.

  20. Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Kiilgaard, Jens Folke; la Cour, Morten;

    2003-01-01

    The retinal pigment epithelium (RPE) of the eye transports water and lactate ions in the direction from retina to choroid. The water transport is important in maintenance of retinal adhesion and the transport of lactate ions serves to regulate the lactate levels and pH of the subretinal space....... This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H(+), lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence...... and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically...

  1. Synthesis, characterization and ion recognition studies of lower rim 1,3-di{rhodamine} conjugate of calix[4]arene

    Indian Academy of Sciences (India)

    Jugun Prakash Chinta; Jayaraman Dessingou; Chebrolu Pulla Rao

    2013-11-01

    An amido-linked rhodamine conjugate of calix[4]arene, L has been synthesized and characterized. Metal ion recognition properties of L have been studied by emission and absorption techniques with 14 different metal ions including the transition ones. Results show that, L exhibits ratiometric emission intensity towards Hg2+, Fe2+, Fe3+, Cu2+, Pb2+ and Zn2+. Composition of the complex formed in the solution has been found to be 1:2 (L:M+), based on the Job’s plot. The L can also act as a chemosensor for Hg2+ through naked eye detection. Fluorescence quenching observed at 485 nm follows an order, Hg2+>>Fe3+∼Cu2+>Zn2+>Pb2+>Ca2+, while the enhancement observed at 580 nm follows, Hg2+>>Fe2+∼Pb2+>Zn2+. Mode of interaction of M+ with L is by the ring opening of spirolactam moiety.

  2. QD-Based FRET Probes at a Glance

    Directory of Open Access Journals (Sweden)

    Armen Shamirian

    2015-06-01

    Full Text Available The unique optoelectronic properties of quantum dots (QDs give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided.

  3. The Role of L- and T-Type Calcium Channels in Local and Remote Calcium Responses in Rat Mesenteric Terminal Arterioles

    DEFF Research Database (Denmark)

    Braunstein, Thomas Hartig; Inoue, Ryuji; Cribbs, Leanne;

    2009-01-01

    Background/Aims: The roles of intercellular communication and T-type versus L-type voltage-dependent Ca(2+) channels (VDCCs) in conducted vasoconstriction to local KCl-induced depolarization were investigated in mesenteric arterioles. Methods: Ratiometric Ca(2+) imaging (R) using Fura-PE3 with...... local (DeltaR = 0.54) and remote (DeltaR = 0.17 at 500 mum) increases in intracellular Ca(2+). Remote Ca(2+) responses were inhibited by the gap junction uncouplers carbenoxolone and palmitoleic acid. Ca(V)1.2, Ca(V)3.1 and Ca(V)3.2 channels were immunolocalized in vascular smooth muscle cells and Ca...... arterioles (at 200-300 mum) using micro-application of VDCC blockers. Conclusion: Both L- and T-type channels mediate Ca(2+) entry during conducted vasoconstriction to local KCl in mesenteric arterioles. However, these channels do not participate in the conduction process per se....

  4. Glutamine-containing “turn-on” fluorescence sensor for the highly sensitive and selective detection of chromium (III) ion in water

    Science.gov (United States)

    Zhao, Meili; Ma, Liguo; Zhang, Min; Cao, Weiguang; Yang, Liting; Ma, Li-Jun

    2013-12-01

    In this study, we reported a new fluorescence sensor for chromium (III) ion, dansyl-L-glutamine (1). The sensor displayed a unique selective fluorescence “turn-on” response to Cr3+ over other common metal ions in water. Notably, 1 still showed a ratiometric response to Cr3+ in UV-vis absorption spectra. The binding mechanism of 1 to Cr3+ was further clarified by using NMR and ESI-MS spectra. The experiment results indicated that the dual-responses of 1 to Cr3+ should attribute to the coordination of deprotonated sulfonamide group with Cr3+ and the protonation of the dimethylamino group due to the coordination of Cr3+ for 1. In addition, two chloride ions also coordinated to the complex of sensor-chromium (III) ion, which further strengthened the conformation of 1-Cr3+.

  5. Assembly and Calcium Binding Properties of Quantum Dot-Calmodulin Calcium Sensor.

    Science.gov (United States)

    Eun, Su-yong; Nguyen-ta, Kim; Yoo, Hoon; Silva, Gabriel A; Kim, Soon-jong

    2016-02-01

    We have developed the first nanoengineered quantum dot molecular complex designed to measure changes of calcium ion (Ca2+) concentration at high spatial and temporal resolutions in real time. The sensor is ratiometric and composed of three components: a quantum dot (QD) emitting at 620 nm as a fluorescence donor, an organic dye (Alexa Fluor 647) as a fluorescence acceptor, and a calmodulin-M13 (CaM-M13) protein part as a calcium sensing component. In this work, we have determined the maximal number of CaM-M13 required for saturating a single QD particle to be approximately 16. The dissociation constant, Kd of the QD-based calcium ion sensor was also estimated to be around 30 microM. PMID:27433729

  6. Laser Induced Breakdown Spectroscopy for Classification of High Energy Materials using Elemental Intensity Ratios

    Directory of Open Access Journals (Sweden)

    S. Sreedhar

    2014-07-01

    Full Text Available A simple, yet efficient, methodology is proposed to classify three high energy materials (HEMs with diverse composition using nanosecond laser induced breakdown spectroscopic data. We have calculated O/N, N/H, and O/H elemental peaks ratios using a ratiometric method. The present work describes a novel way to construct 1D, 2D, and 3D classification model using the above mentioned ratios. Multivariate statistical methods are followed for construction of the classification models. A detailed procedure for classification of three different HEMs is presented here.Defence Science Journal, Vol. 64, No. 4, July 2014, pp.332-338, DOI:http://dx.doi.org/10.14429/dsj.64.4741

  7. Synthesis of Cross-Linked Polymeric Micelle pH Nanosensors

    DEFF Research Database (Denmark)

    Ek, Pramod Kumar; Jølck, Rasmus Irming; Andresen, Thomas Lars

    2015-01-01

    at the micelle shell using CuAAC results in a stabilized micelle pH nanosensor. Compared to the postmicelle modification strategy, the mixed-micellization approach increases the control of the overall composition of the nanosensors.Both approaches provide stable nanosensors with similar pKa profiles and thereby......The design flexibility that polymeric micelles offer in the fabrication of optical nanosensors for ratiometric pH measurements is investigated. pH nanosensors based on polymeric micelles are synthesized either by a mixed-micellization approach or by a postmicelle modification strategy. In the mixed......-micellization approach, self-assembly of functionalized unimers followed by shell cross-linking by copper-catalyzed azide-alkyne cycloaddition (CuAAC) results in stabilized cRGD-functionalized micelle pH nanosensors. In the postmicelle modification strategy, simultaneous cross-linking and fluorophore conjugation...

  8. A hydrogel based nanosensor with an unprecedented broad sensitivity range for pH measurements in cellular compartments

    DEFF Research Database (Denmark)

    Zhang, M.; Søndergaard, Rikke Vicki; Ek, Pramod Kumar;

    2015-01-01

    H-sensitive fluorophores (difluoro-Oregon Green, Oregon Green 488, and fluorescein) and one pH-insensitive fluorophore (Alexa 568) were covalently incorporated into a nanoparticle hydrogel matrix. With this broad range quadruple-labelled nanosensor all physiological relevant pH levels in living cells can be measured......Optical pH nanosensors have been applied for monitoring intracellular pH in real-time for about two decades. However, the pH sensitivity range of most nanosensors is too narrow, and measurements that are on the borderline of this range may not be correct. Furthermore, ratiometric measurements...... of acidic intracellular pH (pH pH 1.4 to 7.0. In this nanosensor, three p...

  9. Absolute photoacoustic thermometry in deep tissue.

    Science.gov (United States)

    Yao, Junjie; Ke, Haixin; Tai, Stephen; Zhou, Yong; Wang, Lihong V

    2013-12-15

    Photoacoustic thermography is a promising tool for temperature measurement in deep tissue. Here we propose an absolute temperature measurement method based on the dual temperature dependences of the Grüneisen parameter and the speed of sound in tissue. By taking ratiometric measurements at two adjacent temperatures, we can eliminate the factors that are temperature irrelevant but difficult to correct for in deep tissue. To validate our method, absolute temperatures of blood-filled tubes embedded ~9 mm deep in chicken tissue were measured in a biologically relevant range from 28°C to 46°C. The temperature measurement accuracy was ~0.6°C. The results suggest that our method can be potentially used for absolute temperature monitoring in deep tissue during thermotherapy.

  10. Monitoring of extracellular pH in young dental biofilms grown in vivo in the presence and absence of sucrose

    DEFF Research Database (Denmark)

    Dige, Irene; Baelum, Vibeke; Nyvad, Bente;

    2016-01-01

    BACKGROUND AND OBJECTIVE: pH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply. DESIGN......: Dental biofilms (48 h) from 10 individuals were collected on glass slabs mounted on intra-oral appliances. During growth, appliances were immersed extra-orally in either physiological saline or 4% sucrose for 2 min, eight times per day. Fluorescence emissions of C-SNARF-4 in deep layers of the biofilms...... kind to apply the combination of pH ratiometry and digital image analysis to systematically record extracellular pH in intact dental biofilms from several individuals for up to 1 h. We observed highly heterogeneous pH landscapes and the presence of acidogenic microenvironments - 'acidogenic hotspots...

  11. Monitoring of extracellular pH in young dental biofilms grown in situ in the presence and absence of sucrose

    DEFF Research Database (Denmark)

    Dige, Irene; Bælum, Vibeke; Schlafer, Sebastian;

    pH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply. 48-h dental biofilms from 10......H in intact dental biofilms from several individuals for up to 1 h. We observed heterogeneous pH landscapes and the presence of highly acidogenic microenvironments within the biofilms. The data suggest that pH drops in young dental biofilm are independent of the sucrose supply during growth....... individuals were collected on glass slabs mounted on intra-oral appliances. During growth, appliances were immersed extra-orally in either physiological saline or 4% sucrose for 2 min eight times per day. For each individual, eight biofilm specimens were analyzed for each experiment, yielding a total of 160...

  12. Fluorescent europium-modified polymer nanoparticles for rapid and sensitive anthrax sensors.

    Science.gov (United States)

    Oh, Wan-Kyu; Jeong, Yoon Seon; Song, Jooyoung; Jang, Jyongsik

    2011-11-15

    Novel fluorescent polyacrylonitrile nanoparticles were synthesized by microemulsion polymerization and Schiff base modification. By further modification with europium, the polyacrylonitrile nanoparticles could be used as a highly sensitive and rapid sensor for Bacillus anthracis spore detection in aqueous solution. The europium-modified polyacrylonitrile nanoparticles were readily combined with dipicolinic acid as a unique biomarker of B. anthracis, leading to high fluorescence emission. These nanoparticles enabled ratiometric detection without instrument-specific calibration due to the internal fluorescence reference. Additionally, the europium-modified polyacrylonitrile nanoparticle sensors exhibited a remarkable limit of detection (10pM) for dipicolinic acid and outstanding selectivity (160×) over aromatic ligands in aqueous solution. The ultrafine nanoparticle sensor showed a high capability for detecting anthrax due to the increased surface area-to-volume ratio and enhanced dispersibility. PMID:21893406

  13. Design and Implementation Model for Linearization Sensor Characteristic by FPAA

    Directory of Open Access Journals (Sweden)

    Alaa Abdul Hussein Salman

    2015-11-01

    Full Text Available Linearization sensors characteristics becomes very interest field for researchers due to the importance in enhance the system performance, measurement accuracy, system design simplicity (hardware and software, reduce system cost, ..etc. in this paper, two approaches has been introduced in order to linearize the sensor characteristics; first is signal condition circuit based on lock up table (LUT which this method performed for linearize NTC sensor characteristic. Second is ratiometric measurement equation which this method performed for linearize LVDT sensor characteristic. The proposed methods has been simulated by MATLAB, and then implemented by using Anadigm AN221E04 Field Programmable Analog Array (FPAA development kit which several experiments performed in order to improve the performance of these approaches.

  14. Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Kiilgaard, Jens Folke; la Cour, Morten;

    2003-01-01

    . This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H(+), lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence...... and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically...... using the fluorescent dye BCECF. In lactate-free solutions, mannitol addition to the retinal bath caused intracellular acidification and cell shrinkage, given by a single osmotic water permeability of 1.2+/-0.1 x 10(-4)cmsec(-1) (osmoll(-1))(-1). In solutions containing 50 mmoll(-1) lactate, however...

  15. pH-Insensitive FRET voltage dyes.

    Science.gov (United States)

    Maher, Michael P; Wu, Nyan-Tsz; Ao, Hong

    2007-08-01

    Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Förster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell. PMID:17517905

  16. Helmet Versus Active Repositioning for Plagiocephaly: A Three-Dimensional Analysis

    DEFF Research Database (Denmark)

    Lipira, A.B.; Gordon, S.; Darvann, Tron Andre;

    2010-01-01

    BACKGROUND AND PURPOSE: Orthotic helmets and active repositioning are the most common treatments for deformational plagiocephaly (DP). Existing evidence is not sufficient to objectively inform decisions between these options. A three-dimensional (3D), whole-head asymmetry analysis was used...... for severity of initial deformity. Surfaces were spatially registered to a symmetric template, which was deformed to achieve detailed right-to-left point correspondence for every point on the head surface. A ratio-metric asymmetry value was calculated for each point relative to its contralateral counterpart....... Maximum and mean asymmetry values were determined. Change in mean and maximum asymmetry with treatment was the basis for group comparison. RESULTS: The helmeted group had a larger reduction than the repositioned group in both maximum (4.0% vs 2.5%; P = .02) and mean asymmetry (0.9% vs 0.5%; P = .02...

  17. Synthesis, selective pH-sensing activity and logic behavior of highly water-soluble 1,8-naphthalimide and dihydroimidazonaphthalimide derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Georgiev, Nikolai I.; Dimov, Stefan M. [Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Street, 1756 Sofia (Bulgaria); Asiri, Abdullah M. [Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Alamry, Khalid A.; Obaid, Abdullah Y. [Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Bojinov, Vladimir B., E-mail: vlbojin@uctm.edu [Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Street, 1756 Sofia (Bulgaria); Chemistry Department, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia)

    2014-05-01

    This paper reports on the design, synthesis and fluorescence pH-sensing activity of a novel highly water-soluble 1,8-naphthalimide and its 9,10-dihydro-7H-imidazo[1,2-b]benz[d,e]isoqionolin-7-one derivative. The changes in the photophysical properties of the compounds as a function of pH were investigated in 100% aqueous medium. The 1,8-naphthalimide dye manifests “off–on” pH sensing properties based on photoinduced electron transfer, while its condensed heterocyclic derivative revealed ratiometric “off–on–off” fluorescence pH probe activity. Due to the two different “off”-states the dihydroimidazonaphthalimide derivative is able to execute the logical functions INH and XNOR and as such, to act as a magnitude digital comparator. The synthesized compounds show excellent selectivity toward protons over the representative transition metal ions (Co{sup 2+}, Cu{sup 2+}, Fe{sup 3+}, Ni{sup 2+}, Cd{sup 2+}, Pb{sup 2+}, Zn{sup 2+}, Hg{sup 2+} and Ag{sup +}) is commonly used buffer solutions. The high water solubility and excellent pH selectivity of both probes as well as the ratiometric pH sensitivity of dihydroimidazonaphthalimide derivative may be beneficially for monitoring pH variations in complex samples. - Highlights: • Two novel highly water-soluble fluorescent dihydroimidazonaphthalimide and 1,8-naphthalimide derivatives are synthesized. • Compounds are designed as fluorescent “off–on” and “off–on–off” molecular pH probes based on PET and ICT. • Probes manifest selective response to protons over representative transition metal ions in 100% aqueous medium. • Logic functions INH and XNOR are achieved for dihydroimidazonaphthalimide derivative. • A combinatorial logic circuit (magnitude digital comparator) is demonstrated.

  18. Multimodality pH imaging in a mouse dorsal skin fold window chamber model

    Science.gov (United States)

    Leung, Hui Min; Schafer, Rachel; Pagel, Mark M.; Robey, Ian F.; Gmitro, Arthur F.

    2013-03-01

    Upregulate levels of expression and activity of membrane H+ ion pumps in cancer cells drives the extracellular pH (pHe,) to values lower than normal. Furthermore, disregulated pH is indicative of the changes in glycolytic metabolism in tumor cells and has been shown to facilitate extracellular tissue remodeling during metastasis Therefore, measurement of pHe could be a useful cancer biomarker for diagnostic and therapy monitoring evaluation. Multimodality in-vivo imaging of pHe in tumorous tissue in a mouse dorsal skin fold window chamber (DSFWC) model is described. A custom-made plastic window chamber structure was developed that is compatible with both imaging optical and MR imaging modalities and provides a model system for continuous study of the same tissue microenvironment on multiple imaging platforms over a 3-week period. For optical imaging of pHe, SNARF-1 carboxylic acid is injected intravenously into a SCID mouse with an implanted tumor. A ratiometric measurement of the fluorescence signal captured on a confocal microscope reveals the pHe of the tissue visible within the window chamber. This imaging method was used in a preliminary study to evaluate sodium bicarbonate as a potential drug treatment to reverse tissue acidosis. For MR imaging of pHe the chemical exchange saturation transfer (CEST) was used as an alternative way of measuring pHe in a DSFWC model. ULTRAVIST®, a FDA approved x-ray/CT contrast agent has been shown to have a CEST effect that is pH dependent. A ratiometric analysis of water saturation at 5.6 and 4.2 ppm chemical shift provides a means to estimate the local pHe.

  19. The saturation of the fluorescence and its consequences for laser-induced fluorescence thermometry in liquid flows

    Science.gov (United States)

    Chaze, William; Caballina, Ophélie; Castanet, Guillaume; Lemoine, Fabrice

    2016-04-01

    The temperature dependence of the fluorescence emission of certain organic dyes such as rhodamine B has been widely utilized for measuring the temperature in liquid flows. Measurements are generally based on two assumptions: The fluorescence signal is proportional to the intensity of the laser excitation, and the temperature sensitivity of the dye is not affected by the laser irradiance. In the ratiometric methods, these assumptions allow justifying that the influence of the laser intensity can be totally eliminated in the intensity ratio of two spectral bands of the fluorescence emission and thus that measurements can be taken with no biases under experimental conditions, where the laser propagation is disturbed by the flow. However, when pulsed lasers are used (mainly in planar LIF measurements), the peak irradiance usually compares or exceeds the saturation intensity of the dyes. The present study assesses the consequences of a saturation of the dye emission on temperature measurements. Tests among fluoresceins and rhodamines reveal that the saturation can be accompanied by a significant loss of temperature sensitivity. The dyes, for which this loss of sensitivity is observed, mainly owe their temperature dependence to the fluorescence quantum yield and have a fluorescence signal decreasing with the temperature. The couple fluorescein/sulforhodamine 640 is finally proposed for an implementation of the ratiometric method, since its relatively high temperature dependence (+3 %/° {C}) is not altered at high laser irradiances. The possibility of measuring instantaneous temperature fields with this pair of dyes using a single laser shot is finally demonstrated on a turbulent heated jet injected into quiescent water.

  20. Simultaneous spectroscopic measurements of the interior temperature and induced cargo release from pore-restricted mesoporous silica nanoparticles

    Science.gov (United States)

    Dong, Juyao; Zink, Jeffrey I.

    2016-05-01

    Temperature changes initiated within nano structures are being increasingly used to externally activate responsive delivery vehicles. Yet, the precise measurement of the nano environment temperature increase and its correlation with the induced macroscopic cargo release are difficult to achieve. In this study, we focus on a photothermally activated drug delivery system based on mesoporous silica nanoparticles, and use an optical nanothermometer - NaYF4:Yb3+,Er3+ crystals - for a ratiometric temperature measurement. Using fluorescent dyes as the payload molecule, both the nanoparticle interior temperature change and the macroscopic cargo release amount are monitored simultaneously by fluorescent spectroscopy. We found that the cargo release lags the temperature increase by about 5 min, revealing the threshold temperature that the particles have to reach before a substantial release could happen. Using this spectroscopic method, we are able to directly compare and correlate a nano environment event with its stimulated macroscopic results.Temperature changes initiated within nano structures are being increasingly used to externally activate responsive delivery vehicles. Yet, the precise measurement of the nano environment temperature increase and its correlation with the induced macroscopic cargo release are difficult to achieve. In this study, we focus on a photothermally activated drug delivery system based on mesoporous silica nanoparticles, and use an optical nanothermometer - NaYF4:Yb3+,Er3+ crystals - for a ratiometric temperature measurement. Using fluorescent dyes as the payload molecule, both the nanoparticle interior temperature change and the macroscopic cargo release amount are monitored simultaneously by fluorescent spectroscopy. We found that the cargo release lags the temperature increase by about 5 min, revealing the threshold temperature that the particles have to reach before a substantial release could happen. Using this spectroscopic method, we are

  1. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    Science.gov (United States)

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

  2. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    Science.gov (United States)

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.

  3. Bio-inspired synthesis of hybrid silica nanoparticles templated from elastin-like polypeptide micelles

    Science.gov (United States)

    Han, Wei; MacEwan, Sarah R.; Chilkoti, Ashutosh; López, Gabriel P.

    2015-07-01

    The programmed self-assembly of block copolymers into higher order nanoscale structures offers many attractive attributes for the development of new nanomaterials for numerous applications including drug delivery and biosensing. The incorporation of biomimetic silaffin peptides in these block copolymers enables the formation of hybrid organic-inorganic materials, which can potentially enhance the utility and stability of self-assembled nanostructures. We demonstrate the design, synthesis and characterization of amphiphilic elastin-like polypeptide (ELP) diblock copolymers that undergo temperature-triggered self-assembly into well-defined spherical micelles. Genetically encoded incorporation of the silaffin R5 peptide at the hydrophilic terminus of the diblock ELP leads to presentation of the silaffin R5 peptide on the coronae of the micelles, which results in localized condensation of silica and the formation of near-monodisperse, discrete, sub-100 nm diameter hybrid ELP-silica particles. This synthesis method, can be carried out under mild reaction conditions suitable for bioactive materials, and will serve as the basis for the development and application of functional nanomaterials. Beyond silicification, the general strategies described herein may also be adapted for the synthesis of other biohybrid nanomaterials as well.The programmed self-assembly of block copolymers into higher order nanoscale structures offers many attractive attributes for the development of new nanomaterials for numerous applications including drug delivery and biosensing. The incorporation of biomimetic silaffin peptides in these block copolymers enables the formation of hybrid organic-inorganic materials, which can potentially enhance the utility and stability of self-assembled nanostructures. We demonstrate the design, synthesis and characterization of amphiphilic elastin-like polypeptide (ELP) diblock copolymers that undergo temperature-triggered self-assembly into well

  4. Leveraging material properties in fluorescence anion sensor arrays: a general approach.

    Science.gov (United States)

    Anzenbacher, Pavel; Liu, Yuanli; Palacios, Manuel A; Minami, Tsuyoshi; Wang, Zhuo; Nishiyabu, Ryuhei

    2013-06-24

    As the demand for probes suitable for sensor development increases, investigation of approaches that utilize known successful receptors gains in general importance. This study describes a two-prong approach that can be used as a guide to developing sensors from known receptors. First, the conversion of a simple receptor, calix[4]pyrrole, into a fluorescent probe to establish a ratiometric signal is described. Secondly, the sensors that employ an output from a single ratiometric calix[4]pyrrole probe are fabricated by using poly(ether-urethane) hydrogel copolymers. These hydrogels are designed to absorb, internalize and transport aqueous electrolytes. A sensor array of ten different poly(ether-urethane) matrices with varying comonomer proportions were doped with a single probe and were exposed to eight different anions: acetate, benzoate, fluoride, chloride, phosphate, pyrophosphate, hydrogen sulfide, and cyanide, eight urine samples and anti-inflammatory drugs (NSAIDs). The poly(ether-urethane) matrices comprise different proportions of anion-binding urethane moieties and different hydrophilicity given by the ratio between ethylene glycol ether and butylene glycol ether. This diversity in the hydration behavior provides different environment polarity, in which the recognition and self-assembly processes display enough diverse behavior to allow for unique response of the probe to the analytes. Furthermore, a single probe is shown to recognize eight different aqueous anions and eight urine samples when embedded in ten different polyurethanes in an array that displays 100 % classification accuracy. To demonstrate the potential of the concept for quantitative studies, an estimation of non-steroidal anti-inflammatory drugs ibuprofen and diclofenac in water and in saliva was performed. A limit of detection of 0.1 ppm and a dynamic range of 0.1-0.6 and 0.05-60 ppm was observed, respectively. Given the general difficulty of chemosensors to recognize aqueous anions

  5. Thiazole derivative-modified upconversion nanoparticles for Hg2+ detection in living cells

    Science.gov (United States)

    Gu, Bin; Zhou, Yi; Zhang, Xiao; Liu, Xiaowang; Zhang, Yuhai; Marks, Robert; Zhang, Hua; Liu, Xiaogang; Zhang, Qichun

    2015-12-01

    Mercury ion (Hg2+) is an extremely toxic ion, which will accumulate in human bodies and cause severe nervous system damage. Therefore, the sensitive and efficient monitoring of Hg2+ in human bodies is of great importance. Upconversion nanoparticle (UCNPs) based nano probes exhibit no autofluorescence, deep penetration depth and chemical stability in biological samples, as well as a large anti-stokes shift. In this study, we have developed thiazole-derivative-functionalized UCNPs, and employed an upconversion emission intensity ratio of 540 nm to 803 nm (I540/I803) as a ratiometric signal to detect Hg2+ in living cells showing excellent photo stability and high selectivity. Our nano probe was characterized using transmission electron microscopy (TEM) and powder X-ray diffraction (PXRD). The low cytotoxicity of our probe was confirmed by an MTT assay and the UCL test in HeLa cells was carried out by confocal microscopy. Our results demonstrated that organic-dye-functionalized UCNPs should be a good strategy for detecting toxic metal ions when studying cellular biosystems.Mercury ion (Hg2+) is an extremely toxic ion, which will accumulate in human bodies and cause severe nervous system damage. Therefore, the sensitive and efficient monitoring of Hg2+ in human bodies is of great importance. Upconversion nanoparticle (UCNPs) based nano probes exhibit no autofluorescence, deep penetration depth and chemical stability in biological samples, as well as a large anti-stokes shift. In this study, we have developed thiazole-derivative-functionalized UCNPs, and employed an upconversion emission intensity ratio of 540 nm to 803 nm (I540/I803) as a ratiometric signal to detect Hg2+ in living cells showing excellent photo stability and high selectivity. Our nano probe was characterized using transmission electron microscopy (TEM) and powder X-ray diffraction (PXRD). The low cytotoxicity of our probe was confirmed by an MTT assay and the UCL test in HeLa cells was carried out by

  6. pH landscapes in a novel five-species model of early dental biofilm.

    Directory of Open Access Journals (Sweden)

    Sebastian Schlafer

    Full Text Available BACKGROUND: Despite continued preventive efforts, dental caries remains the most common disease of man. Organic acids produced by microorganisms in dental plaque play a crucial role for the development of carious lesions. During early stages of the pathogenetic process, repeated pH drops induce changes in microbial composition and favour the establishment of an increasingly acidogenic and aciduric microflora. The complex structure of dental biofilms, allowing for a multitude of different ecological environments in close proximity, remains largely unexplored. In this study, we designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We then performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate. METHODOLOGY/PRINCIPAL FINDINGS: Strains of Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Streptococcus downei and Actinomyces naeslundii were employed in the model. Biofilms were grown in flow channels that allowed for direct microscopic analysis of the biofilms in situ. The architecture and composition of the biofilms were analysed using fluorescence in situ hybridization and confocal laser scanning microscopy. Both biofilm structure and composition were highly reproducible and showed similarity to in-vivo-grown dental plaque. We employed the pH-sensitive ratiometric probe C-SNARF-4 to perform real-time microscopic analyses of the biofilm pH in response to salivary solutions containing glucose. Anaerobic glycolysis in the model biofilms created a mildly acidic environment. Decrease in pH in different areas of the biofilms varied, and distinct extracellular pH-microenvironments were conserved over several hours. CONCLUSIONS/SIGNIFICANCE: The designed biofilm model represents a promising tool to determine the effect of potential therapeutic agents on biofilm growth

  7. Characterization of excited-state reactions with instant spectra of fluorescence kinetics

    International Nuclear Information System (INIS)

    Comprehensible knowledge of the excited-state proton transfer processes in organic compounds is overwhelmingly important not only for physics, but also chemistry and Life Sciences, since they play a key role in main processes of photosynthesis and functioning of biological organisms. Moreover compounds with Excited-State Intramolecular Proton Transfer (ESIPT) are in the focus of the interest of scientists throughout the world, because dual fluorescence spectra of such objects corresponding to two forms of molecular structure (normal and photoproduct) are very sensitive to characteristics of molecular microenvironment. This property allows to use such substances as fluorescent probes for diverse applications in chemistry and Life Sciences. But at the same time studying of proton transfer processes is not simple, because this process is characterized by extremely fast times (on picoseconds time scale and less order) and very often contribution of reverse reactions is essentially complicates an interpretation of observed properties of dual fluorescence. Hence, understanding of a role of reversible reactions is crucial for a comprehensive description of all processes accompanying excited state reactions. We discuss new approach for treatment ESIPT reaction on the basis of experimentally measured instant spectra of dual fluorescence and temporal behavior of ratiometric signal of normal to tautomer form intensities. Simple analytical expressions show in transparent way how to distinguish a degree of reverse reaction contribution to ratiometric signal. A validation of the approach under consideration is fulfilled with two different flavonols – 3-hydroxyflavone and 4′-(Dimethylamino)-3-hydroxyflavone – representing two extreme cases in affecting reversible reaction on dual emission. A comparing of new approach and traditional method when we analyze kinetics of separate the N* and T* fluorescence bands decays, has been carried out. - Highlights: • The excited

  8. Local pH tracking in living cells

    Science.gov (United States)

    Tsou, Chieh-Jui; Hsia, Chih-Hao; Chu, Jia-Yin; Hung, Yann; Chen, Yi-Ping; Chien, Fan-Ching; Chou, Keng C.; Chen, Peilin; Mou, Chung-Yuan

    2015-02-01

    Continuous and simultaneous 3D single-particle movement and local pH detection in HeLa cells were demonstrated for the first time by combining fluorescent mesoporous silica nanoparticles (FMSNs) and a single-particle tracking (SPT) technique with a precision of ~10 nm. FMSNs, synthesized by the co-condensation of both pH-sensitive and reference dyes with a silica/surfactant source, allow long-term reliable ratiometric pH measurements with a precision better than 0.3 pH unit because of their excellent brightness and stability. pH variation in the surrounding area of FMSNs during endocytosis was monitored in real-time. Acidification and low mobility of FMSNs were observed at the early endocytic stage, whereas basification and high mobility of FMSNs were observed at the late stage. Our results indicate that it is possible to monitor local pH changes in the environments surrounding nanoparticles during the cellular uptake process of FMSNs, which provides much needed information for designing an efficient drug delivery nanosystem.Continuous and simultaneous 3D single-particle movement and local pH detection in HeLa cells were demonstrated for the first time by combining fluorescent mesoporous silica nanoparticles (FMSNs) and a single-particle tracking (SPT) technique with a precision of ~10 nm. FMSNs, synthesized by the co-condensation of both pH-sensitive and reference dyes with a silica/surfactant source, allow long-term reliable ratiometric pH measurements with a precision better than 0.3 pH unit because of their excellent brightness and stability. pH variation in the surrounding area of FMSNs during endocytosis was monitored in real-time. Acidification and low mobility of FMSNs were observed at the early endocytic stage, whereas basification and high mobility of FMSNs were observed at the late stage. Our results indicate that it is possible to monitor local pH changes in the environments surrounding nanoparticles during the cellular uptake process of FMSNs, which

  9. Cyclometallated ruthenium complex-modified upconversion nanophosphors for selective detection of Hg2+ ions in water

    Science.gov (United States)

    Li, Xianghong; Wu, Yongquan; Liu, Yi; Zou, Xianmei; Yao, Liming; Li, Fuyou; Feng, Wei

    2013-12-01

    Upconversion detection nanocomposites were assembled for the selective luminescent detection of mercury ions in water. A hydrophobic cyclometallated ruthenium complex [RuII(bpy)2(thpy)]PF6 (abbreviated as Ru1; bpy = 2,2'-bipyridine and thpy = 2-(2-thienyl)pyridine) is employed as a chemodosimeter to assemble on amphiphilic polymer-coating upconversion nanophosphors (UCNPs) based on the hydrophobic-hydrophobic interaction. Upon addition of Hg2+, the nanocomposite not only exhibits a remarkable color change from deep-red to yellow, but also an enhanced upconversion luminescence (UCL) emission by hindering the luminescent resonance energy transfer (LRET) process from the upconversion emission of UCNPs to Ru1. Using the ratiometric UCL emission as a detection signal, the detection limit of Hg2+ for this nanoprobe in aqueous solution is 8.2 ppb, which is much lower than that (329 ppb) determined by UV/Vis technology. Such an Hg2+-tunable LRET process provides a general strategy for fabricating a water-soluble upconversion-based nanoprobe for some special analyte.Upconversion detection nanocomposites were assembled for the selective luminescent detection of mercury ions in water. A hydrophobic cyclometallated ruthenium complex [RuII(bpy)2(thpy)]PF6 (abbreviated as Ru1; bpy = 2,2'-bipyridine and thpy = 2-(2-thienyl)pyridine) is employed as a chemodosimeter to assemble on amphiphilic polymer-coating upconversion nanophosphors (UCNPs) based on the hydrophobic-hydrophobic interaction. Upon addition of Hg2+, the nanocomposite not only exhibits a remarkable color change from deep-red to yellow, but also an enhanced upconversion luminescence (UCL) emission by hindering the luminescent resonance energy transfer (LRET) process from the upconversion emission of UCNPs to Ru1. Using the ratiometric UCL emission as a detection signal, the detection limit of Hg2+ for this nanoprobe in aqueous solution is 8.2 ppb, which is much lower than that (329 ppb) determined by UV/Vis technology

  10. Characterization of excited-state reactions with instant spectra of fluorescence kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Tomin, Vladimir I., E-mail: tomin@apsl.edu.pl; Ushakou, Dzmitryi V.

    2015-10-15

    Comprehensible knowledge of the excited-state proton transfer processes in organic compounds is overwhelmingly important not only for physics, but also chemistry and Life Sciences, since they play a key role in main processes of photosynthesis and functioning of biological organisms. Moreover compounds with Excited-State Intramolecular Proton Transfer (ESIPT) are in the focus of the interest of scientists throughout the world, because dual fluorescence spectra of such objects corresponding to two forms of molecular structure (normal and photoproduct) are very sensitive to characteristics of molecular microenvironment. This property allows to use such substances as fluorescent probes for diverse applications in chemistry and Life Sciences. But at the same time studying of proton transfer processes is not simple, because this process is characterized by extremely fast times (on picoseconds time scale and less order) and very often contribution of reverse reactions is essentially complicates an interpretation of observed properties of dual fluorescence. Hence, understanding of a role of reversible reactions is crucial for a comprehensive description of all processes accompanying excited state reactions. We discuss new approach for treatment ESIPT reaction on the basis of experimentally measured instant spectra of dual fluorescence and temporal behavior of ratiometric signal of normal to tautomer form intensities. Simple analytical expressions show in transparent way how to distinguish a degree of reverse reaction contribution to ratiometric signal. A validation of the approach under consideration is fulfilled with two different flavonols – 3-hydroxyflavone and 4′-(Dimethylamino)-3-hydroxyflavone – representing two extreme cases in affecting reversible reaction on dual emission. A comparing of new approach and traditional method when we analyze kinetics of separate the N* and T* fluorescence bands decays, has been carried out. - Highlights: • The excited

  11. Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance.

    Science.gov (United States)

    Lórenz-Fonfría, Víctor A; Bamann, Christian; Resler, Tom; Schlesinger, Ramona; Bamberg, Ernst; Heberle, Joachim

    2015-10-27

    The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(-) and 1,648(+) cm(-1). These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.

  12. Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome.

    Science.gov (United States)

    Marland, Jamie R K; Smillie, Karen J; Cousin, Michael A

    2016-01-01

    Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy.

  13. Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome.

    Directory of Open Access Journals (Sweden)

    Jamie R K Marland

    Full Text Available Down syndrome (DS is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy.

  14. Using Plasmids as DNA Vaccines for Infectious Diseases.

    Science.gov (United States)

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  15. Integrating anatomy and function for zebrafish circuit analysis.

    Science.gov (United States)

    Arrenberg, Aristides B; Driever, Wolfgang

    2013-01-01

    Due to its transparency, virtually every brain structure of the larval zebrafish is accessible to light-based interrogation of circuit function. Advanced stimulation techniques allow the activation of optogenetic actuators at different resolution levels, and genetically encoded calcium indicators report the activity of a large proportion of neurons in the CNS. Large datasets result and need to be analyzed to identify cells that have specific properties-e.g., activity correlation to sensory stimulation or behavior. Advances in three-dimensional (3D) functional mapping in zebrafish are promising; however, the mere coordinates of implicated neurons are not sufficient. To comprehensively understand circuit function, these functional maps need to be placed into the proper context of morphological features and projection patterns, neurotransmitter phenotypes, and key anatomical landmarks. We discuss the prospect of merging functional and anatomical data in an integrated atlas from the perspective of our work on long-range dopaminergic neuromodulation and the oculomotor system. We propose that such a resource would help researchers to surpass current hurdles in circuit analysis to achieve an integrated understanding of anatomy and function.

  16. Modulation of Neural Network Activity through Single Cell Ablation: An in Vitro Model of Minimally Invasive Neurosurgery.

    Science.gov (United States)

    Soloperto, Alessandro; Bisio, Marta; Palazzolo, Gemma; Chiappalone, Michela; Bonifazi, Paolo; Difato, Francesco

    2016-01-01

    The technological advancement of optical approaches, and the growth of their applications in neuroscience, has allowed investigations of the physio-pathology of neural networks at a single cell level. Therefore, better understanding the role of single neurons in the onset and progression of neurodegenerative conditions has resulted in a strong demand for surgical tools operating with single cell resolution. Optical systems already provide subcellular resolution to monitor and manipulate living tissues, and thus allow understanding the potentiality of surgery actuated at single cell level. In the present work, we report an in vitro experimental model of minimally invasive surgery applied on neuronal cultures expressing a genetically encoded calcium sensor. The experimental protocol entails the continuous monitoring of the network activity before and after the ablation of a single neuron, to provide a robust evaluation of the induced changes in the network activity. We report that in subpopulations of about 1000 neurons, even the ablation of a single unit produces a reduction of the overall network activity. The reported protocol represents a simple and cost effective model to study the efficacy of single-cell surgery, and it could represent a test-bed to study surgical procedures circumventing the abrupt and complete tissue removal in pathological conditions.

  17. Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.

    Science.gov (United States)

    Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

    2016-01-01

    The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

  18. RNA aptamer probes as optical imaging agents for the detection of amyloid plaques.

    Directory of Open Access Journals (Sweden)

    Christian T Farrar

    Full Text Available Optical imaging using multiphoton microscopy and whole body near infrared imaging has become a routine part of biomedical research. However, optical imaging methods rely on the availability of either small molecule reporters or genetically encoded fluorescent proteins, which are challenging and time consuming to develop. While directly labeled antibodies can also be used as imaging agents, antibodies are species specific, can typically not be tagged with multiple fluorescent reporters without interfering with target binding, and are bioactive, almost always eliciting a biological response and thereby influencing the process that is being studied. We examined the possibility of developing highly specific and sensitive optical imaging agents using aptamer technology. We developed a fluorescently tagged anti-Aβ RNA aptamer, β55, which binds amyloid plaques in both ex vivo human Alzheimer's disease brain tissue and in vivo APP/PS1 transgenic mice. Diffuse β55 positive halos, attributed to oligomeric Aβ, were observed surrounding the methoxy-XO4 positive plaque cores. Dot blots of synthetic Aβ aggregates provide further evidence that β55 binds both fibrillar and non-fibrillar Aβ. The high binding affinity, the ease of probe development, and the ability to incorporate multiple and multimodal imaging reporters suggest that RNA aptamers may have complementary and perhaps advantageous properties compared to conventional optical imaging probes and reporters.

  19. Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

    Directory of Open Access Journals (Sweden)

    Brian J Belmont

    Full Text Available Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs, which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i the Tet Repressor protein (TetR genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

  20. Versatile three-dimensional virus-based template for dye-sensitized solar cells with improved electron transport and light harvesting.

    Science.gov (United States)

    Chen, Po-Yen; Dang, Xiangnan; Klug, Matthew T; Qi, Jifa; Dorval Courchesne, Noémie-Manuelle; Burpo, Fred J; Fang, Nicholas; Hammond, Paula T; Belcher, Angela M

    2013-08-27

    By genetically encoding affinity for inorganic materials into the capsid proteins of the M13 bacteriophage, the virus can act as a template for the synthesis of nanomaterial composites for use in various device applications. Herein, the M13 bacteriophage is employed to build a multifunctional and three-dimensional scaffold capable of improving both electron collection and light harvesting in dye-sensitized solar cells (DSSCs). This has been accomplished by binding gold nanoparticles (AuNPs) to the virus proteins and encapsulating the AuNP-virus complexes in TiO2 to produce a plasmon-enhanced and nanowire (NW)-based photoanode. The NW morphology exhibits an improved electron diffusion length compared to traditional nanoparticle-based DSSCs, and the AuNPs increase the light absorption of the dye-molecules through the phenomenon of localized surface plasmon resonance. Consequently, we report a virus-templated and plasmon-enhanced DSSC with an efficiency of 8.46%, which is achieved through optimizing both the NW morphology and the concentration of AuNPs loaded into the solar cells. In addition, we propose a theoretical model that predicts the experimentally observed trends of plasmon enhancement. PMID:23808626

  1. The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons

    Directory of Open Access Journals (Sweden)

    Marina ePolito

    2013-11-01

    Full Text Available The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-SH150 with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, and this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 medium spiny neurons. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission.

  2. Cytosolic NADH-NAD+ Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

    Science.gov (United States)

    Mongeon, Rebecca; Venkatachalam, Veena

    2016-01-01

    Abstract Aim: Cytosolic NADH-NAD+ redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD+ redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. Results: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD+ ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Innovation: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Conclusion: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD+ ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553–563. PMID:26857245

  3. In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons

    KAUST Repository

    Mächler, Philipp

    2015-11-19

    Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. © 2016 Elsevier Inc.

  4. Polycyclic peptide therapeutics.

    Science.gov (United States)

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. PMID:23355488

  5. Imaging Ca2+ dynamics in cone photoreceptor axon terminals of the mouse retina.

    Science.gov (United States)

    Kulkarni, Manoj; Schubert, Timm; Baden, Tom; Wissinger, Bernd; Euler, Thomas; Paquet-Durand, Francois

    2015-01-01

    Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca(2+)), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca(2+) imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca(2+) biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections ("slices") of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca(2+) level. The protocol also allows "in-slice measurement" of absolute Ca(2+) concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca(2+) signaling as well as the potential involvement of Ca(2+) in photoreceptor death and retinal degeneration. PMID:25993489

  6. Visualization of cyclic nucleotide dynamics in neurons

    Directory of Open Access Journals (Sweden)

    Kirill eGorshkov

    2014-12-01

    Full Text Available The second messengers cAMP and cGMP transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks.

  7. Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers.

    Science.gov (United States)

    Uchugonova, Aisada; Cao, Wenluo; Hoffman, Robert M; Koenig, Karsten

    2015-01-01

    Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.

  8. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.

    Science.gov (United States)

    Kamioka, Yuji; Sumiyama, Kenta; Mizuno, Rei; Sakai, Yoshiharu; Hirata, Eishu; Kiyokawa, Etsuko; Matsuda, Michiyuki

    2012-01-01

    Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

  9. Modulation of Neural Network Activity through Single Cell Ablation: An in Vitro Model of Minimally Invasive Neurosurgery.

    Science.gov (United States)

    Soloperto, Alessandro; Bisio, Marta; Palazzolo, Gemma; Chiappalone, Michela; Bonifazi, Paolo; Difato, Francesco

    2016-01-01

    The technological advancement of optical approaches, and the growth of their applications in neuroscience, has allowed investigations of the physio-pathology of neural networks at a single cell level. Therefore, better understanding the role of single neurons in the onset and progression of neurodegenerative conditions has resulted in a strong demand for surgical tools operating with single cell resolution. Optical systems already provide subcellular resolution to monitor and manipulate living tissues, and thus allow understanding the potentiality of surgery actuated at single cell level. In the present work, we report an in vitro experimental model of minimally invasive surgery applied on neuronal cultures expressing a genetically encoded calcium sensor. The experimental protocol entails the continuous monitoring of the network activity before and after the ablation of a single neuron, to provide a robust evaluation of the induced changes in the network activity. We report that in subpopulations of about 1000 neurons, even the ablation of a single unit produces a reduction of the overall network activity. The reported protocol represents a simple and cost effective model to study the efficacy of single-cell surgery, and it could represent a test-bed to study surgical procedures circumventing the abrupt and complete tissue removal in pathological conditions. PMID:27527143

  10. Active Shop Scheduling Of Production Process Based On RFID Technology

    Directory of Open Access Journals (Sweden)

    Cuihua Chao

    2016-01-01

    Full Text Available In industry 4.0 environment, intelligent technology is almost applied to all parts of the manufacturing process, such as process design, job shop scheduling, etc.. This paper presents an efficient approach to job shop scheduling actively by using RFID to collect real-time manufacturing data. Identified the workpiece by RFID which needs to be machined, it can “ask for” the resource actively for the following process. With these active asking-for strategy, a double genetically encoded improved genetic algorithm is proposed for achieving active job shop scheduling solution during the actual manufacturing process. A case was used to evaluate its effectiveness. Meanwhile, , it can effectively and actively carry out job shop scheduling and has much better convergence effect comparing with basic genetic algorithm. And the job shop scheduler in management center can use the proposed algorithm to get the satisfied scheduling result timely by reducing waiting time and making begin time earlier during transmission between manufacturing process, which makes the scheduling result feasible and accurate.

  11. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  12. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans

    Science.gov (United States)

    Wojtovich, Andrew P.; Wei, Alicia Y.; Sherman, Teresa A.; Foster, Thomas H.; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the ‘singlet oxygen generator’ miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

  13. Ontogeny of innate T lymphocytes - some innate lymphocytes are more innate then others

    Directory of Open Access Journals (Sweden)

    David eVermijlen

    2014-10-01

    Full Text Available Innate lymphocytes have recently received a lot of attention. However, there are different ideas about the definition of what is innate in lymphocytes. Lymphocytes without V(DJ-rearranged antigen receptors are now termed innate lymphoid cells (ILCs and include cells formerly known as NK cells. Also, lymphocytes that are innate should be able to recognize microbial or stress-induced patterns and react rapidly without prior sensitization, as opposed to adaptive immune responses. Formally, genuine innate lymphocytes would be present before or at birth. Here we review the ontogeny of human and mouse innate T lymphocyte populations. We focus on γδ T cells, which are prototype lymphocytes that often use their V(DJ rearrangement machinery to generate genetically encoded predetermined recombinations of antigen receptors. We make parallels between the development of γδ T cells with that of innate aβ T cells (invariant (iNKT and mucosa-associated invariant T (MAIT cells and compare this with the ontogeny of innate B cells and innate lymphoid cells (ILCs, including NK cells. We conclude that some subsets are more innate than others, i.e. innate lymphocytes that are made primarily early in utero during gestation while others are made after birth. In practice, a ranking of innateness by ontogeny has implications for the reconstitution of innate lymphocyte subsets after hematopoietic stem cell transplantation (HSCT.

  14. High-throughput automated home-cage mesoscopic functional imaging of mouse cortex.

    Science.gov (United States)

    Murphy, Timothy H; Boyd, Jamie D; Bolaños, Federico; Vanni, Matthieu P; Silasi, Gergely; Haupt, Dirk; LeDue, Jeff M

    2016-01-01

    Mouse head-fixed behaviour coupled with functional imaging has become a powerful technique in rodent systems neuroscience. However, training mice can be time consuming and is potentially stressful for animals. Here we report a fully automated, open source, self-initiated head-fixation system for mesoscopic functional imaging in mice. The system supports five mice at a time and requires minimal investigator intervention. Using genetically encoded calcium indicator transgenic mice, we longitudinally monitor cortical functional connectivity up to 24 h per day in >7,000 self-initiated and unsupervised imaging sessions up to 90 days. The procedure provides robust assessment of functional cortical maps on the basis of both spontaneous activity and brief sensory stimuli such as light flashes. The approach is scalable to a number of remotely controlled cages that can be assessed within the controlled conditions of dedicated animal facilities. We anticipate that home-cage brain imaging will permit flexible and chronic assessment of mesoscale cortical function. PMID:27291514

  15. Channel-Mediated Lactate Release by K+-Stimulated Astrocytes

    KAUST Repository

    Sotelo-Hitschfeld, T.

    2015-03-11

    Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K+ or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

  16. [life origin and early ecolution on Earth].

    Science.gov (United States)

    Bregestowski, P D

    2015-01-01

    The most widely accepted modern scenario of prebiotic evolution that led to the emergence of the first cells on our planet is the "RNA World"--a hypothetical period of the early Earth's biosphere, when the information transfer and all the processes necessary for the functioning of the primary systems were provided by replicating RNA molecules. The essence of the "RNA World" hypothesis is based on two postulates: 1) at the initial stages of the origin of life, RNA molecules performed all functions necessary for reproduction and replication of biological molecules: informational, catalytic and structural; 2) at a certain stage of evolution arose separation of RNA and DNA, appeared genetically encoded proteins and occurred a transition to the modern world of living systems functioning. However, the analysis shows that the hypothesis of "RNA World" has a number of unsurmountable problems of chemical and informational nature. The biggest of them are: a) the unreliability of the initial components synthesis; b) a catastrophic rise of polynucleotide chains instability with their elongation; c) catastrophically low probability of formation of sequences possessing meaningful information; d) lack of a mechanism determining the regularities division of the membrane vesicles permeable to nitrogen bases and other RNA components; e) lack of driving forces for the transition from the RNA world to the much more complex world based on DNA and RNA. Therefore, the "RNA World" scenario seems unlikely.

  17. 改进遗传算法在人群行为仿真中的应用%The simulation of improved genetic algorithms of crowd behavior

    Institute of Scientific and Technical Information of China (English)

    娄银霞

    2013-01-01

    For the combinatorial optimization problem of crowd behavior simulation multi-objective and consuained,proposed a improved genetic algorithm in the application of group behavior simulation.By setting the special data structure to simulate the process of a variety of constraint rules,genetic encoding of genetic algorithm,fittness evaluation function implement of crowd behavior simulation.Simulation experiment verified that the method can greatly reduce the search space,and enables optimal results.%针对目前人群行为仿真中多目标,有约束的组合优化问题,提出了一种改进遗传算法在人群行为仿真的应用方案.通过设定特殊的数据结构、仿真过程中的各种约束规则、遗传算法中的基因编码、适应度评价函数实现了人群行为仿真.仿真实验验证了该算法可以大大减少搜索空间,并能使结果达到最优.

  18. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.

    Science.gov (United States)

    Takemoto, Kiwamu; Matsuda, Tomoki; Sakai, Naoki; Fu, Donald; Noda, Masanori; Uchiyama, Susumu; Kotera, Ippei; Arai, Yoshiyuki; Horiuchi, Masataka; Fukui, Kiichi; Ayabe, Tokiyoshi; Inagaki, Fuyuhiko; Suzuki, Hiroshi; Nagai, Takeharu

    2013-01-01

    Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed. PMID:24043132

  19. Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy

    Science.gov (United States)

    Baumann, Fabian; Bauer, Magnus S.; Milles, Lukas F.; Alexandrovich, Alexander; Gaub, Hermann E.; Pippig, Diana A.

    2016-01-01

    Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

  20. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes

    Science.gov (United States)

    Iwane, Yoshihiko; Hitomi, Azusa; Murakami, Hiroshi; Katoh, Takayuki; Goto, Yuki; Suga, Hiroaki

    2016-04-01

    In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

  1. Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation.

    Science.gov (United States)

    Srivastava, Poonam; Yang, Hao; Ellis-Guardiola, Ken; Lewis, Jared C

    2015-07-24

    Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

  2. Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans.

    Science.gov (United States)

    Wojtovich, Andrew P; Wei, Alicia Y; Sherman, Teresa A; Foster, Thomas H; Nehrke, Keith

    2016-01-01

    Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the 'singlet oxygen generator' miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology. PMID:27440050

  3. Loss of Pde6 reduces cell body Ca(2+) transients within photoreceptors.

    Science.gov (United States)

    Ma, E Y; Lewis, A; Barabas, P; Stearns, G; Suzuki, S; Krizaj, D; Brockerhoff, S E

    2013-01-01

    Modulation of Ca(2+) within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca(2+) indicators to define changes in perikaryal Ca(2+) concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca(2+)]i levels within the cell body. Arguing against Ca(2+) overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca(2+) fluxes revealed that degeneration of pde6c(w59) mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca(2+)]i. Analysis of [Ca(2+)]i in dissociated Pde6β(rd1)mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca(2+) elevations ('flashes') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca(2+) fluxes diminished. This study thus provides insight into Ca(2+) dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca(2+)]i that occurs in normal cones. PMID:24030149

  4. Loss of Pde6 reduces cell body Ca2+ transients within photoreceptors

    Science.gov (United States)

    Ma, E Y; Lewis, A; Barabas, P; Stearns, G; Suzuki, S; Krizaj, D; Brockerhoff, S E

    2013-01-01

    Modulation of Ca2+ within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca2+ indicators to define changes in perikaryal Ca2+ concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca2+]i levels within the cell body. Arguing against Ca2+ overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca2+ fluxes revealed that degeneration of pde6cw59 mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca2+]i. Analysis of [Ca2+]i in dissociated Pde6βrd1mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca2+ elevations (‘flashes') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca2+ fluxes diminished. This study thus provides insight into Ca2+ dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca2+]i that occurs in normal cones. PMID:24030149

  5. Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes.

    Science.gov (United States)

    Morgan, Bruce; Van Laer, Koen; Owusu, Theresa N E; Ezeriņa, Daria; Pastor-Flores, Daniel; Amponsah, Prince Saforo; Tursch, Anja; Dick, Tobias P

    2016-06-01

    Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H2O2 probes, roGFP2-Tsa2ΔCR and roGFP2-Tsa2ΔCPΔCR, that afford such sensitivity. These probes are ∼50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H2O2 homeostasis. The roGFP2-Tsa2ΔCR probe revealed real-time interplay between basal H2O2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H2O2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H2O2 levels and cellular growth rate. PMID:27089028

  6. Fluorescent and Bioluminescent Reporter Myxoviruses

    Science.gov (United States)

    Rostad, Christina A.; Currier, Michael C.; Moore, Martin L.

    2016-01-01

    The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo. PMID:27527209

  7. Visualizing spatiotemporal dynamics of multicellular cell-cycle progressions with fucci technology.

    Science.gov (United States)

    Sakaue-Sawano, Asako; Miyawaki, Atsushi

    2014-05-01

    The visualization of cell-cycle behavior of individual cells within complex tissues presents an irresistible challenge to biologists studying multicellular structures. However, the transition from G1 to S in the cell cycle is difficult to monitor despite the fact that the process involves the critical decision to initiate a new round of DNA replication. Here, we use ubiquitination oscillators that control cell-cycle transitions to develop genetically encoded fluorescent probes for cell-cycle progression. Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes exploit the regulation of cell-cycle-dependent ubiquitination to effectively label individual nuclei in G1 phase red, and those in S/G2/M phases green. Cultured cells and transgenic mice constitutively expressing the probes have been generated, such that every cell nucleus shows either red or green fluorescence. This protocol details two experiments that use biological samples expressing Fucci probes. One experiment involves time-lapse imaging of cells stably expressing a Fucci derivative (Fucci2), which allows for the exploration of the spatiotemporal patterns of cell-cycle dynamics during structural and behavioral changes of cultured cells. The other experiment involves large-field, high-resolution imaging of fixed sections of Fucci transgenic mouse embryos, which provides maps that illustrate cell proliferation versus differentiation in various developing organs.

  8. Adipose Clocks: Burning the Midnight Oil.

    Science.gov (United States)

    Henriksson, Emma; Lamia, Katja A

    2015-10-01

    Circadian clocks optimize the timing of physiological processes in synchrony with daily recurring and therefore predictable changes in the environment. Until the late 1990s, circadian clocks were thought to exist only in the central nervous systems of animals; elegant studies in cultured fibroblasts and using genetically encoded reporters in Drosophila melanogaster and in mice showed that clocks are ubiquitous and cell autonomous. These findings inspired investigations of the advantages construed by enabling each organ to independently adjust its function to the time of day. Studies of rhythmic gene expression in several organs suggested that peripheral organ clocks might play an important role in optimizing metabolic physiology by synchronizing tissue-intrinsic metabolic processes to cycles of nutrient availability and energy requirements. The effects of clock disruption in liver, pancreas, muscle, and adipose tissues support that hypothesis. Adipose tissues coordinate energy storage and utilization and modulate behavior and the physiology of other organs by secreting hormones known as "adipokines." Due to behavior- and environment-driven diurnal variations in supply and demand for chemical and thermal energy, adipose tissues might represent an important peripheral location for coordinating circadian energy balance (intake, storage, and utilization) over the whole organism. Given the complexity of adipose cell types and depots, the sensitivity of adipose tissue biology to age and diet composition, and the plethora of known and yet-to-be-discovered adipokines and lipokines, we have just begun to scratch the surface of understanding the role of circadian clocks in adipose tissues.

  9. A green fluorescent protein with photoswitchable emission from the deep sea.

    Directory of Open Access Journals (Sweden)

    Alexander Vogt

    Full Text Available A colorful variety of fluorescent proteins (FPs from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

  10. Microfluidic devices for imaging neurological response of Drosophila melanogaster larva to auditory stimulus.

    Science.gov (United States)

    Ghaemi, Reza; Rezai, Pouya; Iyengar, Balaji G; Selvaganapathy, Ponnambalam Ravi

    2015-02-21

    Two microfluidic devices (pneumatic chip and FlexiChip) have been developed for immobilization and live-intact fluorescence functional imaging of Drosophila larva's Central Nervous System (CNS) in response to controlled acoustic stimulation. The pneumatic chip is suited for automated loading/unloading and potentially allows high throughput operation for studies with a large number of larvae while the FlexiChip provides a simple and quick manual option for animal loading and is suited for smaller studies. Both chips were capable of significantly reducing the endogenous CNS movement while still allowing the study of sound-stimulated CNS activities of Drosophila 3rd instar larvae using genetically encoded calcium indicator GCaMP5. Temporal effects of sound frequency (50-5000 Hz) and intensity (95-115 dB) on CNS activities were investigated and a peak neuronal response of 200 Hz was identified. Our lab-on-chip devices can not only aid further studies of Drosophila larva's auditory responses but can be also adopted for functional imaging of CNS activities in response to other sensory cues. Auditory stimuli and the corresponding response of the CNS can potentially be used as a tool to study the effect of chemicals on the neurophysiology of this model organism.

  11. Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags

    Energy Technology Data Exchange (ETDEWEB)

    Abdelkader, Elwy H.; Yao, Xuejun [Australian National University, Research School of Chemistry (Australia); Feintuch, Akiva [Weizmann Institute of Science, Department of Chemical Physics (Israel); Adams, Luke A.; Aurelio, Luigi; Graham, Bim [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Goldfarb, Daniella [Weizmann Institute of Science, Department of Chemical Physics (Israel); Otting, Gottfried, E-mail: gottfried.otting@anu.edu.au [Australian National University, Research School of Chemistry (Australia)

    2016-01-15

    Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd{sup 3+}-Gd{sup 3+} distances measured by double electron–electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm{sup 3+} and C3-Tb{sup 3+} tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb{sup 3+} and Tm{sup 3+} rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.

  12. The Human SepSecS-tRNA[superscript Sec] Complex Reveals the Mechanism of Selenocysteine Formation

    Energy Technology Data Exchange (ETDEWEB)

    Palioura, Sotiria; Sherrer, R. Lynn; Steitz, Thomas A.; Söll, Dieter; Simonovic, Miljan; (Yale); (UIC)

    2009-08-13

    Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNA-dependent mechanism. The crystal structure of human tRNA{sup Sec} in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA{sup Sec} formation. Two tRNA{sup Sec} molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-T{Upsilon}C arm (where {Upsilon} indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA{sup Sec}, but not free phosphoserine, to be oriented properly for the reaction to occur.

  13. A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

    Directory of Open Access Journals (Sweden)

    Jakobsdottir Klara B

    2002-06-01

    Full Text Available Abstract Background The jellyfish green fluorescent protein (GFP can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. Results We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. Conclusion This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

  14. X-ray irradiation activates K+ channels via H2O2 signaling.

    Science.gov (United States)

    Gibhardt, Christine S; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  15. Guidelines for the use of protein domains in acidic phospholipid imaging

    Science.gov (United States)

    Platre, Matthieu Pierre; Jaillais, Yvon

    2015-01-01

    Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS) and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the years, several techniques have emerged in either fixed or live cells to analyze the subcellular localization and dynamics of acidic phospholipids. In this chapter, we review one of them: the use of genetically encoded biosensors that are based on the expression of specific lipid binding domains (LBDs) fused to fluorescent proteins. We discuss how to design such sensors, including the criteria for selecting the lipid binding domains of interest and to validate them. We also emphasize the care that must be taken during data analysis as well as the main limitations and advantages of this approach. PMID:26552684

  16. Orthogonal Optical Control of a G Protein-Coupled Receptor with a SNAP-Tethered Photochromic Ligand.

    Science.gov (United States)

    Broichhagen, Johannes; Damijonaitis, Arunas; Levitz, Joshua; Sokol, Kevin R; Leippe, Philipp; Konrad, David; Isacoff, Ehud Y; Trauner, Dirk

    2015-10-28

    The covalent attachment of synthetic photoswitches is a general approach to impart light sensitivity onto native receptors. It mimics the logic of natural photoreceptors and significantly expands the reach of optogenetics. Here we describe a novel photoswitch design-the photoswitchable orthogonal remotely tethered ligand (PORTL)-that combines the genetically encoded SNAP-tag with photochromic ligands connected to a benzylguanine via a long flexible linker. We use the method to convert the G protein-coupled receptor mGluR2, a metabotropic glutamate receptor, into a photoreceptor (SNAG-mGluR2) that provides efficient optical control over the neuronal functions of mGluR2: presynaptic inhibition and control of excitability. The PORTL approach enables multiplexed optical control of different native receptors using distinct bioconjugation methods. It should be broadly applicable since SNAP-tags have proven to be reliable, many SNAP-tagged receptors are already available, and photochromic ligands on a long leash are readily designed and synthesized. PMID:27162996

  17. Using optogenetics to translate the "inflammatory dialogue" between heart and brain in the context of stress

    Institute of Scientific and Technical Information of China (English)

    Jinbo Cheng; Jie Zhang; Caiyi Lu; Liping Wang

    2012-01-01

    Inflammatory processes are an integral part of the stress response and are likely to result from a programmed adaptation that is vital to the organism's survival and well-being.The whole inflammatory response is mediated by largely overlapping circuits in the limbic forebrain,hypothalamus and brainstem,but is also under the control of the neuroendocrine and autonomic nervous systems.Genetically predisposed individuals who fail to tune the respective contributions of the two systems in accordance with stressor modality and intensity after adverse experiences can be at risk for stress-related psychiatric disorders and cardiovascular diseases.Altered glucocorticoid (GC) homeostasis due to GC resistance leads to the failure of neural and negative feedback regulation of the hypothalamic-pituitary-adrenal axis during chronic inflammation,and this might be the mechanism underlying the ensuing brain and heart diseases and the high prevalence of co-morbidity between the two systems.By the combined use of light and genetically-encoded lightsensitive proteins,optogenetics allows cell-type-specific,fast (millisecond-scale) control of precisely defined events in biological systems.This method is an important breakthrough to explore the causality between neural activity patterns and behavioral profiles relevant to anxiety,depression,autism and schizophrenia.Optogenetics also helps to understand the "inflammatory dialogue",the inflammatory processes in psychiatric disorders and cardiovascular diseases,shared by heart and brain in the context of stress.

  18. New Tools for Investigating Astrocyte-to-Neuron Communication

    Directory of Open Access Journals (Sweden)

    Dongdong eLi

    2013-10-01

    Full Text Available Grey matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca2+-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated.Studying astrocytes in their natural environment is challenging because: i astrocytes are electrically silent; ii astrocytes and neurons express an overlapping repertoire of transmembrane receptors; iii astrocyte processes in contact with synapses are below confocal and two-photon microscope resolution; iv bulk-loading techniques using fluorescent Ca2+ indicators lack cellular specificity.In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca2+ indicators (GECIs, light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: i the complexity of astrocyte Ca2+ signalling revealed by GECIs; ii new pharmacogenetic and optogenetic approaches to activate specific Ca2+ signalling pathways in astrocytes; iii classical and new techniques to monitor vesicle fusion in cultured astrocytes; iv possible strategies to express specifically reporter genes in astrocytes.

  19. Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

    Energy Technology Data Exchange (ETDEWEB)

    Akerboom, Jasper; Velez Rivera, Jonathan D.; Rodriguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Hernandez, Hector H.; Tian, Lin; Hires, S. Andrew; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.; (MIT); (Puerto Rico); (HHMI)

    2009-03-16

    The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca{sup 2+}-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

  20. Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein.

    Science.gov (United States)

    Dang, Thanh X; Milligan, Ronald A; Tweten, Rodney K; Wilson-Kubalek, Elizabeth M

    2005-11-01

    To facilitate purification and subsequent structural studies of recombinant proteins the most widely used genetically encoded tag is the histidine tag (His-tag) which specifically binds to N-nitrilotriacetic-acid-chelated nickel ions. Lipids derivatized with a nickel-chelating head group can be mixed with galactosylceramide glycolipids to prepare lipid nanotubes that bind His-tagged proteins. In this study, we use His-tagged perfringolysin O (PFO), a soluble toxin secreted by the bacterial pathogen Clostridium perfringens, as a model protein to test the utility of nickel-lipid nanotubes as a tool for structural studies of His-tagged proteins. PFO is a member of the cholesterol dependent cytolysin family (CDC) of oligomerizing, pore-forming toxins found in a variety of Gram-positive bacterial pathogens. CDC pores have been difficult to study by X-ray crystallography because they are membrane associated and vary in size. We demonstrate that both a wild-type and a mutant form of PFO form helical arrays on nickel-lipid containing nanotubes. Cryo-electron microscopy and image analysis of the helical arrays were used to reconstruct a 3D density map of wild-type PFO. This study suggests that the use of nickel-lipid nanotubes may offer a general approach for structural studies of recombinant proteins and may provide insights into the molecular interactions of proteins that have a natural affinity for a membrane surface.