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Sample records for dual fret sensors

  1. 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

  2. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  3. 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.

  4. Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors.

    Science.gov (United States)

    Clifton, Ben E; Whitfield, Jason H; Sanchez-Romero, Inmaculada; Herde, Michel K; Henneberger, Christian; Janovjak, Harald; Jackson, Colin J

    2017-01-01

    Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.

  5. 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.

  6. Compound parabolic concentrator optical fiber tip for FRET-based fluorescent sensors

    DEFF Research Database (Denmark)

    Hassan, Hafeez Ul; Nielsen, Kristian; Aasmul, Soren

    2015-01-01

    The Compound Parabolic Concentrator (CPC) optical fiber tip shape has been proposed for intensity based fluorescent sensors working on the principle of FRET (Förster Resonance Energy Transfer). A simple numerical Zemax model has been used to optimize the CPC tip geometry for a step-index multimod...

  7. Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity

    Directory of Open Access Journals (Sweden)

    Alberola-Ila José

    2005-07-01

    Full Text Available Abstract Background Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. Conclusion EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.

  8. Dual Cryogenic Capacitive Density Sensor

    Science.gov (United States)

    Youngquist, Robert; Mata, Carlos; Vokrot, Peter; Cox, Robert

    2009-01-01

    A dual cryogenic capacitive density sensor has been developed. The device contains capacitive sensors that monitor two-phase cryogenic flow density to within 1% accuracy, which, if temperature were known, could be used to determine the ratio of liquid to gas in the line. Two of these density sensors, located a known distance apart, comprise the sensor, providing some information on the velocity of the flow. This sensor was constructed as a proposed mass flowmeter with high data acquisition rates. Without moving parts, this device is capable of detecting the density change within a two-phase cryogenic flow more than 100 times a second. Detection is enabled by a series of two sets of five parallel plates with stainless steel, cryogenically rated tubing. The parallel plates form the two capacitive sensors, which are measured by electrically isolated digital electronics. These capacitors monitor the dielectric of the flow essentially the density of the flow and can be used to determine (along with temperature) the ratio of cryogenic liquid to gas. Combining this information with the velocity of the flow can, with care, be used to approximate the total two-phase mass flow. The sensor can be operated at moderately high pressures and can be lowered into a cryogenic bath. The electronics have been substantially improved over the older sensors, incorporating a better microprocessor, elaborate ground loop protection and noise limiting circuitry, and reduced temperature sensitivity. At the time of this writing, this design has been bench tested at room temperature, but actual cryogenic tests are pending

  9. Compound parabolic concentrator optical fiber tip for FRET-based fluorescent sensors

    DEFF Research Database (Denmark)

    Hassan, Hafeez Ul; Nielsen, Kristian; Aasmul, Soren;

    2015-01-01

    polymer optical fiber for an excitation and emission wavelength of 550 nm and 650nm, respectively. The model suggests an increase of a factor of 1.6 to 4 in the collected fluorescent power for an ideal CPC tip, as compared to the plane-cut fiber tip for fiber lengths between 5 and 45mm......The Compound Parabolic Concentrator (CPC) optical fiber tip shape has been proposed for intensity based fluorescent sensors working on the principle of FRET (Förster Resonance Energy Transfer). A simple numerical Zemax model has been used to optimize the CPC tip geometry for a step-index multimode...

  10. 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.

  11. Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology.

    Science.gov (United States)

    Lanquar, Viviane; Grossmann, Guido; Vinkenborg, Jan L; Merkx, Maarten; Thomine, Sébastien; Frommer, Wolf B

    2014-04-01

    Zinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn(2+) homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine. Our work combines the use of genetically encoded Förster resonance energy transfer (FRET) sensors and a novel microfluidic technology, the RootChip, to monitor the dynamics of cytosolic Zn(2+) concentrations in Arabidopsis root cells. Our experiments provide estimates of cytosolic free Zn(2+) concentrations in Arabidopsis root cells grown under sufficient (0.4 nM) and excess (2 nM) Zn(2+) supply. In addition, monitoring the dynamics of cytosolic [Zn(2+) ] in response to external supply suggests the involvement of high- and low-affinity uptake systems as well as release from internal stores. In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  12. Dual Axis Light Sensor for Tracking Sun

    Science.gov (United States)

    Shibata, Miki; Tambo, Toyokazu

    We have developed convenient light sensors to control a platform of solar cell panel. Dual axis light sensor in the present paper has structure of 5 PD (photodiode) light sensor which is composed of 5 photodiodes attached on a frustum of pyramid(1). Light source can be captured in front of the sensor by rotating the X and Y axis as decreasing the output deviation between two pairs of outside photodiodes. We here report the mechanism of sun tacking using the dual axis 5 PD light sensor and the fundamental results performed in the dark room.

  13. Dual Brushless Resolver Rate Sensor

    Science.gov (United States)

    Howard, David E. (Inventor)

    1997-01-01

    A resolver rate sensor is disclosed in which dual brushless resolvers are mechanically coupled to the same output shaft. Diverse inputs are provided to each resolver by providing the first resolver with a DC input and the second resolver with an AC sinusoidal input. A trigonometric identity in which the sum of the squares of the sin and cosine components equal one is used to advantage in providing a sensor of increased accuracy. The first resolver may have a fixed or variable DC input to permit dynamic adjustment of resolver sensitivity thus permitting a wide range of coverage. In one embodiment of the invention the outputs of the first resolver are directly inputted into two separate multipliers and the outputs of the second resolver are inputted into the two separate multipliers, after being demodulated in a pair of demodulator circuits. The multiplied signals are then added in an adder circuit to provide a directional sensitive output. In another embodiment the outputs from the first resolver is modulated in separate modulator circuits and the output from the modulator circuits are used to excite the second resolver. The outputs from the second resolver are demodulated in separate demodulator circuit and added in an adder circuit to provide a direction sensitive rate output.

  14. 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.

  15. Dual Time Stepping Algorithms With the High Order Harmonic Balance Method for Contact Interfaces With Fretting-Wear

    CERN Document Server

    Salles, Loic; Gouskov, Alexandre; Jean, Pierrick; Thouverez, Fabrice

    2014-01-01

    Contact interfaces with dry friction are frequently used in turbomachinery. Dry friction damping produced by the sliding surfaces of these interfaces reduces the amplitude of bladed-disk vibration. The relative displacements at these interfaces lead to fretting-wear which reduces the average life expectancy of the structure. Frequency response functions are calculated numerically by using the multi-harmonic balance method (mHBM). The dynamic Lagrangian frequency-time method is used to calculate contact forces in the frequency domain. A new strategy for solving nonlinear systems based on dual time stepping is applied. This method is faster than using Newton solvers. It was used successfully for solving Nonlinear CFD equations in the frequency domain. This new approach allows identifying the steady state of worn systems by integrating wear rate equations a on dual time scale. The dual time equations are integrated by an implicit scheme. Of the different orders tested, the first order scheme provided the best re...

  16. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

    Science.gov (United States)

    Demeautis, Claire; Sipieter, François; Roul, Julien; Chapuis, Catherine; Padilla-Parra, Sergi; Riquet, Franck B.; Tramier, Marc

    2017-01-01

    Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM. PMID:28106114

  17. Graphene quantum dots FRET based sensor for early detection of heart attack in human.

    Science.gov (United States)

    Bhatnagar, Deepika; Kumar, Vanish; Kumar, Ashok; Kaur, Inderpreet

    2016-05-15

    Cardiac immunosensor for early detection of heart attack (myocardial infarction) was developed using amine functionalized graphene quantum dots (afGQDs) conjugated with antibody anti-cardiac Troponin I (anti-cTnI) to detect cardiac marker antigen Troponin I (cTnI) in blood based on fluorescence resonance energy transfer (FRET) between conjugate and graphene (quencher) only in 10 min. The anti-cTnI was covalently conjugated to afGQDs through carbodiimide coupling reaction. The conjugate was characterized by zeta potential UV-vis spectroscopy and field emission scanning electron microscopy (FESEM). The sensing performance of the sensor was studied with respect to changes in the photon count and photoluminescence of GQDs based on interaction of target cTnI with its specific anti-cTnI antibody. The sensor is highly specific and shows negligible response to non-specific antigens. The sensor displayed a linear response to cTnI from 0.001 to 1000 ng mL(-1) with a limit of detection of 0.192 pg mL(-1).

  18. Dynamic monitoring of Gi/o-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors.

    Science.gov (United States)

    Storch, Ursula; Straub, Julie; Erdogmus, Serap; Gudermann, Thomas; Mederos Y Schnitzler, Michael

    2017-06-01

    Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.

  19. 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.

  20. A virus-MIPs fluorescent sensor based on FRET for highly sensitive detection of JEV.

    Science.gov (United States)

    Liang, Caishuang; Wang, Huan; He, Kui; Chen, Chunyan; Chen, Xiaoming; Gong, Hang; Cai, Changqun

    2016-11-01

    Major stumbling blocks in the recognition and detection of virus are the unstable biological recognition element or the complex detection means. Here a fluorescent sensor based on virus-molecular imprinted polymers (virus-MIPs) was designed for specific recognition and highly sensitive detection of Japanese encephalitis virus (JEV). The virus-MIPs were anchored on the surface of silica microspheres modified by fluorescent dye, pyrene-1-carboxaldehyde (PC). The fluorescence intensity of PC can be enhanced by the principle of fluorescence resonance energy transfer (FRET), where virus acted as energy donor and PC acted as energy acceptor. The enhanced fluorescence intensity was proportional to the concentration of virus in the range of 24-960pM, with a limit of detection (LOD, 3σ) of 9.6pM, and the relative standard deviation was 1.99%. In additional, the specificity study confirmed the resultant MIPs has high-selectivity for JEV. This sensor would become a new key for the detection of virus because of its high sensitive, simple operation, high stability and low cost.

  1. Dynamic imaging of glucose flux impedance using FRET sensors in wild-type Arabidopsis plants.

    Science.gov (United States)

    Chaudhuri, Bhavna; Hörmann, Friederike; Frommer, Wolf B

    2011-04-01

    Quantitative and dynamic analysis of metabolites and signalling molecules is limited by technical challenges in obtaining temporally resolved information at the cellular and compartmental level. Real-time information on signalling and metabolite levels with subcellular granularity can be obtained with the help of genetically encoded FRET (Förster resonance energy transfer) nanosensors. FRET nanosensors represent powerful tools for gene discovery, and analysis of regulatory networks, for example by screening mutants. However, RNA silencing has impaired our ability to express FRET nanosensors functionally in Arabidopsis plants. This drawback was overcome here by expressing the nanosensors in RNA silencing mutants. However, the use of silencing mutants requires the generation of homozygous lines deficient in RNA silencing as well as the mutation of interest and co-expression of the nanosensor. Here it is shown that dynamic changes in cytosolic glucose levels can readily be quantified in wild-type Arabidopsis plants at early stages of development (7-15 d) before silencing had a major effect on fluorescence intensity. A detailed protocol for screening 10-20 mutant seedlings per day is provided. The detailed imaging protocol provided here is suitable for analysing sugar flux in young wild-type plants as well as mutants affected in sugar signalling, metabolism, or transport using a wide spectrum of FRET nanosensors.

  2. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

    Directory of Open Access Journals (Sweden)

    Jakobus van Unen

    Full Text Available G-protein coupled receptors (GPCRs can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

  3. A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

    Directory of Open Access Journals (Sweden)

    Nidhi Mathur

    2008-01-01

    Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

  4. Handheld Fluorescence Resonance Energy Transfer (FRET)-Aptamer Sensor for Bone Markers

    Science.gov (United States)

    Bruno, John G.

    2015-01-01

    Astronauts lose significant bone mass during lengthy space flights. NASA wishes to monitor this bone loss in order to develop nutritional and exercise countermeasures. Operational Technologies Corporation (OpTech) has developed a handheld device that quantifies bone loss in a spacecraft environment. The innovation works by adding fluorescent dyes and quenchers to aptamers to enable pushbutton, one-step bind-and-detect FRET assays that can be freeze-dried, rehydrated with body fluids, and used to quantify bone loss.

  5. FRET Sensor for Erythrosine Dye Based on Organic Nanoparticles: Application to Analysis of Food Stuff.

    Science.gov (United States)

    Mahajan, Prasad G; Bhopate, Dhanaji P; Kolekar, Govind B; Patil, Shivajirao R

    2016-07-01

    An aqueous suspension of fluorescent nanoparticles (PHNNPs) of naphthol based fluorescent organic compound 1-[(Z)-(2-phenylhydrazinylidene) methyl] naphthalene -2-ol (PHN) were prepared using reprecipitation method shows bathochromically shifted aggregation induced enhanced emission (AIEE) in the spectral region where erythrosine (ETS) food dye absorbs strongly. The average size of 72.6 nm of aqueous suspension of PHNNPs obtained by Dynamic light scattering results shows a narrow particle size distribution. The negative zeta potential of nano probe (-22.6 mV) responsible to adsorb oppositely charged analyte on its surface and further permit to bind nano probe and analyte within the close distance proximity required for efficient fluorescence resonance energy transfer (FRET) to take place from donor (PHNNPs) to acceptor (ETS). Systematic FRET experiments performed by measuring fluorescence quenching of PHNNPs with successive addition of ETS solution exploited the use of the PHNNPs as a novel nano probe for the detection of ETS in aqueous solution with extremely lower limit of detection equal to 3.6 nM (3.1 ng/mL). The estimation of photo kinetic and thermodynamic parameters such as quenching rate constant, enthalpy change (∆H), Gibbs free energy change (∆G) and entropy change (∆S) was obtained by the quenching results obtained at different constant temperatures which were found to fit the well-known Stern-Volmer relation. The mechanism of binding and fluorescence quenching of PHNNPs by ETS food dye is proposed on the basis of results obtained in photophysical studies, thermodynamic parameter, energy transfer efficiency, critical energy transfer distance (R0) and distance of approach between donor-acceptor molecules (r). The proposed FRET method based on fluorescence quenching of PHNNPs was successfully applied to develop an analytical method for estimation of ETS from food stuffs without interference of other complex ingredients. Graphical Abstract A

  6. Conversion of a putative Agrobacterium sugar-binding protein into a FRET sensor with high selectivity for sucrose.

    Science.gov (United States)

    Lager, Ida; Looger, Loren L; Hilpert, Melanie; Lalonde, Sylvie; Frommer, Wolf B

    2006-10-13

    Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes. Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose nanosensors. Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 mum affinity. This FLIPsuc-4mu protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose. Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4mu for sucrose, maltose, and glucose. Mutant nanosensors showed up to 50- and 11-fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells.

  7. Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor.

    Science.gov (United States)

    Hou, Bi-Huei; Takanaga, Hitomi; Griesbeck, Oliver; Frommer, Wolf B

    2009-08-01

    Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.

  8. SPHERICAL COVERAGE DUAL MODE SENSOR FOR UAS SEPARATION ASSURANCE Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Proposed is a dual-mode sensor for use aboard unmanned aircraft for safe operation in the NAS that: 1. Incorporates high resolution Millimeter Wave radar with high...

  9. An Integrated DNA Modified Dual-microelectrode Sensor Probe

    Institute of Scientific and Technical Information of China (English)

    Xiang Qin LIN; Li Ping LU; Xiao Hua JIANG

    2005-01-01

    A unique method for preparing a coaxial dual-microelectrode sensor by vaporizing the nano-thickness Au layer on the DNA modified carbon fiber micro-column electrode was illustrated.The dual-electrode showed particular merit for determination in biological systems.

  10. 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.

  11. Initial research of dual wavelength fibre optic perimeter sensor

    Science.gov (United States)

    Zyczkowski, M.; Kondrat, M.; Ciurapinski, W.

    2005-10-01

    The dual wavelength fibre optic perimeter sensor bases on input signals measurements in an arrangement of fibre optic Michelson and Sagnac interferometers with a 3 × 3 coupler and two semiconductor lasers. For 3 km long sensor we obtained 20-50m resolution of determination of disturbance point.

  12. A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells.

    Directory of Open Access Journals (Sweden)

    Alejandro San Martín

    Full Text Available Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3-5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg

  13. Dual SAW sensor technique for determining mass and modulus changes.

    Science.gov (United States)

    Hietala, S L; Hietala, V M; Brinker, C J

    2001-01-01

    Surface acoustic wave (SAW) sensors, which are sensitive to a variety of surface changes, have been widely used for chemical and physical sensing. The ability to control or compensate for the many surface forces has been instrumental in collecting valid data. In cases in which it is not possible to neglect certain effects, such as frequency drift with temperature, methods such as the "dual sensor" technique have been utilized. This paper describes a novel use of a dual sensor technique, using two sensor materials (quartz and GaAs) to separate out the contributions of mass and modulus of the frequency change during gas adsorption experiments. The large modulus change in the film calculated using this technique and predicted by the Gassmann equation provide a greater understanding of the challenges of SAW sensing.

  14. A displacement sensor of dual-light based on FPGA

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The dual-light displacement sensor is presented to obtain a higher accuracy compared with the single. The structure and principle of the system are also introduced, and the hardware and software are brought in too. The function of the system is feasible through the experiments and simulating the data process based on FPGA.

  15. GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor.

    Science.gov (United States)

    Takanaga, Hitomi; Chaudhuri, Bhavna; Frommer, Wolf B

    2008-04-01

    Genetically encoded FRET glucose nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05-11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes as well, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens.

  16. GLUT1 and GLUT9 as the major contributors to glucose influx in HEPG2 cells identified by a high sensitivity intramolecular FRET glucose sensor

    Science.gov (United States)

    Takanaga, Hitomi; Chaudhuri, Bhavna; Frommer, Wolf B.

    2008-01-01

    Genetically encoded FRET-glucose nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05–11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes also, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens. PMID:18177733

  17. Dual mode acoustic wave sensor for precise pressure reading

    Science.gov (United States)

    Mu, Xiaojing; Kropelnicki, Piotr; Wang, Yong; Randles, Andrew Benson; Chuan Chai, Kevin Tshun; Cai, Hong; Gu, Yuan Dong

    2014-09-01

    In this letter, a Microelectromechanical system acoustic wave sensor, which has a dual mode (lateral field exited Lamb wave mode and surface acoustic wave (SAW) mode) behavior, is presented for precious pressure change read out. Comb-like interdigital structured electrodes on top of piezoelectric material aluminium nitride (AlN) are used to generate the wave modes. The sensor membrane consists of single crystalline silicon formed by backside-etching of the bulk material of a silicon on insulator wafer having variable device thickness layer (5 μm-50 μm). With this principle, a pressure sensor has been fabricated and mounted on a pressure test package with pressure applied to the backside of the membrane within a range of 0 psi to 300 psi. The temperature coefficient of frequency was experimentally measured in the temperature range of -50 °C to 300 °C. This idea demonstrates a piezoelectric based sensor having two modes SAW/Lamb wave for direct physical parameter—pressure readout and temperature cancellation which can operate in harsh environment such as oil and gas exploration, automobile and aeronautic applications using the dual mode behavior of the sensor and differential readout at the same time.

  18. Spatially distributed fiber sensor with dual processed outputs

    Science.gov (United States)

    Xu, X.; Spillman, William B., Jr.; Claus, Richard O.; Meissner, K. E.; Chen, K.

    2005-05-01

    Given the rapid aging of the world"s population, improvements in technology for automation of patient care and documentation are badly needed. We have previously demonstrated a 'smart bed' that can non-intrusively monitor a patient in bed and determine a patient's respiration, heart rate and movement without intrusive or restrictive medical measurements. This is an application of spatially distributed integrating fiber optic sensors. The basic concept is that any patient movement that also moves an optical fiber within a specified area will produce a change in the optical signal. Two modal modulation approaches were considered, a statistical mode (STM) sensor and a high order mode excitation (HOME) sensor. The present design includes an STM sensor combined with a HOME sensor, using both modal modulation approaches. A special lens system allows only the high order modes of the optical fiber to be excited and coupled into the sensor. For handling output from the dual STM-HOME sensor, computer processing methods are discussed that offer comprehensive perturbation analysis for more reliable patient monitoring.

  19. Dual-use micromechanical inertial sensors

    Science.gov (United States)

    Elwell, John M., Jr.

    1995-03-01

    A new industry, which will provide low-cost silicon-based inertial sensors to the commercial and military markets. is being created. Inertial measurement units are used extensively in military systems, and new versions are expected to find their way into commercial products, such as automobiles, as production costs fall as technology advances. An automotive inertial measurement unit can be expected to perform a complete range of control, diagnostic, and navigation functions. These functions are expected to provide significant active safety, performance, comfort, convenience, and fuel economy advantages to the automotive consumer. An inertial measurement unit applicable to the automobile industry would meet many of the performance requirements for the military in important areas, such as antenna and image stabilization, autopilot control, and the guidance of smart weapons. Such a new industrial base will significantly reduce the acquisition cost of many future tactical weapons systems. An alliance, consisting of the Charles Stark Draper Laboratory and Rockwell International, has been created to develop inertial products for this new industry.

  20. Dual cameras acquisition and display system of retina-like sensor camera and rectangular sensor camera

    Science.gov (United States)

    Cao, Nan; Cao, Fengmei; Lin, Yabin; Bai, Tingzhu; Song, Shengyu

    2015-04-01

    For a new kind of retina-like senor camera and a traditional rectangular sensor camera, dual cameras acquisition and display system need to be built. We introduce the principle and the development of retina-like senor. Image coordinates transformation and interpolation based on sub-pixel interpolation need to be realized for our retina-like sensor's special pixels distribution. The hardware platform is composed of retina-like senor camera, rectangular sensor camera, image grabber and PC. Combined the MIL and OpenCV library, the software program is composed in VC++ on VS 2010. Experience results show that the system can realizes two cameras' acquisition and display.

  1. Dual frequency cavitation event sensor with iodide dosimeter.

    Science.gov (United States)

    Ebrahiminia, Ali; Mokhtari-Dizaji, Manijhe; Toliyat, Tayebeh

    2016-01-01

    The inertial cavitation activity depends on the sonication parameters. The purpose of this work is development of dual frequency inertial cavitation meter for therapeutic applications of ultrasound waves. In this study, the chemical effects of sonication parameters in dual frequency sonication (40 kHz and 1 MHz) were investigated in the progressive wave mode using iodide dosimetry. For this purpose, efficacy of different exposure parameters such as intensity, sonication duration, sonication mode, duty factor and net ultrasound energy on the inertial cavitation activity have been studied. To quantify cavitational effects, the KI dosimeter solution was sonicated and its absorbance at a wavelength of 350 nm was measured. The absorbance values in continuous sonication mode was significantly higher than the absorbance corresponding to the pulsed mode having duty factors of 20-80% (plevel intensity (sensor can be useful for ultrasonic treatments.

  2. Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity.

    Directory of Open Access Journals (Sweden)

    Jeffrey Klarenbeek

    Full Text Available Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio. The design is based on mTurquoise2, currently the brightest and most bleaching-resistant donor, and a new acceptor cassette that consists of a tandem of two cp173Venus fluorophores. We also report variants with a single point mutation, Q270E, in the Epac moiety, which decreases the dissociation constant of cAMP from 9.5 to 4 μM, and thus increases the affinity ~ 2.5-fold. Finally, we also prepared and characterized dedicated variants with non-emitting (dark acceptors for single-wavelength FLIM acquisition that display an exceptional near-doubling of fluorescence lifetime upon saturation of cAMP levels. We believe this generation of cAMP outperforms all other sensors and therefore recommend these sensors for all future studies.

  3. Survey of high pressure using dual cora sensors and the differentiator electric bridge

    Science.gov (United States)

    Sun, Cheng-Yuan

    1994-03-01

    In this paper, the possible survey of a high-pressure field by using dual core fiber sensors and a differentiator electric bridge is described. Also, the interaction between high-pressure field and a dual core fiber nonlinear coupler is discussed, using the analyzed theory. We have obtained the formula of the theory and some correlative experimental results. We can measure high-pressure (2.2 * 106 V) by using dual core fiber sensors and a differentiator bridge.

  4. Dual-Beam Antenna Design for Autonomous Sensor Network Applications

    Directory of Open Access Journals (Sweden)

    Jean-Marie Floc'h

    2012-01-01

    Full Text Available This paper describes our contribution in the ANR project called CAPNET dedicated to the site security (autonomous sensor network. The network is autonomous in term of energy and it is very easy to deploy on the site (the time to deploy each node of the network is around 10 minutes. The first demonstrator was deployed in the fire base station of Brest, France with 10 nodes with a security perimeter around 1.5 km. Our contribution takes place in the field of antennas, with the development of two systems: a single-beam antenna reserved for the supervisor or the last node of the network, and a dual-beam antenna dedicated to the node in linear configuration. For the design and optimization of antennas, we use HFSS CAD software from ANSOFT. The antennas have been designed and successfully measured.

  5. Analysis of observations with dual sensor superconducting gravimeters

    Science.gov (United States)

    Kroner, C.; Dierks, O.; Neumeyer, J.; Wilmes, H.

    2005-12-01

    Among the 21 superconducting gravimeters presently operating worldwide four instruments exist that are equipped with two vertically aligned sensor units. Three of the instruments are installed in Germany (Bad Homburg, Moxa, Wettzell) and one in South Africa (Sutherland). Comparisons of the data sets obtained with the dual sensor systems yield information on instrumental effects and sensitivity as well as on the efficiency of reductions of environmental effects applied to the data. The latter is an important constraint when looking for small geodynamic signals like Slichter and core modes or aperiodic variations. From analyses of the two data sets of each instrument a small but significant difference of 1-3% in the response of the sensor units on barometric pressure variations is found. Likewise, the records of lower and upper sensor vary slightly but not systematically with regard to the noise levels in the different frequency ranges. The tidal analyses yield an agreement of the tidal parameters generally well within the standard deviations determined from the least squares adjustment in the tidal analysis. The deviations are in the range between 0×10-4 and 3×10-4 for the amplitude factor and the phases differ between 0.0005° and 0.01° for the four main tidal constituents O1, K1, M2, and S2. The comparison of the gravity residuals of the two sensors with each other as well as with their sum and difference in the time and frequency domain shows the existence of identical signals in the records of the two sensors in the whole range of observation. This probably means that either the environmental reductions applied are not sufficient or there are additional disturbing effects in the data which have not been taken care of yet. From the study it emerges that it is not possible to get entirely rid of the tidal signals in the data. This is probably also due to the fact that despite reductions the data sets contain additional signals and slightly different noise at

  6. Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.

    Directory of Open Access Journals (Sweden)

    Andrew Woehler

    Full Text Available We have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+], [cAMP], and PKA activity.

  7. Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.

    Directory of Open Access Journals (Sweden)

    Bobin George Abraham

    Full Text Available Fluorescence Resonance Energy Transfer (FRET using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM.

  8. Dual Matter-Wave Inertial Sensors in Weightlessness

    CERN Document Server

    Barrett, Brynle; Chichet, Laure; Battelier, Baptiste; Lévèque, Thomas; Landragin, Arnaud; Bouyer, Philippe

    2016-01-01

    Quantum technology based on cold-atom interferometers is showing great promise for fields such as inertial sensing and fundamental physics. However, the best precision achievable on Earth is limited by the free-fall time of the atoms, and their full potential can only be realized in Space where interrogation times of many seconds will lead to unprecedented sensitivity. Various mission scenarios are presently being pursued which plan to implement matter-wave inertial sensors. Toward this goal, we realize the first onboard operation of simultaneous $^{87}$Rb $-$ $^{39}$K interferometers in the weightless environment produced during parabolic flight. The large vibration levels ($10^{-2}~g/\\sqrt{\\rm Hz}$), acceleration range ($0-1.8~g$) and rotation rates ($5$ deg/s) during flight present significant challenges. We demonstrate the capability of our dual-quantum sensor by measuring the E\\"{o}tv\\"{o}s parameter with systematic-limited uncertainties of $1.1 \\times 10^{-3}$ and $3.0 \\times 10^{-4}$ during standard- a...

  9. Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

    Science.gov (United States)

    Miyamoto, Akitoshi; Miyauchi, Hiroshi; Kogure, Takako; Miyawaki, Atsushi; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2015-04-24

    Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction.

  10. Dual permeability FEM models for distributed fiber optic sensors development

    Science.gov (United States)

    Aguilar-López, Juan Pablo; Bogaard, Thom

    2017-04-01

    Fiber optic cables are commonly known for being robust and reliable mediums for transferring information at the speed of light in glass. Billions of kilometers of cable have been installed around the world for internet connection and real time information sharing. Yet, fiber optic cable is not only a mean for information transfer but also a way to sense and measure physical properties of the medium in which is installed. For dike monitoring, it has been used in the past for detecting inner core and foundation temperature changes which allow to estimate water infiltration during high water events. The DOMINO research project, aims to develop a fiber optic based dike monitoring system which allows to directly sense and measure any pore pressure change inside the dike structure. For this purpose, questions like which location, how many sensors, which measuring frequency and which accuracy are required for the sensor development. All these questions may be initially answered with a finite element model which allows to estimate the effects of pore pressure change in different locations along the cross section while having a time dependent estimation of a stability factor. The sensor aims to monitor two main failure mechanisms at the same time; The piping erosion failure mechanism and the macro-stability failure mechanism. Both mechanisms are going to be modeled and assessed in detail with a finite element based dual permeability Darcy-Richards numerical solution. In that manner, it is possible to assess different sensing configurations with different loading scenarios (e.g. High water levels, rainfall events and initial soil moisture and permeability conditions). The results obtained for the different configurations are later evaluated based on an entropy based performance evaluation. The added value of this kind of modelling approach for the sensor development is that it allows to simultaneously model the piping erosion and macro-stability failure mechanisms in a time

  11. Autonomous sensor-based dual-arm satellite grappling

    Science.gov (United States)

    Wilcox, Brian; Tso, Kam; Litwin, Todd; Hayati, Samad; Bon, Bruce

    1989-01-01

    Dual-arm satellite grappling involves the integration of technologies developed in the Sensing and Perception (S&P) Subsystem for object acquisition and tracking, and the Manipulator Control and Mechanization (MCM) Subsystem for dual-arm control. S&P acquires and tracks the position, orientation, velocity, and angular velocity of a slowly spinning satellite, and sends tracking data to the MCM subsystem. MCM grapples the satellite and brings it to rest, controlling the arms so that no excessive forces or torques are exerted on the satellite or arms. A 350-pound satellite mockup which can spin freely on a gimbal for several minutes, closely simulating the dynamics of a real satellite is demonstrated. The satellite mockup is fitted with a panel under which may be mounted various elements such as line replacement modules and electrical connectors that will be used to demonstrate servicing tasks once the satellite is docked. The subsystems are housed in three MicroVAX II microcomputers. The hardware of the S&P Subsystem includes CCD cameras, video digitizers, frame buffers, IMFEX (a custom pipelined video processor), a time-code generator with millisecond precision, and a MicroVAX II computer. Its software is written in Pascal and is based on a locally written vision software library. The hardware of the MCM Subsystem includes PUMA 560 robot arms, Lord force/torque sensors, two MicroVAX II computers, and unimation pneumatic parallel grippers. Its software is written in C, and is based on a robot language called RCCL. The two subsystems are described and test results on the grappling of the satellite mockup with rotational rates of up to 2 rpm are provided.

  12. Reliable dual-redundant sensor failure detection and identification for the NASA F-8 DFBW aircraft

    Science.gov (United States)

    Deckert, J. C.; Desai, M. N.; Deyst, J. J., Jr.; Willsky, A. S.

    1978-01-01

    A technique was developed which provides reliable failure detection and identification (FDI) for a dual redundant subset of the flight control sensors onboard the NASA F-8 digital fly by wire (DFBW) aircraft. The technique was successfully applied to simulated sensor failures on the real time F-8 digital simulator and to sensor failures injected on telemetry data from a test flight of the F-8 DFBW aircraft. For failure identification the technique utilized the analytic redundancy which exists as functional and kinematic relationships among the various quantities being measured by the different control sensor types. The technique can be used not only in a dual redundant sensor system, but also in a more highly redundant system after FDI by conventional voting techniques reduced to two the number of unfailed sensors of a particular type. In addition the technique can be easily extended to the case in which only one sensor of a particular type is available.

  13. First-Order Acoustic Wave Equation Reverse Time Migration Based on the Dual-Sensor Seismic Acquisition System

    Science.gov (United States)

    You, Jiachun; Liu, Xuewei; Wu, Ru-Shan

    2017-03-01

    We analyze the mathematical requirements for conventional reverse time migration (RTM) and summarize their rationale. The known information provided by current acquisition system is inadequate for the second-order acoustic wave equations. Therefore, we introduce a dual-sensor seismic acquisition system into the coupled first-order acoustic wave equations. We propose a new dual-sensor reverse time migration called dual-sensor RTM, which includes two input variables, the pressure and vertical particle velocity data. We focus on the performance of dual-sensor RTM in estimating reflection coefficients compared with conventional RTM. Synthetic examples are used for the study of estimating coefficients of reflectors with both dual-sensor RTM and conventional RTM. The results indicate that dual-sensor RTM with two inputs calculates amplitude information more accurately and images structural positions of complex substructures, such as the Marmousi model, more clearly than that of conventional RTM. This shows that the dual-sensor RTM has better accuracy in backpropagation and carries more information in the directivity because of particle velocity injection. Through a simple point-shape model, we demonstrate that dual-sensor RTM decreases the effect of multi-pathing of propagating waves, which is helpful for focusing the energy. In addition, compared to conventional RTM, dual-sensor RTM does not cause extra memory costs. Dual-sensor RTM is, therefore, promising for the computation of multi-component seismic data.

  14. Composed planar Hall effect sensors with dual-mode operation

    OpenAIRE

    Vladislav Mor; Debangsu Roy; Moty Schultz; Lior Klein

    2016-01-01

    We present a composed planar Hall effect sensor with two modes of operation: (a) an ON mode where the composed sensor responds to magnetic field excitations similarly to the response of a regular planar Hall effect sensor, and (b) an OFF mode where the response is negligible. The composed planar Hall effect sensor switches from the OFF mode to the ON mode when it is exposed to a magnetic field which exceeds a certain threshold determined by the sensor design. The features of this sensor make ...

  15. 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.

  16. Large Scale Bacterial Colony Screening of Diversified FRET Biosensors.

    Directory of Open Access Journals (Sweden)

    Julia Litzlbauer

    Full Text Available Biosensors based on Förster Resonance Energy Transfer (FRET between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors.

  17. A fluorescence resonance energy transfer (FRET) based "Turn-On" nanofluorescence sensor using a nitrogen-doped carbon dot-hexagonal cobalt oxyhydroxide nanosheet architecture and application to α-glucosidase inhibitor screening.

    Science.gov (United States)

    Li, Guoliang; Kong, Weiheng; Zhao, Mei; Lu, Shuaimin; Gong, Peiwei; Chen, Guang; Xia, Lian; Wang, Hua; You, Jinmao; Wu, Yongning

    2016-05-15

    The medicines targeted at α-glucosidase played an important role in anti-diabetes and anti-HIV therapy. Unfortunately, the method based on fluorescent assay strategy for α-glucosidase inhibitor screening remains poorly investigated. In this study, a novel "Turn On" fluorescence sensor platform has been developed for trace α-glucosidase inhibitor screening from natural medicines. Firstly, carbon dots were prepared by one-pot synthesis and used as the signal output. Combining with the carbon dots, cobalt oxyhydroxide (CoOOH) nanoflakes were employed to build the fluorescence resonance energy transfer (FRET) based sensor platform. Secondly, L-ascorbic acid-2-O-α-D-glucopyranosyl (AAG) was innovatively introduced as α-glucosidase substrate. With hydrolysis of AAG by α-glucosidase, ascorbic acids (AA) were released that can rapidly reduce CoOOH nanoflakes to Co(2+), and then FRET was stopped accompanying with the fluorescence recovery of CDs. The sensor platform was ultrasensitive to AA with a detection limit of 5 nM, ensuring the sensitive monitoring of enzyme activity. Acarbose was used as the inhibitor model and its inhibition rate is proportional to the logarithm of concentration in range of 10(-9)-10(-3)M with the correlation coefficient of R(2)=0.996, and an ultralow limit of detection of ~1×10(-9)M was obtained. The inhibiting ability of seven compounds isolated from natural medicines was also evaluated. The constructed sensor platform was proven to be sensitive and selective as well as cost-effective, facile and reliable, making it promising as a candidate for trace α-glucosidase inhibitor screening.

  18. Composed planar Hall effect sensors with dual-mode operation

    Science.gov (United States)

    Mor, Vladislav; Roy, Debangsu; Schultz, Moty; Klein, Lior

    2016-02-01

    We present a composed planar Hall effect sensor with two modes of operation: (a) an ON mode where the composed sensor responds to magnetic field excitations similarly to the response of a regular planar Hall effect sensor, and (b) an OFF mode where the response is negligible. The composed planar Hall effect sensor switches from the OFF mode to the ON mode when it is exposed to a magnetic field which exceeds a certain threshold determined by the sensor design. The features of this sensor make it useful as a switch triggered by magnetic field and as a sensing device with memory, as its mode of operation indicates exposure to a magnetic field larger than a certain threshold without the need to be activated during the exposure itself.

  19. Composed planar Hall effect sensors with dual-mode operation

    Directory of Open Access Journals (Sweden)

    Vladislav Mor

    2016-02-01

    Full Text Available We present a composed planar Hall effect sensor with two modes of operation: (a an ON mode where the composed sensor responds to magnetic field excitations similarly to the response of a regular planar Hall effect sensor, and (b an OFF mode where the response is negligible. The composed planar Hall effect sensor switches from the OFF mode to the ON mode when it is exposed to a magnetic field which exceeds a certain threshold determined by the sensor design. The features of this sensor make it useful as a switch triggered by magnetic field and as a sensing device with memory, as its mode of operation indicates exposure to a magnetic field larger than a certain threshold without the need to be activated during the exposure itself.

  20. IIem-spFRET: improved Iem-spFRET method for robust FRET measurement

    Science.gov (United States)

    Zhang, Jiang; Lin, Fangrui; Chai, Liuying; Wei, Lichun; Chen, Tongsheng

    2016-10-01

    We recently developed a quantitative Förster resonance energy transfer (FRET) measurement method based on emission-spectral unmixing (Iem-spFRET). We here developed an improved Iem-spFRET method (termed as IIem-spFRET) for more robust FRET measurement in living cells. First, two background (BG) spectral fingerprints measured from blank living cells are introduced to remove BG and autofluorescence. Second, we introduce a ρ factor denoting the ratio of two molar extinction coefficient ratios (γ) of acceptor to donor at two excitations into IIem-spFRET for direct measurement of the γ values using a tandem construct with unknown FRET efficiency (E). We performed IIem-spFRET on our microscope-spectrometer platform to measure the γ values of Venus (V) to Cerulean (C) and the E values of C32V, CVC, VCV, and VCVV constructs, respectively, in living Huh7 cells. For the C32V or CVC cells, the Iem-spFRET and IIem-spFRET methods measured consistent E values. However, for the cells especially with low expressing levels of VCV or VCVV, the E values measured by Iem-spFRET showed large deviations and fluctuations, whereas the IIem-spFRET method greatly improved the measured E values. Collectively, IIem-spFRET is a powerful and robust tool for quantitatively measuring FRET signal in living cells.

  1. CD/AuNPs/MWCNTs based electrochemical sensor for quercetin dual-signal detection.

    Science.gov (United States)

    Kan, Xianwen; Zhang, Tingting; Zhong, Min; Lu, Xiaojing

    2016-03-15

    A dual-signal strategy was developed in the present work for quercetin (QR) electrochemical recognition and detection. Mercapto-β-cyclodextrin (HS-β-CD) self-assembled on gold nanoparticles and multi-walled carbon nanotubes modified electrode surface to fabricate an electrochemical sensor. Scanning electron microscope, electrochemical impedance spectroscopy, and cyclic voltammetry were employed to characterize the preparation process of the sensor. Hydroquinone (HQ) was chosen as an electrochemical marker for QR detection due to its small molecular size for the formation of inclusion with HS-β-CD. The results of UV-vis and differential pulse voltammetry demonstrate that the added QR can replace the included HQ in CD cavities, resulting in the dual-signal in electrochemical experiments composed of the decrease of oxidized current of HQ and the increase of oxidized current of QR. Compared with the sensor for QR detection in the absence of HQ, the sensor based dual-signal strategy exhibited a higher sensitivity with a wider detection range from 5.0 × 10(-9) to 7.0 × 10(-6)mol/L. With good selectivity, reproducibility, and stability, the sensor was applied for real samples detection with satisfactory results. The proposed dual-signal strategy can be readily extended to the selective recognition and sensitive detection of other molecules.

  2. A dual-mode proximity sensor with integrated capacitive and temperature sensing units

    Science.gov (United States)

    Qiu, Shihua; Huang, Ying; He, Xiaoyue; Sun, Zhiguang; Liu, Ping; Liu, Caixia

    2015-10-01

    The proximity sensor is one of the most important devices in the field of robot application. It can accurately provide the proximity information to assistant robots to interact with human beings and the external environment safely. In this paper, we have proposed and demonstrated a dual-mode proximity sensor composed of capacitive and resistive sensing units. We defined the capacitive type proximity sensor perceiving the proximity information as C-mode and the resistive type proximity sensor detecting as R-mode. Graphene nanoplatelets (GNPs) were chosen as the R-mode sensing material because of its high performance. The dual-mode proximity sensor presents the following features: (1) the sensing distance of the dual-mode proximity sensor has been enlarged compared with the single capacitive proximity sensor in the same geometrical pattern; (2) experiments have verified that the proposed sensor can sense the proximity information of different materials; (3) the proximity sensing capability of the sensor has been improved by two modes perceive collaboratively, for a plastic block at a temperature of 60 °C: the R-mode will perceive the proximity information when the distance d between the sensor and object is 6.0-17.0 mm and the C-mode will do that when their interval is 0-2.0 mm additionally two modes will work together when the distance is 2.0-6.0 mm. These features indicate our transducer is very valuable in skin-like sensing applications.

  3. A Three-Axis Force Sensor for Dual Finger Haptic Interfaces

    Directory of Open Access Journals (Sweden)

    Fabio Salsedo

    2012-10-01

    Full Text Available In this work we present the design process, the characterization and testing of a novel three-axis mechanical force sensor. This sensor is optimized for use in closed-loop force control of haptic devices with three degrees of freedom. In particular the sensor has been conceived for integration with a dual finger haptic interface that aims at simulating forces that occur during grasping and surface exploration. The sensing spring structure has been purposely designed in order to match force and layout specifications for the application. In this paper the design of the sensor is presented, starting from an analytic model that describes the characteristic matrix of the sensor. A procedure for designing an optimal overload protection mechanism is proposed. In the last part of the paper the authors describe the experimental characterization and the integrated test on a haptic hand exoskeleton showing the improvements in the controller performances provided by the inclusion of the force sensor.

  4. Rational design, synthesis of reaction-based dual-channel cyanide sensor in aqueous solution

    Science.gov (United States)

    Li, Jun-Jian; Wei, Wei; Qi, Xiao-Liang; Xu, Xiao; Liu, Yu-Cheng; Lin, Qiu-Han; Dong, Wei

    2016-01-01

    A new dual-channel sensor for the detection of cyanide was developed based on the conjugated of naphthalene and malononitrile. Upon the addition of CN-, the sensor displayed very large blue-shift in both fluorescence (80 nm) and absorption (120 nm) spectra. The sensor of cyanide was performed via the nucleophilic attack of cyanide anion to vinylic groups of the sensor with a 1:1 binding stoichiometry and the color changed of the sensor is mainly due to the intramolecular charge transfer process improvement. The intramolecular charge transfer progress was blocked with color changed and fluorescence blue-shift. The mechanism of sensor reaction with CN- ion was studied using 1H NMR and mass spectrometry.

  5. Fretting of AISI 9310 and selected fretting resistant surface treatments

    Science.gov (United States)

    Bill, R. C.

    1977-01-01

    Fretting wear experiments were conducted with uncoated AISI 9310 mating surfaces, and with combinations incorporating a selected coating to one of the mating surfaces. Wear measurements and SEM observations indicated that surface fatigue, as made evident by spallation and surface crack formation, is an important mechanism in promoting fretting wear to uncoated 9310. Increasing humidity resulted in accelerated fretting, and a very noticeable difference in nature of the fretting debris. Of the coatings evaluated, aluminum bronze with a polyester additive was most effective at reducing wear and minimizing fretting damage to the mating uncoated surface, by means of a selflubricating film that developed on the fretting surfaces. Chromium plate performed as an effective protective coating, itself resisting fretting and not accelerating damage to the uncoated surface.

  6. From Dual-Mode Triboelectric Nanogenerator to Smart Tactile Sensor: A Multiplexing Design.

    Science.gov (United States)

    Li, Tao; Zou, Jingdian; Xing, Fei; Zhang, Meng; Cao, Xia; Wang, Ning; Wang, Zhong Lin

    2017-04-25

    Triboelectric nanogenerators (TENGs) can be applied for the next generation of artificial intelligent products, where skin-like tactile sensing advances the ability of robotics to feel and interpret environment. In this paper, a flexible and thin tactile sensor was developed on the basis of dual-mode TENGs. The effective transduction of touch and pressure stimulus into independent and interpretable electrical signals permits the instantaneous sensing of location and pressure with a plane resolution of 2 mm, a high-pressure-sensing sensitivity up to 28 mV·N(-1), and a linear pressure detection ranging from 40 to 140 N. Interestingly, this self-powered dual-mode sensor can even interpret contact and hardness of objects by analyzing the shape of the current peak, which makes this low-cost TENG-based sensor promising for applications in touch screens, electronic skins, healthcare, and environmental survey.

  7. Theoretical modeling of a self-referenced dual mode SPR sensor utilizing indium tin oxide film

    Science.gov (United States)

    Srivastava, Sachin K.; Verma, Roli; Gupta, Banshi D.

    2016-06-01

    A prism based dual mode SPR sensor was theoretically modeled to work as a self-referenced sensor in spectral interrogation scheme. Self-referenced sensing was achieved by sandwiching an indium tin oxide thin film in between the prism base and the metal layer. The proposed sensor possesses two plasmon modes similar to long and short range SPRs (LR- and SR-SPRs) and we have analogically used LRSPR and SRSPR for them. However, these modes do not possess usual long range character due to the losses introduced by the imaginary part of indium tin oxide (ITO) dielectric function. One of the two plasmon modes responds to change in analyte refractive index while the other remains fixed. The influence of various design parameters on the performance of the sensor was evaluated. The performance of the proposed sensor was compared, via control simulations, with established dual mode geometries utilizing silicon dioxide (SiO2), Teflon AF-1600 and Cytop. The design parameters of the established geometries were optimized to obtain self-referenced sensing operation. Trade-offs between the resonance spectral width, minimum reflectivity, shift in resonance wavelength and angle of incidence were examined for optimal design. The present study will be useful in the fabrication of self-referenced sensors where the ambient conditions are not quite stable.

  8. Highly sensitive label-free dual sensor array for rapid detection of wound bacteria.

    Science.gov (United States)

    Sheybani, Roya; Shukla, Anita

    2017-06-15

    Wound infections are a critical healthcare concern worldwide. Rapid and effective antibiotic treatments that can mitigate infection severity and prevent the spread of antibiotic resistance are contingent upon timely infection detection. In this work, dual electrochemical pH and cell-attachment sensor arrays were developed for the real-time spatial and temporal monitoring of potential wound infections. Biocompatible polymeric device coatings were integrated to stabilize the sensors and promote bacteria attachment while preventing non-specific cell and protein fouling. High sensitivity (bacteria concentration of 10(2) colony forming units (CFU)/mL and -88.1±6.3mV/pH over a pH range of 1-13) and stability over 14 days were achieved without the addition of biological recognition elements. The dual sensor array was demonstrated to successfully monitor the growth of both gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) over time through lag and log growth phases and following antibiotic administration and in simulated shallow wounds conditions. The versatile fabrication methods utilized in sensor development, superior sensitivity, prolonged stability, and lack of non-specific sensor fouling may enable long-term in situ sensor array operation in low resource settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Dual resonant frequencies effects on an induction-based oil palm fruit sensor.

    Science.gov (United States)

    Harun, Noor Hasmiza; Misron, Norhisam; Mohd Sidek, Roslina; Aris, Ishak; Wakiwaka, Hiroyuki; Tashiro, Kunihisa

    2014-11-19

    As the main exporter in the oil palm industry, the need to improve the quality of palm oil has become the main interest among all the palm oil millers in Malaysia. To produce good quality palm oil, it is important for the miller to harvest a good oil palm Fresh Fruit Bunch (FFB). Conventionally, the main reference used by Malaysian harvesters is the manual grading standard published by the Malaysian Palm Oil Board (MPOB). A good oil palm FFB consists of all matured fruitlets, aged between 18 to 21 weeks of antheses (WAA). To expedite the harvesting process, it is crucial to implement an automated detection system for determining the maturity of the oil palm FFB. Various automated detection methods have been proposed by researchers in the field to replace the conventional method. In our preliminary study, a novel oil palm fruit sensor to detect the maturity of oil palm fruit bunch was proposed. The design of the proposed air coil sensor based on the inductive sensor was further investigated mainly in the context of the effect of coil diameter to improve its sensitivity. In this paper, the sensitivity of the inductive sensor was further examined with a dual flat-type shape of air coil. The dual air coils were tested on fifteen samples of fruitlet from two categories, namely ripe and unripe. Samples were tested within 20 Hz to 10 MHz while evaluations on both peaks were done separately before the gap between peaks was analyzed. A comparative analysis was conducted to investigate the improvement in sensitivity of the induction-based oil palm fruit sensor as compared to previous works. Results from the comparative study proved that the inductive sensor using a dual flat-type shape air coil has improved by up to 167%. This provides an indication in the improvement in the coil sensitivity of the palm oil fruit sensor based on the induction concept.

  10. Dual Resonant Frequencies Effects on an Induction-Based Oil Palm Fruit Sensor

    Directory of Open Access Journals (Sweden)

    Noor Hasmiza Harun

    2014-11-01

    Full Text Available As the main exporter in the oil palm industry, the need to improve the quality of palm oil has become the main interest among all the palm oil millers in Malaysia. To produce good quality palm oil, it is important for the miller to harvest a good oil palm Fresh Fruit Bunch (FFB. Conventionally, the main reference used by Malaysian harvesters is the manual grading standard published by the Malaysian Palm Oil Board (MPOB. A good oil palm FFB consists of all matured fruitlets, aged between 18 to 21 weeks of antheses (WAA. To expedite the harvesting process, it is crucial to implement an automated detection system for determining the maturity of the oil palm FFB. Various automated detection methods have been proposed by researchers in the field to replace the conventional method. In our preliminary study, a novel oil palm fruit sensor to detect the maturity of oil palm fruit bunch was proposed. The design of the proposed air coil sensor based on the inductive sensor was further investigated mainly in the context of the effect of coil diameter to improve its sensitivity. In this paper, the sensitivity of the inductive sensor was further examined with a dual flat-type shape of air coil. The dual air coils were tested on fifteen samples of fruitlet from two categories, namely ripe and unripe. Samples were tested within 20 Hz to 10 MHz while evaluations on both peaks were done separately before the gap between peaks was analyzed. A comparative analysis was conducted to investigate the improvement in sensitivity of the induction-based oil palm fruit sensor as compared to previous works. Results from the comparative study proved that the inductive sensor using a dual flat-type shape air coil has improved by up to 167%. This provides an indication in the improvement in the coil sensitivity of the palm oil fruit sensor based on the induction concept.

  11. A flexible dual-mode proximity sensor based on cooperative sensing for robot skin applications

    Science.gov (United States)

    Huang, Ying; Cai, Xia; Kan, Wenqing; Qiu, Shihua; Guo, Xiaohui; Liu, Caixia; Liu, Ping

    2017-08-01

    A flexible dual-mode proximity sensor has been designed and implemented, which is capable of combining capacitive-resistive detection in this paper. The capacitive type proximity sensor detecting is defined as mode-C, and the resistive type proximity sensor detecting is defined as mode-R. The characteristics of the proximity sensor are as follows: (1) the theoretical mode is developed which indicates that this proximity sensor can reflect proximity information accurately; (2) both sensing modes are vertically integrated into a sandwich-like chip with an 8 mm × 12 mm unit area. The thickness of a mode-R sensing material (graphene nanoplatelets) and mode-C dielectric (the mixture of carbon black and silicone rubber) is 1 mm and 2.5 mm, respectively; (3) for mode-R, the linearity of temperature-resistance curve can achieve 0.998 in the temperature range from 25°C to 65°C. And for mode-C, various materials can be successfully detected with fast response and high reversibility. Meanwhile, the study compensated the influence of object temperature to ensure mode-C properly works. A cooperative sensing test shows that R-C dual modes sense effectively which can enlarge the sensing distance compared with the single mode proximity sensor. The fabrication of this sensor is convenient, and the integrity of a flexible sandwich-like structure based on dual modes is beneficial to form arrays, which is suitable to be used in skin-like sensing applications.

  12. Affordable dual-sensing proximity sensor for touchless interactive systems

    KAUST Repository

    Nassar, Joanna M.

    2016-09-13

    We report an ultra-low cost flexible proximity sensor using only off-the-shelf recyclable materials such as aluminum foil, napkin and double-sided tape. Unlike previous reports, our device structure exhibits two sensing capabilities in one platform, with outstanding long detection range of 20 cm and pressure sensitivity of 0.05 kPa. This is the first ever demonstration of a low-cost, accessible, and batch manufacturing process for pressure and proximity sensing on a singular platform. The mechanical flexibility of the sensor makes it possible to mount on various irregular platforms, which is vital in many areas, such as robotics, machine automation, vehicular technology and inspection tools.

  13. A fluorescence ratiometric sensor for hypochlorite based on a novel dual-fluorophore response approach.

    Science.gov (United States)

    Long, Lingliang; Zhang, Dongdong; Li, Xiufen; Zhang, Jinfang; Zhang, Chi; Zhou, Liping

    2013-05-02

    A fluorescence ratiometric sensor for OCl(-) has been developed based on a novel dual fluorophore response approach. The sensor molecule contains a coumarin fluorophore and a rhodamine fluorophore, and the two fluorophores are directly linked to an OCl(-) recognition group. The structure of the sensor was characterized by ESI-MS, NMR, and X-ray crystallographic analysis. Upon treatment with OCl(-), both fluorophores in the sensor responded simultaneously at two separate optical windows, with large enhancement of the fluorescence ratio (I578/I501) from 0.01 to 39.55. The fluorescence ratios for the sensor showed a good linearity with the concentration of OCl(-) in the range of 0.2-40 μM and the detection limits is 0.024 μM (SN(-1)=3). Investigation of reaction products indicated that the sensor reaction with OCl(-) produced two new fluorescent molecules, which were responsible for the fluorescence changes in two optical windows. In addition, the sensor showed high selectivity to OCl(-) over other reactive oxygen species, reactive nitrogen species, cations, and anions. The sensor has also been successfully applied to detection of OCl(-) in natural water samples with satisfactory recovery.

  14. Evaluation of the prototype dual-axis wall attitude measurement sensor

    Science.gov (United States)

    Wong, Douglas T.

    1994-01-01

    A prototype dual-axis electrolytic tilt sensor package for angular position measurements was built and evaluated in a laboratory environment. The objective was to investigate the use of this package for making wind tunnel wall attitude measurements for the National Transonic Facility (NTF) at NASA Langley Research Center (LaRC). The instrumentation may replace an existing, more costly, and less rugged servo accelerometer package (angle-of-attack package) currently in use. The dual-axis electrolytic tilt sensor package contains two commercial electrolytic tilt sensors thermally insulated with NTF foam, all housed within a stainless steel package. The package is actively heated and maintained at 160 F using foil heating elements. The laboratory evaluation consisted of a series of tests to characterize the linearity, repeatability, cross-axis interaction, lead wire effect, step response, thermal time constant, and rectification errors. Tests revealed that the total RMS errors for the x-axis sensor is 0.084 degree, and 0.182 degree for the y-axis sensor. The RMS errors are greater than the 0.01 degree specification required for NTF wall attitude measurements. It is therefore not a viable replacement for the angle-of-attack package in the NTF application. However, with some physical modifications, it can be used as an inexpensive 5-degree range dual-axis inclinometer with overall accuracy approaching 0.01 degree under less harsh environments. Also, the data obtained from the tests can be valuable for wind tunnel applications of most types of electrolytic tilt sensors.

  15. Single-fluorophore membrane transport activity sensors with dual-emission read-out.

    Science.gov (United States)

    Ast, Cindy; De Michele, Roberto; Kumke, Michael U; Frommer, Wolf B

    2015-06-19

    We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer is important for the successful generation of single-fluorophore-based dual-emission sensors.

  16. FRET-based dual-emission and pH-responsive nanocarriers for enhanced delivery of protein across intestinal epithelial cell barrier.

    Science.gov (United States)

    Lu, Kun-Ying; Lin, Cheng-Wei; Hsu, Chun-Hua; Ho, Yi-Cheng; Chuang, Er-Yuan; Sung, Hsing-Wen; Mi, Fwu-Long

    2014-10-22

    The oral route is a convenient and commonly employed way for drug delivery. However, therapeutic proteins have poor bioavailability upon oral administration due to the impermeable barrier from intestinal epithelial tight junction (TJ). Moreover, the pH of the small intestine varies among different regions of the intestinal tract where digestion and absorption occur at different levels. In this study, a tunable dual-emitting and pH-responsive nanocarrier that can alter the fluorescent color and emission intensity in response to pH changes and can trigger the opening of intestinal epithelial TJ at different levels were developed from chitosan-N-arginine and poly(γ-glutamic acid)-taurine conjugates. As pH increased from 6.0 to 8.0, the binding affinity of the oppositely charged polyions decreased, whereas the ratio of the intensity of the donor-to-acceptor emission intensity (ID/IA) increased by 27-fold. The fluorescent and pH-responsive nanocarrier was able to monitor the pH change of intestinal environment and to control the release of an anti-angiogenic protein in response to the pH gradient. The nanocarrier triggered the opening of intestinal epithelial TJ and consequently enhanced the permeation of the released protein through the intestinal epithelial barrier model (Caco-2 cell monolayer) to inhibit tube formation of human umbilical vein endothelial cells.

  17. A communication theoretical analysis of FRET-based mobile ad hoc molecular nanonetworks.

    Science.gov (United States)

    Kuscu, Murat; Akan, Ozgur B

    2014-09-01

    Nanonetworks refer to a group of nanosized machines with very basic operational capabilities communicating to each other in order to accomplish more complex tasks such as in-body drug delivery, or chemical defense. Realizing reliable and high-rate communication between these nanomachines is a fundamental problem for the practicality of these nanonetworks. Recently, we have proposed a molecular communication method based on Förster Resonance Energy Transfer (FRET) which is a nonradiative excited state energy transfer phenomenon observed among fluorescent molecules, i.e., fluorophores. We have modeled the FRET-based communication channel considering the fluorophores as single-molecular immobile nanomachines, and shown its reliability at high rates, and practicality at the current stage of nanotechnology. In this study, for the first time in the literature, we investigate the network of mobile nanomachines communicating through FRET. We introduce two novel mobile molecular nanonetworks: FRET-based mobile molecular sensor/actor nanonetwork (FRET-MSAN) which is a distributed system of mobile fluorophores acting as sensor or actor node; and FRET-based mobile ad hoc molecular nanonetwork (FRET-MAMNET) which consists of fluorophore-based nanotransmitter, nanoreceivers and nanorelays. We model the single message propagation based on birth-death processes with continuous time Markov chains. We evaluate the performance of FRET-MSAN and FRET-MAMNET in terms of successful transmission probability and mean extinction time of the messages, system throughput, channel capacity and achievable communication rates.

  18. Cysteine-cystine redox cycling in a gold-gold dual-plate generator-collector microtrench sensor.

    Science.gov (United States)

    Hammond, Jules L; Gross, Andrew J; Estrela, Pedro; Iniesta, Jesus; Green, Stephen J; Winlove, C Peter; Winyard, Paul G; Benjamin, Nigel; Marken, Frank

    2014-07-15

    Thiols and disulfides are ubiquitous and important analytical targets. However, their redox properties, in particular on gold sensor electrodes, are complex and obscured by strong adsorption. Here, a gold-gold dual-plate microtrench dual-electrode sensor with feedback signal amplification is demonstrated to give well-defined (but kinetically limited) steady-state voltammetric current responses for the cysteine-cystine redox cycle in nondegassed aqueous buffer media at pH 7 down to micromolar concentration levels.

  19. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Science.gov (United States)

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.

    2015-01-01

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  20. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Directory of Open Access Journals (Sweden)

    Bernhard Hochreiter

    2015-10-01

    Full Text Available Fluorescence- or Förster resonance energy transfer (FRET is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a cleavage; (b conformational-change; (c mechanical force and (d changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  1. Fluorescent proteins as genetically encoded FRET biosensors in life sciences.

    Science.gov (United States)

    Hochreiter, Bernhard; Garcia, Alan Pardo; Schmid, Johannes A

    2015-10-16

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  2. Dual matter-wave inertial sensors in weightlessness

    Science.gov (United States)

    Barrett, Brynle; Antoni-Micollier, Laura; Chichet, Laure; Battelier, Baptiste; Lévèque, Thomas; Landragin, Arnaud; Bouyer, Philippe

    2016-12-01

    Quantum technology based on cold-atom interferometers is showing great promise for fields such as inertial sensing and fundamental physics. However, the finite free-fall time of the atoms limits the precision achievable on Earth, while in space interrogation times of many seconds will lead to unprecedented sensitivity. Here we realize simultaneous 87Rb-39K interferometers capable of operating in the weightless environment produced during parabolic flight. Large vibration levels (10-2 g Hz-1/2), variations in acceleration (0-1.8 g) and rotation rates (5° s-1) onboard the aircraft present significant challenges. We demonstrate the capability of our correlated quantum system by measuring the Eötvös parameter with systematic-limited uncertainties of 1.1 × 10-3 and 3.0 × 10-4 during standard- and microgravity, respectively. This constitutes a fundamental test of the equivalence principle using quantum sensors in a free-falling vehicle. Our results are applicable to inertial navigation, and can be extended to the trajectory of a satellite for future space missions.

  3. Chemiresistive/SERS dual sensor based on densely packed gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Sanda Boca

    2015-12-01

    Full Text Available Chemiresistors are a class of sensitive electrical devices capable of detecting (biochemicals by simply monitoring electrical resistance. Sensing based on surface enhanced Raman scattering (SERS represents a radically different approach, in which molecules are optically detected according to their vibrational spectroscopic fingerprint. Despite different concepts are involved, one can find in the literature examples from both categories reporting sensors made of gold nanoparticles. The same building blocks appear because both sensor classes share a common principle: nanometric interparticle gaps are needed, for electron tunneling in chemiresistors, and for enhancing electromagnetic fields by plasmon coupling in SERS-based sensors. By exploiting such nano-gaps in self-assembled films of gold nanoparticles, we demonstrate the proof of concept of a dual electrical/optical sensor, with both chemiresistive and SERS capabilities. The proposed device is realized by self-assembling 15 nm gold nanoparticles into few micrometers-wide strips across commercially available interdigitated electrodes. The dual-mode operation of the device is demonstrated by the detection of a biologically relevant model analyte, 4-mercaptophenyl boronic acid.

  4. High Sensitivity Refractive Index Sensor Based on Dual-Core Photonic Crystal Fiber with Hexagonal Lattice

    Directory of Open Access Journals (Sweden)

    Haiyang Wang

    2016-10-01

    Full Text Available A refractive index sensor based on dual-core photonic crystal fiber (PCF with hexagonal lattice is proposed. The effects of geometrical parameters of the PCF on performances of the sensor are investigated by using the finite element method (FEM. Two fiber cores are separated by two air holes filled with the analyte whose refractive index is in the range of 1.33–1.41. Numerical simulation results show that the highest sensitivity can be up to 22,983 nm/RIU(refractive index unit when the analyte refractive index is 1.41. The lowest sensitivity can reach to 21,679 nm/RIU when the analyte refractive index is 1.33. The sensor we proposed has significant advantages in the field of biomolecule detection as it provides a wide-range of detection with high sensitivity.

  5. High Sensitivity Refractive Index Sensor Based on Dual-Core Photonic Crystal Fiber with Hexagonal Lattice.

    Science.gov (United States)

    Wang, Haiyang; Yan, Xin; Li, Shuguang; An, Guowen; Zhang, Xuenan

    2016-10-08

    A refractive index sensor based on dual-core photonic crystal fiber (PCF) with hexagonal lattice is proposed. The effects of geometrical parameters of the PCF on performances of the sensor are investigated by using the finite element method (FEM). Two fiber cores are separated by two air holes filled with the analyte whose refractive index is in the range of 1.33-1.41. Numerical simulation results show that the highest sensitivity can be up to 22,983 nm/RIU(refractive index unit) when the analyte refractive index is 1.41. The lowest sensitivity can reach to 21,679 nm/RIU when the analyte refractive index is 1.33. The sensor we proposed has significant advantages in the field of biomolecule detection as it provides a wide-range of detection with high sensitivity.

  6. Interferometric fiber-optic bending / nano-displacement sensor using plastic dual-core fiber

    CERN Document Server

    Qu, H; Skorobogatiy, M

    2014-01-01

    We demonstrate an interferometric fiber-optic bending/micro-displacement sensor based on a plastic dual-core fiber with one end coated with a silver mirror. The two fiber cores are first excited with the same laser beam, the light in each core is then back-reflected at the mirror-coated fiber-end, and, finally, the light from the two cores is made to interfere at the coupling end. Bending of the fiber leads to shifting interference fringes that can be interrogated with a slit and a single photodetector. We find experimentally that the resolution of our bending sensor is ~3x10-4 m-1 for sensing of bending curvature, as well as ~70 nm for sensing of displacement of the fiber tip. We demonstrate operation of our sensor using two examples. One is weighting of the individual micro-crystals of salt, while the other one is monitoring dynamics of isopropanol evaporation.

  7. Energy-Efficiency of Dual-Switched Branch Diversity Receiver in Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Ghaida A. AL-Suhail

    2011-12-01

    Full Text Available In this paper, we develop an analytical energy efficiency model using dual switched branch diversity receiver in wireless sensor networks in fading environments. To adapt energy efficiency of sensor node to channel variations, the optimal packet length at the data link layer is considered. Within this model, the energy efficiency can be effectively improved for switch-and-stay combiner (SSC receiver with optimal switching threshold. Moreover, to improve energy efficiency, we use error control of Bose-Chaudhuri-Hochquengh (BCH coding for SSC-BPSK receiver node compared to one of non-diversity NCFSK receiver of sensor node. The results show that the BCH code for channel coding can improve the energy efficiency significantly for long link distance and various values of high energy consumptions over Rayleigh fading channel.

  8. Deployment of dual-sensor ALIS for humanitarian demining in Cambodia

    Science.gov (United States)

    Sato, M.; Takahashi, K.

    2013-06-01

    We are in the process of developing a high-resolution landmine scanning system "ALIS" which produces horizontal slices of the shallow subsurface for visualization of buried explosives and inert clutter. As many AP mines contain minimum amounts of metal, metal detectors need to be combined with a complimentary subsurface imaging sensor. Ground Penetrating Radar (GPR) is widely accepted for subsurface sensing in the fields of geology, archaeology and utility detection. The demining application requires real-time imaging results with centimetre resolution in a highly portable package. The key requirement for sharp images of the subsurface is the precise tracking of the geophysical sensor(s) during data collection. We should also notice that GPR system is a very wide band radar system, and equivalent to UWB radar, which has recently been developed for short-range high-accuracy radar. We are testing simplified but effective signal processing for imaging mines. We are currently testing a dual sensor ALIS which is a realtime sensor tracking system based on a CCD camera and image processing. In this paper we introduce the GPR systems which we have developed for detection of buried antipersonnel mines and small size explosives. ALIS has been deployed in Cambodia since 2009 and detected more than 70 mines in mine fields, and returned more than 13ha cleaned fields to local farmers. We also report the current status of ALIS in Cambodia.

  9. Laterally Driven Resonant Pressure Sensor with Etched Silicon Dual Diaphragms and Combined Beams

    Directory of Open Access Journals (Sweden)

    Xiaohui Du

    2016-01-01

    Full Text Available A novel structure of the resonant pressure sensor is presented in this paper, which tactfully employs intercoupling between dual pressure-sensing diaphragms and a laterally driven resonant strain gauge. After the resonant pressure sensor principle is introduced, the coupling mechanism of the diaphragms and resonator is analyzed and the frequency equation of the resonator based on the triangle geometry theory is developed for this new coupling structure. The finite element (FE simulation results match the theoretical analysis over the full scale of the device. This pressure sensor was first fabricated by dry/wet etching and thermal silicon bonding, followed by vacuum-packaging using anodic bonding technology. The test maximum error of the fabricated sensor is 0.0310%F.S. (full scale in the range of 30 to 190 kPa, its pressure sensitivity is negative and exceeding 8 Hz/kPa, and its Q-factor reaches 20,000 after wafer vacuum-packaging. A novel resonant pressure sensor with high accuracy is presented in this paper.

  10. Laterally Driven Resonant Pressure Sensor with Etched Silicon Dual Diaphragms and Combined Beams.

    Science.gov (United States)

    Du, Xiaohui; Liu, Yifang; Li, Anlin; Zhou, Zhou; Sun, Daoheng; Wang, Lingyun

    2016-01-26

    A novel structure of the resonant pressure sensor is presented in this paper, which tactfully employs intercoupling between dual pressure-sensing diaphragms and a laterally driven resonant strain gauge. After the resonant pressure sensor principle is introduced, the coupling mechanism of the diaphragms and resonator is analyzed and the frequency equation of the resonator based on the triangle geometry theory is developed for this new coupling structure. The finite element (FE) simulation results match the theoretical analysis over the full scale of the device. This pressure sensor was first fabricated by dry/wet etching and thermal silicon bonding, followed by vacuum-packaging using anodic bonding technology. The test maximum error of the fabricated sensor is 0.0310%F.S. (full scale) in the range of 30 to 190 kPa, its pressure sensitivity is negative and exceeding 8 Hz/kPa, and its Q-factor reaches 20,000 after wafer vacuum-packaging. A novel resonant pressure sensor with high accuracy is presented in this paper.

  11. A hybrid Fabry-Perot/Michelson interferometer sensor using a dual asymmetric core microstructured fiber

    Science.gov (United States)

    Frazão, O.; Silva, S. F.; Viegas, J.; Baptista, J. M.; Santos, J. L.; Roy, P.

    2010-02-01

    A hybrid Fabry-Perot/Michelson interferometer sensor using a dual asymmetric core microstructured fiber is demonstrated. The hybrid interferometer presents three waves. Two parallel Fabry-Perot cavities with low finesse are formed between the splice region and the end of a dual-core microstructured fiber. A Michelson configuration is obtained by the two small cores of the microstructured fiber. The spectral response of the hybrid interferometer presents two pattern fringes with different frequencies due to the respective optical path interferometers. The hybrid interferometer was characterized in strain and temperature presenting different sensitivity coefficients for each topology. Due to these characteristics, this novel sensing head is able to measure strain and temperature, simultaneously.

  12. Magnetic field sensor based on the Ampere's force using dual-polarization DBR fiber laser

    Science.gov (United States)

    Yao, Shuang; Zhang, Yang; Guan, Baiou

    2015-08-01

    A novel magnetic field sensor using distributed Bragg reflector (DBR) fiber laser by Ampere's force effect is proposed and experimentally demonstrated. The key sensing element, that is the dual-polarization DBR fiber laser, is fixed on the middle part of two copper plates which carry the current. Ampere's force is applied onto the coppers due to an external magnetic field generated by a DC solenoid. Thus, the lateral force from the coppers is converted to a corresponding beat frequency signal shift produced by the DBR laser. The electric current sensing is also realized by the same configuration and same principle simultaneously in an intuitive manner. Good agreement between the theory calculation and the experimental results is obtained, which shows a good linearity. This sensor's sensitivity to the magnetic field and to the electric current finally reaches ~258.92 kHz/mT and ~1.08727 MHz/A, respectively.

  13. A Novel Dual Separate Paths (DSP) Algorithm Providing Fault-Tolerant Communication for Wireless Sensor Networks.

    Science.gov (United States)

    Tien, Nguyen Xuan; Kim, Semog; Rhee, Jong Myung; Park, Sang Yoon

    2017-07-25

    Fault tolerance has long been a major concern for sensor communications in fault-tolerant cyber physical systems (CPSs). Network failure problems often occur in wireless sensor networks (WSNs) due to various factors such as the insufficient power of sensor nodes, the dislocation of sensor nodes, the unstable state of wireless links, and unpredictable environmental interference. Fault tolerance is thus one of the key requirements for data communications in WSN applications. This paper proposes a novel path redundancy-based algorithm, called dual separate paths (DSP), that provides fault-tolerant communication with the improvement of the network traffic performance for WSN applications, such as fault-tolerant CPSs. The proposed DSP algorithm establishes two separate paths between a source and a destination in a network based on the network topology information. These paths are node-disjoint paths and have optimal path distances. Unicast frames are delivered from the source to the destination in the network through the dual paths, providing fault-tolerant communication and reducing redundant unicast traffic for the network. The DSP algorithm can be applied to wired and wireless networks, such as WSNs, to provide seamless fault-tolerant communication for mission-critical and life-critical applications such as fault-tolerant CPSs. The analyzed and simulated results show that the DSP-based approach not only provides fault-tolerant communication, but also improves network traffic performance. For the case study in this paper, when the DSP algorithm was applied to high-availability seamless redundancy (HSR) networks, the proposed DSP-based approach reduced the network traffic by 80% to 88% compared with the standard HSR protocol, thus improving network traffic performance.

  14. A portable FRET analyzer for rapid detection of sugar content.

    Science.gov (United States)

    Kim, Haseong; Kim, Hyo Sang; Ha, Jae-Seok; Lee, Seung-Goo

    2015-05-21

    Fluorescence resonance energy transfer (FRET) is widely used as a core process in biometric sensors to detect small molecules such as sugars, calcium ions, or amino acids. However, FRET based biosensors with innate weak signal intensity require the use of expensive, high-sensitive equipment. In the present study, these shortcomings were overcome with the fabrication of a sensitive, inexpensive, and portable analyzer which provides quantitative detection of small molecules in a liquid sample. The usability of the developed analyzer was successfully tested by measuring sucrose and maltose contents in commercially available beverage samples, with better performance than the conventional monochromator-type spectrofluorometer. It is anticipated that miniaturization of the equipment and improving the FRET based biosensors will contribute to the practical use of this hand-held analyzer in conditions where high-end equipment is not available.

  15. Dual-wavelength optical fluidic glucose sensor using time series analysis of d(+)-glucose measurement

    Science.gov (United States)

    Tang, Jing-Yau; Chen, Nan-Yueh; Chen, Ming-Kun; Wang, Min-Haw; Jang, Ling-Sheng

    2016-10-01

    This paper presents a rising-edge time-series analysis (TSA) method that can be applied to a dual-wavelength optical fluidic glucose sensor (DWOFGS). In the experiment, the concentration of glucose in phosphate buffered saline (PBS) was determined by measuring the absorbance of the solution as determined by variation in the rising edge of the photodiode (PD) voltage response waveform. The DWOFGS principle is based on near-infrared (NIR) absorption spectroscopy at selected dual wavelengths (1450 and 1650 nm) in the first overtone band. The DWOFGS comprises two light-emitting diodes (LEDs) and two PD detectors. No additional fibers or lenses are required in our device. The output light level of the LEDs is adjusted to a light intensity suitable to the glucose absorption rate in an electronic circuit. Four light absorbance paths enable detection of d(+)-glucose concentrations from 0 to 20 wt % in steps of 5 wt %. The glucose light absorbance process was calculated based on the rising edge of the PD waveform under a low-intensity light source using TSA. The TSA method can be used to obtain the glucose level in PBS and reduce measurement background noise. The application of the rising-edge TSA method improves sensor sensitivity, increases the accuracy of the data analysis, and lowers measurement equipment costs.

  16. Novel pressure sensor by diode-pumped birefringent Nd:YAG dual-frequency laser

    Science.gov (United States)

    Huang, Chunning; Li, Yan; Zhang, Shulian; Guo, Hui

    2002-09-01

    The prototype of a novel sensor based on laser frequency splitting technology is presented in this paper and the results of a series of experiments are reported. A scheme of the novel pressure sensor by diode-pumped birefringent Nd:YAG dual-frequency laser is brought forward. As a result of the stress birefringence the laser's longitudinal mode is split to two with frequency difference. Both the theoretical analysis and the experiments indicate that there is a direct ratio relation between the frequency difference and the pressure imposed on the Nd:YAG crystal. Therefore when the external pressure is sensed by the Nd:YAG dual-frequency laser and the beat frequency is measured by the frequency counter, the pressure value can be obtained from the magnitude of the beat frequency. The laser can operate under single mode or complex modes with little error to the experiment results. Some research work under different pumping ways is finished and it indicates that the beat frequency has little relation with pumping ways. The experiment has a good linearity (R greater than 0.999) with satisfied precision, sensitivity (5.27 MHz/kPa) and stability, which provides a good academic and experimental foundation for further research.

  17. Optimization of a fiber grating film sensor based on dual peak resonance

    Institute of Scientific and Technical Information of China (English)

    GU Zheng-tian; Xu Yan-ping; DENG Chuan-lu

    2008-01-01

    Based on the dual peak resonance of long-period fider grating(LPFG),a novel film sensor is presented.in which films sensitive to the surrounding gases are coated on the cladding of the fiber grating region,and the intervals of the dual peak resonant wavelengths change with the film refractive index.According to the coupled-mode theory,a triple-clad numerical model is developed to analyze the relation between the sensitivity S and the thin film optical parameters(the film thickness h3 and the refractive index n3and the fiber grating parameters (the grating period A and the coreindex modulation (o)).By using optimization method,the optimal film optical parameters and the grating structure parameters are obtained.Numerical simulation shows that the sensitivity of this scheme to refractive index of the films is predicted to be more than 10-7.The theomtic analysis provides straightforward foundation for the aetual highly sensitive fdm sensors.

  18. Assessing Impact of Dual Sensor Enhanced Flight Vision Systems on Departure Performance

    Science.gov (United States)

    Kramer, Lynda J.; Etherington, Timothy J.; Severance, Kurt; Bailey, Randall E.

    2016-01-01

    Synthetic Vision (SV) and Enhanced Flight Vision Systems (EFVS) may serve as game-changing technologies to meet the challenges of the Next Generation Air Transportation System and the envisioned Equivalent Visual Operations (EVO) concept - that is, the ability to achieve the safety and operational tempos of current-day Visual Flight Rules operations irrespective of the weather and visibility conditions. One significant obstacle lies in the definition of required equipage on the aircraft and on the airport to enable the EVO concept objective. A motion-base simulator experiment was conducted to evaluate the operational feasibility and pilot workload of conducting departures and approaches on runways without centerline lighting in visibility as low as 300 feet runway visual range (RVR) by use of onboard vision system technologies on a Head-Up Display (HUD) without need or reliance on natural vision. Twelve crews evaluated two methods of combining dual sensor (millimeter wave radar and forward looking infrared) EFVS imagery on pilot-flying and pilot-monitoring HUDs. In addition, the impact of adding SV to the dual sensor EFVS imagery on crew flight performance and workload was assessed. Using EFVS concepts during 300 RVR terminal operations on runways without centerline lighting appears feasible as all EFVS concepts had equivalent (or better) departure performance and landing rollout performance, without any workload penalty, than those flown with a conventional HUD to runways having centerline lighting. Adding SV imagery to EFVS concepts provided situation awareness improvements but no discernible improvements in flight path maintenance.

  19. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection

    Science.gov (United States)

    Yao, B. C.; Wu, Y.; Yu, C. B.; He, J. R.; Rao, Y. J.; Gong, Y.; Fu, F.; Chen, Y. F.; Li, Y. R.

    2016-03-01

    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel ‘FRET on Fiber’ concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based ‘FRET on fiber’ configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated ‘FRET on Fiber’ sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response

  20. Attitude Heading Reference System Using MEMS Inertial Sensors with Dual-Axis Rotation

    Directory of Open Access Journals (Sweden)

    Li Kang

    2014-09-01

    Full Text Available This paper proposes a low cost and small size attitude and heading reference system based on MEMS inertial sensors. A dual-axis rotation structure with a proper rotary scheme according to the design principles is applied in the system to compensate for the attitude and heading drift caused by the large gyroscope biases. An optimization algorithm is applied to compensate for the installation angle error between the body frame and the rotation table’s frame. Simulations and experiments are carried out to evaluate the performance of the AHRS. The results show that the proper rotation could significantly reduce the attitude and heading drifts. Moreover, the new AHRS is not affected by magnetic interference. After the rotation, the attitude and heading are almost just oscillating in a range. The attitude error is about 3° and the heading error is less than 3° which are at least 5 times better than the non-rotation condition.

  1. Attitude heading reference system using MEMS inertial sensors with dual-axis rotation.

    Science.gov (United States)

    Kang, Li; Ye, Lingyun; Song, Kaichen; Zhou, Yang

    2014-09-29

    This paper proposes a low cost and small size attitude and heading reference system based on MEMS inertial sensors. A dual-axis rotation structure with a proper rotary scheme according to the design principles is applied in the system to compensate for the attitude and heading drift caused by the large gyroscope biases. An optimization algorithm is applied to compensate for the installation angle error between the body frame and the rotation table's frame. Simulations and experiments are carried out to evaluate the performance of the AHRS. The results show that the proper rotation could significantly reduce the attitude and heading drifts. Moreover, the new AHRS is not affected by magnetic interference. After the rotation, the attitude and heading are almost just oscillating in a range. The attitude error is about 3° and the heading error is less than 3° which are at least 5 times better than the non-rotation condition.

  2. Highly sensitive curvature sensor based on a multicladding fiber sandwiched dual no-core fibers structure.

    Science.gov (United States)

    Qi, Yanhui; Ma, Lin; Kang, Zexin; Bai, Yunlong; Yin, Bin; Jian, Shuisheng

    2014-10-01

    In this paper, we present a simple, compact, and highly sensitive optical fiber curvature sensor. It consists of dual no-core fibers for coupling energy from the lead-in single-mode fiber (SMF), and recoupling the energy into the lead-out SMF. The sensing section is constituted by multicladding fiber with a length of 5 cm. In the experiment, the spectrum shows a blueshift, and the energy corresponding to the resonant wavelength shows a cosine series with a bending of the sensing segment. The curvature sensitivity around the wavelength of 1550 nm shows -39.02 nm/m(-1) within the curvature range from 0.3 to 2.14 m(-1). The temperature sensitivity is 78.2 pm/°C in the temperature range of 10 °C-60 °C. It implies the possibility of temperature measurement.

  3. Notch Effect and Frequency Compensation of Dual-Sensor OBC Data in Shallow Water

    Institute of Scientific and Technical Information of China (English)

    Dongkai Wang; Siyou Tong; Huaishan Liu; Weiqiang Zhu

    2015-01-01

    Reverberation is significant in shallow water and produces obvious notches in OBC spec-tra. It also degrades the quality of sections and increases the difficulty of processing and interpretation. This article presents the relationship between notch, shooting depth, and seabed depth based on the seismic convolution model. Forward modelling based on wave equation theory is used to verify this re-lationship. Dual-sensor summation is applied to suppress receiver-side multiples and remove notches according to the opposite response of geophones and hydrophones to down-going wave fields based on a detailed analysis of the OBC technique. The good results obtained in practical applications reveal the effectiveness of this method.

  4. Dual-mode bioenabled nano-plasmonic sensors for biological and chemical detection

    Science.gov (United States)

    Kong, Xianming; Squire, Kenny; Xi, Yuting; LeDuff, Paul; Rorrer, Gregory L.; Wang, Alan X.

    2016-03-01

    Plasmonic biosensors have greatly overcome the limitations of conventional optical sensors in terms of sensitivity, tunability, photo-stability, and in vivo applicability. In this paper, we present plasmonic biosensors using bioenabled nanomaterials diatom biosilica, with active surface functionalities as affordable and eco-friendly integration platforms of Ag nanoparticles for label-free detection of biomolecules. Dual-mode plasmon sensing mechanisms, including surface-enhanced Raman scattering (SERS) and refractive-index (RI) sensing will be simultaneously implemented on the plasmonic-biosilica nanostructures to obtain quantitative biosensing with structural resolution of the biomolecules. We have achieved ultra-sensitive detection of Rhodamine 6G (R6G) at concentrations as low as 10-10 M. Furthermore, this substrate was used to detect TNT, illustrating the potential application as viable substrates for monitoring pollutant and toxics in environment.

  5. Assessing Dual Sensor Enhanced Flight Vision Systems to Enable Equivalent Visual Operations

    Science.gov (United States)

    Kramer, Lynda J.; Etherington, Timothy J.; Severance, Kurt; Bailey, Randall E.; Williams, Steven P.; Harrison, Stephanie J.

    2016-01-01

    Flight deck-based vision system technologies, such as Synthetic Vision (SV) and Enhanced Flight Vision Systems (EFVS), may serve as a revolutionary crew/vehicle interface enabling technologies to meet the challenges of the Next Generation Air Transportation System Equivalent Visual Operations (EVO) concept - that is, the ability to achieve the safety of current-day Visual Flight Rules (VFR) operations and maintain the operational tempos of VFR irrespective of the weather and visibility conditions. One significant challenge lies in the definition of required equipage on the aircraft and on the airport to enable the EVO concept objective. A motion-base simulator experiment was conducted to evaluate the operational feasibility, pilot workload and pilot acceptability of conducting straight-in instrument approaches with published vertical guidance to landing, touchdown, and rollout to a safe taxi speed in visibility as low as 300 ft runway visual range by use of onboard vision system technologies on a Head-Up Display (HUD) without need or reliance on natural vision. Twelve crews evaluated two methods of combining dual sensor (millimeter wave radar and forward looking infrared) EFVS imagery on pilot-flying and pilot-monitoring HUDs as they made approaches to runways with and without touchdown zone and centerline lights. In addition, the impact of adding SV to the dual sensor EFVS imagery on crew flight performance, workload, and situation awareness during extremely low visibility approach and landing operations was assessed. Results indicate that all EFVS concepts flown resulted in excellent approach path tracking and touchdown performance without any workload penalty. Adding SV imagery to EFVS concepts provided situation awareness improvements but no discernible improvements in flight path maintenance.

  6. Chemiresistive and Gravimetric Dual-Mode Gas Sensor toward Target Recognition and Differentiation.

    Science.gov (United States)

    Chen, Yan; Zhang, Hao; Feng, Zhihong; Zhang, Hongxiang; Zhang, Rui; Yu, Yuanyuan; Tao, Jin; Zhao, Hongyuan; Guo, Wenlan; Pang, Wei; Duan, Xuexin; Liu, Jing; Zhang, Daihua

    2016-08-24

    We demonstrate a dual-mode gas sensor for simultaneous and independent acquisition of electrical and mechanical signals from the same gas adsorption event. The device integrates a graphene field-effect transistor (FET) with a piezoelectric resonator in a seamless manner by leveraging multiple structural and functional synergies. Dual signals resulting from independent physical processes, i.e., mass attachment and charge transfer can reflect intrinsic properties of gas molecules and potentially enable target recognition and quantification at the same time. Fabrication of the device is based on standard Integrated Circuit (IC) foundry processes and fully compatible with system-on-a-chip (SoC) integration to achieve extremely small form factors. In addition, the ability of simultaneous measurements of mass adsorption and charge transfer guides us to a more precise understanding of the interactions between graphene and various gas molecules. Besides its practical functions, the device serves as an effective tool to quantitatively investigate the physical processes and sensing mechanisms for a large library of sensing materials and target analytes.

  7. Temperature dependent dual hydrogen sensor response of Pd nanoparticle decorated Al doped ZnO surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, D.; Barman, P. B.; Hazra, S. K., E-mail: surajithazra@yahoo.co.in [Department of Physics and Materials Science, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh-173234 (India); Dutta, D. [IC Design and Fabrication Centre, Department of Electronics and Telecommunication Engineering, Jadavpur University, Kolkata-700032 (India); Kumar, M.; Som, T. [SUNAG Laboratory, Institute of Physics, Sachivalaya Marg, Bhubaneswar 751005 (India)

    2015-10-28

    Sputter deposited Al doped ZnO (AZO) thin films exhibit a dual hydrogen sensing response in the temperature range 40 °C–150 °C after surface modifications with palladium nanoparticles. The unmodified AZO films showed no response in hydrogen in the temperature range 40 °C–150 °C. The operational temperature windows on the low and high temperature sides have been estimated by isolating the semiconductor-to-metal transition temperature zone of the sensor device. The gas response pattern was modeled by considering various adsorption isotherms, which revealed the dominance of heterogeneous adsorption characteristics. The Arrhenius adsorption barrier showed dual variation with change in hydrogen gas concentration on either side of the semiconductor-to-metal transition. A detailed analysis of the hydrogen gas response pattern by considering the changes in nano palladium due to hydrogen adsorption, and semiconductor-to-metal transition of nanocrystalline Al doped ZnO layer due to temperature, along with material characterization studies by glancing incidence X-ray diffraction, atomic force microscopy, and transmission electron microscopy, are presented.

  8. Fano resonance in asymmetric-period two-dimensional plasmonic absorbers for dual-band uncooled infrared sensors

    Science.gov (United States)

    Ogawa, Shinpei; Takagawa, Yousuke; Kimata, Masafumi

    2016-11-01

    The spectral discrimination function of uncooled infrared (IR) sensors has significant advantages for applications such as fire detection, gas analysis, and biological analysis. We have previously demonstrated wavelength-selective uncooled IR sensors using two-dimensional plasmonic absorbers (2-D PLAs) over a wide range spanning the middle- and long-wavelength IR regions. 2-D PLAs are highly promising in terms of practical application due to the ease of fabrication and robustness for structural fluctuations. However, dual-band operation based on this concept has not yet been investigated, even though the ability to absorb in two different wavelength bands is extremely important for object recognition. Thus, a dual-band uncooled IR sensor was developed that employs Fano resonance in the plasmonic structures. To achieve dual-band detection, asymmetric periods in the orthogonal x- and y-directions were introduced into 2-D PLAs. Theoretical investigations predicted an asymmetric absorbance line shape dependent on the polarization attributed to Fano resonance. The spectral responsivity of the developed sensor demonstrated that selective detection occurred in two different wavelength bands due to polarization-dependent Fano resonance. The results obtained in this study will be applicable to the development of advanced sensors capable of multiband detection in the IR region.

  9. SLIM for multispectral FRET imaging

    Science.gov (United States)

    Rück, A.; Dolp, F.; Steiner, R.; Steinmetz, C.; von Einem, B.; von Arnim, C. A. F.

    2008-02-01

    Spectral fluorescence lifetime imaging (SLIM) is an advanced imaging technique, which combines spectral with time resolved detection. Real spectral information is achieved by using a grating in front of a PML-array, which allows time-correlated single photon counting (TCSPC). Whereas spectrally resolved fluorescence imaging alone has a reasonable sensitivity, the specificity of fluorescence detection can be improved by considering the fluorescence lifetime. The various possibilities which SLIM offers to improve FRET (resonant energy transfer) will be discussed as well as successfully realized applications. These include FRET measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

  10. Systematic theoretical analysis of dual-parameters RF readout by a novel LC-type passive sensor

    CERN Document Server

    Tan, Qiulin; Luo, Tao; Wei, Tanyong; Shen, Sanming; Wu, Dezhi; Zhang, Wendong; Xiong, Jijun

    2016-01-01

    This paper systematically studied the simultaneous measurement of two parameters by a LC-type passive sensor from the theoretical perspective. Based on the lumped circuit model of the typical LC-type passive dual-parameter sensor system, the influencing factors of the signal strength of the sensor as well as the influencing factors of signal crosstalk were both analyzed. It is found that the influencing factors of the RF readout signal strength of the sensor are mainly quality factors (Q factors) of the LC tanks, coupling coefficients, and the resonant frequency interval of the two LC tanks. And the influencing factors of the signal crosstalk are mainly coupling coefficient between the sensor inductance coils and the resonant frequency interval of the two LC tanks. The specific influence behavior of corresponding influencing factors on the signal strength and crosstalk is illustrated by a series of curves from numerical results simulated by using MATLAB software. Additionally, a decoupling scheme for solving ...

  11. Dual redundant sensor FDI techniques applied to the NASA F8C DFBW aircraft. [Failure Detection and Identification

    Science.gov (United States)

    Desai, M. N.; Deckert, J. C.; Deyst, J. J.; Willsky, A. S.; Chow, E. Y.

    1976-01-01

    An onboard failure detection and identification (FDI) technique for dual redundant sensors on the NASA F8C digital fly-by-wire (DFBW) aircraft is presented. The failure of one of a pair of sensors of the same type is detected by a direct redundancy trigger which observes the difference between the outputs of these two sensors. Identification of the failed sensor is accomplished utilizing the analytic redundancy that exists as kinematic and functional relationships among the variables being measured by dissimilar instruments. In addition, identification of generic failures, common to both instruments of a given type, is accomplished by using a time trigger to periodically initiate analytic redundancy failure identification tests for individual sensors. The basic form of these tests is the comparison of the measurement of a variable using the suspect instrument with another measurement of the same variable obtained using other instrument types.

  12. Dual frequency microstrip antenna sensor for water content measurements independent of temperature variation

    Science.gov (United States)

    Ghretli, Mohamed; Khalid, Kaida; Valeriu Grozescu, Ionel; Sahri, Hamami; Abbas, Zulkifly

    2007-04-01

    Temperature variation causes errors in all indirect moisture measurement methods. To increase the accuracy of moisture content determination and to reduce the influence of temperature, a two-parameter measurement is used. The method uses the magnitude of reflected waves at two microwave frequencies in the X-band region. A dual frequency sensor system is developed to measure moisture content of dielectric-lossy liquids. The experiment is based on measurements of far-field reflection magnitudes at two different frequencies 8.48 GHz and 10.69 GHz using circular microstrip antennas. A calibration equation is sought that instantly gives temperature-independent moisture content of the samples under consideration. The sensor is integrated with a data acquisition card to record the detected reflection signals. The data analysis and error-correction technique are implemented using custom designed software. The system is tested using diluted rubber latex with moisture content ranging from 39.8% to 91.2% wet basis. The moisture content was predicted with a standard error less than 1.3% for the temperature range of 25 °C to 63 °C compared to the standard oven-drying technique.

  13. Development of a MEMS dual-axis differential capacitance floating element shear stress sensor

    Energy Technology Data Exchange (ETDEWEB)

    Barnard, Casey [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Griffin, Benjamin [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)

    2015-09-01

    A single-axis MEMS wall shear stress sensor with differential capacitive transduction method is produced. Using a synchronous modulation and demodulation interface circuit, the system is capable of making real time measurements of both mean and fluctuating wall shear stress. A sensitivity of 3.44 mV/Pa is achieved, with linearity in response demonstrated up to testing limit of 2 Pa. Minimum detectable signals of 340 μPa at 100 Hz and 120 μPa at 1 kHz are indicated, with a resonance of 3.5 kHz. Multiple full scale wind tunnel tests are performed, producing spectral measurements of turbulent boundary layers in wind speeds ranging up to 0.5 Ma (18 Pa of mean wall shear stress). The compact packaging allows for minimally invasive installation, and has proven relatively robust over multiple testing events. Temperature sensitivity, likely due to poor CTE matching of packaged materials, is an ongoing concern being addressed. These successes are being directly leveraged into a development plan for a dual-axis wall shear stress sensor, capable of producing true vector estimates at the wall.

  14. Vertical Guidance Performance Analysis of the L1-L5 Dual-Frequency GPS/WAAS User Avionics Sensor

    Directory of Open Access Journals (Sweden)

    Shau-Shiun Jan

    2010-03-01

    Full Text Available This paper investigates the potential vertical guidance performance of global positioning system (GPS/wide area augmentation system (WAAS user avionics sensor when the modernized GPS and Galileo are available. This paper will first investigate the airborne receiver code noise and multipath (CNMP confidence (σair. The σair will be the dominant factor in the availability analysis of an L1-L5 dual-frequency GPS/WAAS user avionics sensor. This paper uses the MATLAB Algorithm Availability Simulation Tool (MAAST to determine the required values for the σair, so that an L1-L5 dual-frequency GPS/WAAS user avionics sensor can meet the vertical guidance requirements of APproach with Vertical guidance (APV II and CATegory (CAT I over conterminous United States (CONUS. A modified MAAST that includes the Galileo satellite constellation is used to determine under what user configurations WAAS could be an APV II system or a CAT I system over CONUS. Furthermore, this paper examines the combinations of possible improvements in signal models and the addition of Galileo to determine if GPS/WAAS user avionics sensor could achieve 10 m Vertical Alert Limit (VAL within the service volume. Finally, this paper presents the future vertical guidance performance of GPS user avionics sensor for the United States’ WAAS, Japanese MTSAT-based satellite augmentation system (MSAS and European geostationary navigation overlay service (EGNOS.

  15. Novel design of a refractive index sensor based on a dual-core micro-structured optical fiber

    CERN Document Server

    Tsigaridas, G; Persephonis, P

    2014-01-01

    In the present work a new model of a refractive index (RI) sensor is exhibited. This is based on a dual core micro-structured optical fiber (MOF), where two holes are introduced at the core centers. In this way, the model enhances the interaction of the fiber modes propagated in the core region, providing the possibility of increasing the dimensions of the fiber sensor. Thus, the filling of the fiber holes with the fluid under study is facilitated, and generally the practical use of the system as a refractive index sensor is simplified. The influence of the core separation and the diameter of the central holes on the sensitivity of the sensor have been studied by a numerical simulation. The results are in agreement with the expected behavior as it is determined by the physics of the problem. Based on the same operating principle, it is verified that a dual-core conventional optical fiber with micro-fluidic channels at the center of the cores, can also operates as an RI sensor.

  16. Vertical guidance performance analysis of the L1-L5 dual-frequency GPS/WAAS user avionics sensor.

    Science.gov (United States)

    Jan, Shau-Shiun

    2010-01-01

    This paper investigates the potential vertical guidance performance of global positioning system (GPS)/wide area augmentation system (WAAS) user avionics sensor when the modernized GPS and Galileo are available. This paper will first investigate the airborne receiver code noise and multipath (CNMP) confidence (σair). The σair will be the dominant factor in the availability analysis of an L1-L5 dual-frequency GPS/WAAS user avionics sensor. This paper uses the MATLAB Algorithm Availability Simulation Tool (MAAST) to determine the required values for the σair, so that an L1-L5 dual-frequency GPS/WAAS user avionics sensor can meet the vertical guidance requirements of APproach with Vertical guidance (APV) II and CATegory (CAT) I over conterminous United States (CONUS). A modified MAAST that includes the Galileo satellite constellation is used to determine under what user configurations WAAS could be an APV II system or a CAT I system over CONUS. Furthermore, this paper examines the combinations of possible improvements in signal models and the addition of Galileo to determine if GPS/WAAS user avionics sensor could achieve 10 m Vertical Alert Limit (VAL) within the service volume. Finally, this paper presents the future vertical guidance performance of GPS user avionics sensor for the United States' WAAS, Japanese MTSAT-based satellite augmentation system (MSAS) and European geostationary navigation overlay service (EGNOS).

  17. Dual detection of nitrate and mercury in water using disposable electrochemical sensors.

    Science.gov (United States)

    Bui, Minh-Phuong N; Brockgreitens, John; Ahmed, Snober; Abbas, Abdennour

    2016-11-15

    Here we report a disposable, cost effective electrochemical paper-based sensor for the detection of both nitrate and mercury ions in lake water and contaminated agricultural runoff. Disposable carbon paper electrodes were functionalized with selenium particles (SePs) and gold nanoparticles (AuNPs). The AuNPs served as a catalyst for the reduction of nitrate ions using differential pulse voltammetry techniques. The AuNPs also served as a nucleation sites for mercury ions. The SePs further reinforced this mercury ion nucleation due to their high binding affinity to mercury. Differential pulse stripping voltammetry techniques were used to further enhance mercury ion accumulation on the modified electrode. The fabricated electrode was characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and electrochemistry techniques. The obtained results show that the PEG-SH/SePs/AuNPs modified carbon paper electrode has a dual functionality in that it can detect both nitrate and mercury ions without any interference. The modified carbon paper electrode has improved the analytical sensitivity of nitrate and mercury ions with limits of detection of 8.6µM and 1.0ppb, respectively. Finally, the modified electrode was used to measure nitrate and mercury in lake water samples.

  18. A Dual-Band Antenna for RF Energy Harvesting Systems in Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    A. Bakkali

    2016-01-01

    Full Text Available In this paper, we focus on ambient radio frequency energy available from commercial broadcasting stations in order to provide a system based on RF energy harvesting using a new design of receiving antenna. Several antenna designs have been proposed for use in RF energy harvesting systems, as a pertinent receiving antenna design is highly required since the antenna features can affect the amount of energy harvested. The proposed antenna is aimed at greatly increasing the energy harvesting efficiency over Wi-Fi bands: 2.45 GHz and 5 GHz. This provides a promising alternative energy source in order to power sensors located in harsh environments or remote places, where other energy sources are impracticable. The dual-band antenna can be easily integrated with RF energy harvesting system on the same circuit board. Simulations and measurements were carried out to evaluate the antenna performances and investigate the effects of different design parameters on the antenna performance. The receiving antenna meets the required bandwidth specification and provides peak gain of more than 4 dBi across the operating band.

  19. Dual Wake-up Low Power Listening for Duty Cycled Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Jongkeun Na

    2008-01-01

    Full Text Available Energy management is an interesting research area for wireless sensor networks. Relevant dutycycling (or sleep scheduling algorithm has been actively studied at MAC, routing, and application levels. Low power listening (LPL MAC is one of effective dutycycling techniques. This paper proposes a novel approach called dual wake-up LPL (DW-LPL. Existing LPL scheme uses a preamble detection method for both broadcast and unicast, thus suffers from severe overhearing problem at unicast transmission. DW-LPL uses a different wake-up method for unicast while using LPL-like method for broadcast; DW-LPL introduces a receiver-initiated method in which a sender waits a signal from receiver to start unicast transmission, which incurs some signaling overhead but supports flexible adaptive listening as well as overhearing removal effect. Through analysis and Mote (Telosb experiment, we show that DW-LPL provides more energy saving than LPL and our adaptive listening scheme is effective for energy conservation in practical network topologies and traffic patterns.

  20. Dual Wake-up Low Power Listening for Duty Cycled Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Lim Sangsoon

    2008-01-01

    Full Text Available Abstract Energy management is an interesting research area for wireless sensor networks. Relevant dutycycling (or sleep scheduling algorithm has been actively studied at MAC, routing, and application levels. Low power listening (LPL MAC is one of effective dutycycling techniques. This paper proposes a novel approach called dual wake-up LPL (DW-LPL. Existing LPL scheme uses a preamble detection method for both broadcast and unicast, thus suffers from severe overhearing problem at unicast transmission. DW-LPL uses a different wake-up method for unicast while using LPL-like method for broadcast; DW-LPL introduces a receiver-initiated method in which a sender waits a signal from receiver to start unicast transmission, which incurs some signaling overhead but supports flexible adaptive listening as well as overhearing removal effect. Through analysis and Mote (Telosb experiment, we show that DW-LPL provides more energy saving than LPL and our adaptive listening scheme is effective for energy conservation in practical network topologies and traffic patterns.

  1. Fretting fatigue in AISI 1015 steel

    Indian Academy of Sciences (India)

    R Gnanamoorthy; R Rosi Reddy

    2002-04-01

    A small osillatory movement between two contacting surfaces is termed as fretting and on many occasions it acts as the crack initiation site leading to catastrophic failure of the overall structure. The occurrence of fretting is observed in many engineering structures such as shaft flanges, gas turbines, steel ropes etc. An experimental facility, which can simulate the fretting fatigue in many engineering applications, is the primary requirement of the research program. A laboratory fretting fatigue test facility capable of varying many influencing parameters of fretting fatigue such as slip amplitutde, frequency, contact pressure, etc is designed and developed. Preliminary investigations on plain and fretting fatigue behaviour of AISI 1015 structural steel are reported in this paper. A strength reduction factor of about 1.30 was obtained due to fretting for the test material under the present experimental conditions. Influence of contact load on fretting was also studied. Increasing fretting contact load decreased the fatigue life in the range investigated. Failure analysis showed typical stage I oblique crack growth followed by stage II straight crack perpedicular to the fretting zone.

  2. A disposable amperometric dual-sensor for the detection of hemoglobin and glycated hemoglobin in a finger prick blood sample.

    Science.gov (United States)

    Moon, Jong-Min; Kim, Dong-Min; Kim, Moo Hyun; Han, Jin-Yeong; Jung, Dong-Keun; Shim, Yoon-Bo

    2017-05-15

    A disposable microfluidic amperometric dual-sensor was developed for the detection of glycated hemoglobin (HbA1C) and total hemoglobin (Hb), separately, in a finger prick blood sample. The accurate level of total Hb was determined through the measurements of the cathodic currents of total Hb catalyzed by a toluidine blue O (TBO)-modified working electrode. Subsequently, after washing unbound Hb in the fluidic channel of dual sensor with PBS, the cathodic current by only HbA1C captured on aptamer was monitored using another aptamer/TBO-modified working electrode in the channel. To modify the sensor probe, poly(2,2´:5´,5″-terthiophene-3´-p-benzoic acid) and a multi-wall carbon nanotube (MWCNT) composite layer (pTBA@MWCNT) was electropolymerized on a screen printed carbon electrode (SPCE), followed by immobilization of TBO for the total Hb probe and aptamer/TBO for the HbA1C probe, respectively. The characterization of each sensor surface was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance (QCM), field-emission scanning electron microscopy (FE-SEM), and transmission electron microscopy (TEM). The experimental conditions affecting the analytical signal were optimized in terms of the amount of TBO, pH, temperature, binding time, applied potential, and the content ratio of monomer and MWCNT. The dynamic ranges of Hb and HbA1C were from 0.1 to 10µM and from 0.006 to 0.74µM, with detection limits of 82(±4.2)nM and 3.7(±0.8)nM, respectively. The reliability of the proposed microfluidic dual-sensor for a finger prick blood sample (1µL) was evaluated in parallel with a conventional method (HPLC) for point-of-care analysis.

  3. NIR FRET Fluorophores for Use as an Implantable Glucose Biosensor

    Directory of Open Access Journals (Sweden)

    Majed DWEIK

    2008-12-01

    Full Text Available Development of an in vivo optical sensor requires the utilization of Near Infra Red (NIR fluorophores due to their ability to operate within the biological tissue window. Alexa Fluor 750 (AF750 and Alexa Fluor 680 (AF680 were examined as potential NIR fluorophores for an in vivo fluorescence resonance energy transfer (FRET glucose biosensor. AF680 and AF750 found to be a FRET pair and percent energy transfer was calculated. Next, the tested dye pair was utilized in a competitive binding assay in order to detect glucose. Concanavalin A (Con A and dextran have binding affinity, but in the presence of glucose, glucose displaces dextran due to its higher affinity to Con A than dextran. Finally, the percent signal transfer through porcine skin was examined. The results showed with approximately 4.0 mm porcine skin thickness, 1.98 % of the fluorescence was transmitted and captured by the detector.

  4. A handheld mid-infrared methane sensor using a dual-step differential method for additive/multiplicative noise suppression

    Science.gov (United States)

    Zheng, Yue; Dang, Peipei; Zheng, Chuantao; Ye, Weilin; Wang, Yiding

    2016-11-01

    A miniature mid-infrared (mid-IR) methane (CH4) sensor system was developed by employing a wide-band wire-source and a semi-ellipsoid multi-pass gas cell. A dual-step differential method instead of the traditional one-step differential method was adopted by this sensor to tune measuring range/zero point and to suppress the additive/multiplicative noise. This method included a first subtraction operation between the two output signals (including a detection signal and a reference signal) from the dual-channel detector and a second subtraction operation on the amplitudes of the first-subtraction signal and the reference signal, followed by a ratio operation between the amplitude of the second-subtraction signal and the reference signal. Detailed experiments were performed to assess the performance of the sensor system. The detection range is 0-50 k ppm, and as the concentration gets larger than 12 k ppm, the relative detection error falls into the range of -3% to +3%. The Allan deviation is about 4.65 ppm with an averaging time of 1 s, and such value can be further improved to 0.45 ppm with an averaging time of 124 s. Due to the cost-effective incandescence wire-source, the small-size ellipsoid multi-pass gas cell and the miniature structure of the sensor, the developed standalone device shows potential applications of CH4 detection under coal-mine environment.

  5. EXPERIMENTAL INVESTIGTION OF THE FRETTING PHENOMENON

    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI

    2015-12-01

    Full Text Available Fretting is now fully identified as a small amplitude oscilatory motion which induces a harmonic tangential force between two surfaces in contact.It is related to three main loadings, i.e. fretting-wear, fretting-fatigue and fretting corrosion.Fretting regimes were first mapped by Vingsbo. In a similar way, three fretting regimes will be considered: stick regime,slip regime and mixed regime. The mixed regime was made up of initial gross slip followed by partial slip condition after a few hundred cycles. Obviously the partial slip transition develops the highest stress levels which can induce fatigue crack nucleation depending on the fatigue properties of the two contacting first bodies. Therefore prediction of the frontier between partial slip and gross slip is required.

  6. Sensor for In-Motion Continuous 3D Shape Measurement Based on Dual Line-Scan Cameras.

    Science.gov (United States)

    Sun, Bo; Zhu, Jigui; Yang, Linghui; Yang, Shourui; Guo, Yin

    2016-11-18

    The acquisition of three-dimensional surface data plays an increasingly important role in the industrial sector. Numerous 3D shape measurement techniques have been developed. However, there are still limitations and challenges in fast measurement of large-scale objects or high-speed moving objects. The innovative line scan technology opens up new potentialities owing to the ultra-high resolution and line rate. To this end, a sensor for in-motion continuous 3D shape measurement based on dual line-scan cameras is presented. In this paper, the principle and structure of the sensor are investigated. The image matching strategy is addressed and the matching error is analyzed. The sensor has been verified by experiments and high-quality results are obtained.

  7. Simultaneous, inherently temperature and strain insensitive bio-sensors based on dual-resonance long-period gratings

    CERN Document Server

    Tripathi, Saurabh Mani; Bock, Wojtek J; Mikulic, Predrag

    2016-01-01

    Addressing the temperature and strain induced cross-talks simultaneously, we propose an inherently strain and temperature insensitive fiber-optic bio-sensor. The insensitivity has been achieved by properly adjusting the dopants and their concentrations in the optical fiber core region, and by optimizing the grating period and the strength of concatenated dual-resonance long-period-gratings. The simulations have been carried out using the same fiber parameters as used in our earlier experimental studies, which matched excellently with the experimental results. The proposed sensor has a theoretical refractive-index sensitivity of 4607 nm/RIU, which can be used to detect changes as small as 2.2 x10^-7 in ambient refractive indices using a detection system with spectral resolution of 1 pm. Our work finds application in developing precision biosensors with inherent insensitivity towards temperature and axial strain fluctuations. The sensor is currently under fabrication at our lab.

  8. Sensor for In-Motion Continuous 3D Shape Measurement Based on Dual Line-Scan Cameras

    Directory of Open Access Journals (Sweden)

    Bo Sun

    2016-11-01

    Full Text Available The acquisition of three-dimensional surface data plays an increasingly important role in the industrial sector. Numerous 3D shape measurement techniques have been developed. However, there are still limitations and challenges in fast measurement of large-scale objects or high-speed moving objects. The innovative line scan technology opens up new potentialities owing to the ultra-high resolution and line rate. To this end, a sensor for in-motion continuous 3D shape measurement based on dual line-scan cameras is presented. In this paper, the principle and structure of the sensor are investigated. The image matching strategy is addressed and the matching error is analyzed. The sensor has been verified by experiments and high-quality results are obtained.

  9. A Wide Linear Range Eddy Current Displacement Sensor Equipped with Dual-Coil Probe Applied in the Magnetic Suspension Flywheel

    Directory of Open Access Journals (Sweden)

    Tong Wen

    2012-08-01

    Full Text Available The Eddy Current Displacement Sensor (ECDS is widely used in the Magnetic Suspension Flywheel (MSFW to measure the tiny clearance between the rotor and the magnetic bearings. The linear range of the ECDS is determined by the diameter of its probe coil. Wide clearances must be measured in some new MSFWs recently designed for the different space missions, but the coil diameter is limited by some restrictions. In this paper, a multi-channel ECDS equipped with dual-coil probes is proposed to extend the linear range to satisfy the demands of such MSFWs. In order to determine the best configuration of the dual-coil probe, the quality factors of the potential types of the dual-coil probes, the induced eddy current and the magnetic intensity on the surface of the measuring object are compared with those of the conventional single-coil probe. The linear range of the ECDS equipped with the selected dual-coil probe is extended from 1.1 mm to 2.4 mm under the restrictions without adding any cost for additional compensation circuits or expensive coil materials. The effectiveness of the linear range extension ability and the dynamic response of the designed ECDS are confirmed by the testing and the applications in the MSFW.

  10. Dual-band infrared perfect absorber for plasmonic sensor based on the electromagnetically induced reflection-like effect

    Science.gov (United States)

    Liu, Yang; Zhang, Ying Qiao; Jin, Xing Ri; Zhang, Shou; Lee, Young Pak

    2016-07-01

    We present a scheme for realizing a narrow-dual-band perfect absorber based on the plasmonic analogy of the electromagnetically induced reflection (EIR)-like effect. In our scheme, two short gold bars are excited strongly by incident plane wave serving as the bright mode. The middle gold bar is excited by two short gold bars. Due to the strong hybridization between the two short gold bars and the middle gold bar, two absorption peaks occur. The corresponding absorption rates are both over 99%. The quality factors of the two absorption peaks are 41.76 (198.47 THz) and 71.42 (207.79 THz), respectively, and the narrow-distance of the two absorption peaks is 9.32 THz. Therefore, they are narrow enough for the absorber to be a filter and a dual-band plasmonic sensor.

  11. Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor.

    Science.gov (United States)

    Ahmad, Mohammad; Ameen, Seema; Siddiqi, Tariq Omar; Khan, Parvez; Ahmad, Altaf

    2016-12-15

    Glycine betaine (GB) is one of the key compatible solutes that accumulate in the cell at exceedingly high level under the conditions of high salinity. It plays a crucial role in the maintenance of osmolarity of the cell without affecting the physiological processes. Analysis of stress-induced physiological conditions in living cells, therefore, requires real-time monitoring of cellular GB level. Glycine Betaine Optical Sensor (GBOS), a genetically-encoded FRET-based nanosensor developed in this study, allows the real-time monitoring of GB levels inside living cells. This nanosensor has been developed by sandwiching GB binding protein (ProX) between the Förster resonance energy transfer (FRET) pair, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Conformational change in ProX, which was used as sensory domain, reported the change in the level of this compatible solute in in vitro and in vivo conditions. Binding of the GB to the sensory domain fetches close to both the fluorescent moieties that result in the form of increased FRET ratio. So, any change in the concentration of GB is correlated with change in FRET ratio. This sensor also reported the GB cellular dynamics in real-time in Escherichia coli cells after the addition of its precursor, choline. The GBOS was also expressed in yeast and mammalian cells to monitor the intracellular GB. Therefore, the GBOS represents a unique FRET-based nanosensor which allows the non-invasive ratiometric analysis of the GB in living cells.

  12. Standard guide for fretting fatigue testing

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This guide defines terminology and covers general requirements for conducting fretting fatigue tests and reporting the results. It describes the general types of fretting fatigue tests and provides some suggestions on developing and conducting fretting fatigue test programs. 1.2 Fretting fatigue tests are designed to determine the effects of mechanical and environmental parameters on the fretting fatigue behavior of metallic materials. This guide is not intended to establish preference of one apparatus or specimen design over others, but will establish guidelines for adherence in the design, calibration, and use of fretting fatigue apparatus and recommend the means to collect, record, and reporting of the data. 1.3 The number of cycles to form a fretting fatigue crack is dependent on both the material of the fatigue specimen and fretting pad, the geometry of contact between the two, and the method by which the loading and displacement are imposed. Similar to wear behavior of materials, it is important t...

  13. Tunable Polymer Fiber Bragg Grating (FBG) Inscription: Fabrication of Dual-FBG Temperature Compensated Polymer Optical Fiber Strain Sensors

    DEFF Research Database (Denmark)

    Yuan, Scott Wu; Stefani, Alessio; Bang, Ole

    2012-01-01

    We demonstrate stable wavelength tunable inscription of polymer optical fiber Bragg gratings (FBGs). By straining the fiber during FBG inscription, we linearly tune the center wavelength over 7 nm with less than 1% strain. Above 1% strain, the tuning curve saturates and we show a maximum tuning...... of 12 nm with 2.25% strain. We use this inscription method to fabricate a dual-FBG strain sensor in a poly (methyl methacrylate) single-mode microstructured polymer optical fiber and demonstrate temperature compensated strain sensing around 850 nm....

  14. High precision dual-axis tracking solar wireless charging system based on the four quadrant photoelectric sensor

    Science.gov (United States)

    Liu, Zhilong; Wang, Biao; Tong, Weichao

    2015-08-01

    This paper designs a solar automatic tracking wireless charging system based on the four quadrant photoelectric sensor. The system track the sun's rays automatically in real time to received the maximum energy and wireless charging to the load through electromagnetic coupling. Four quadrant photoelectric sensor responsive to the solar spectrum, the system could get the current azimuth and elevation angle of the light by calculating the solar energy incident on the sensor profile. System driver the solar panels by the biaxial movement mechanism to rotate and tilt movement until the battery plate and light perpendicular to each other. Maximize the use of solar energy, and does not require external power supply to achieve energy self-sufficiency. Solar energy can be collected for portable devices and load wireless charging by close electromagnetic field coupling. Experimental data show that: Four quadrant photoelectric sensor more sensitive to light angle measurement. when track positioning solar light, Azimuth deviation is less than 0.8°, Elevation angle deviation is less than 0.6°. Use efficiency of a conventional solar cell is only 10% -20%.The system uses a Four quadrant dual-axis tracking to raise the utilization rate of 25% -35%.Wireless charging electromagnetic coupling efficiency reached 60%.

  15. A wide dynamic range CMOS image sensor with dual charge storage in a pixel and a multiple sampling technique

    Science.gov (United States)

    Shafie, Suhaidi; Kawahito, Shoji

    2008-02-01

    This paper presents a dynamic range expansion technique of CMOS image sensors with dual charge storage in a pixel and multiple exposures. Each pixel contains two photodiodes, PD1 and PD2 whose sensitivity can be set independently by the accumulation time. The difference of charge accumulation time in both photodiode can be manipulated to expand the dynamic range of the sensor. It allows flexible control of the dynamic range since the accumulation time in PD2 is adjustable. The multiple exposure technique used in the sensor reduces the motion blur in the synthesized wide dynamic range image when capturing fast-moving objects. It also reduces the signal-to-nose ratio dip at the switching point of the PD1 signal to the PD2 signals in the synthesized wide dynamic range image. A wide dynamic range camera with 320x240 pixels image sensor has been tested. It is found that the sampling of 4 times for the short accumulation time signals is sufficient for the reduction of motion blur in the synthesized wide dynamic range image, and the signal-to-noise ratio dip at the switching point of the PD1 signal to the PD2 signal is improved by 6 dB using 4 short-time exposures.

  16. A Study for Optimum Survey Method of Underwater Structure Using the Dual Sonar Sensor

    Directory of Open Access Journals (Sweden)

    Youngseok Kim

    2017-01-01

    Full Text Available We have developed dual sonar equipment and an improved operating method for improving resolution in order to solve the problems of limitations of the optical equipment and the application method of SSS (side scan sonar in the investigation of damage of underwater structures. We analyzed the influence factors of the resolution of sonar data through the comparison of resolution and data quality in indoor test. Also we confirmed the problems about the overlapping area of the dual sonar. Depth and distance were analyzed as major influencing factors for survey angle. Specimens were scanned while adjusting distance and towfish angle according to depth change in order to verify applicability of the developed dual sonar in the field experiment. Optimal resolution was found to be 3 cm in specimen spacing, and 20 sample data items were extracted. We developed the regression model based on the multiple regression analysis and developed the RealDualSONAR-DAQ tool, the dual sonar optimum operating method program based on proposed correlation equations. We can use the developed tools to get the value of the major influencing factors for dual sonar operation and obtain high quality sonar data to analyze damage of underwater structures.

  17. Axisymmetric fretting analysis in coated cylinder

    Indian Academy of Sciences (India)

    M Ramesh; Satish V Kailas; K R Y Simha

    2008-06-01

    Fretting is essentially a contact fatigue phenomenon, although bulk stresses and material properties contribute to final failure. The near surface state of stress developed under oscillatory contact between machine elements plays a major role in deciding the severity of fretting. It is possible to enhance tribological properties by coating the surface. There is rather scanty literature available on fretting analysis of coated components. Presence of such coatings has a large influence on the near surface state of stress. The effect of coatings on the severity of fretting is the focus of this paper. Results obtained for both hard and soft coatings are compared with the results obtained for the homogeneous case. The component geometry and loading are chosen to be cylindrical to enable 3D elastic axisymmetric fretting analysis. The results are compared with 2D models (strip and half-plane) to examine their utility and validity for understanding axisymmetric fretting. Contact pressure and frictional shear loading cases are solved separately and superposed appropriately depending on the coefficient of friction considered. Results for different values of coefficient of friction and elastic mismatch are illustrated through contour plots of stresses and strains. These results are expected to be helpful for identifying fretting failure zones and fracture mechanisms in coated components. Analytical results presented here could serve as useful benchmarks for calibrating numerical codes and experimental techniques.

  18. Application of PIC microcontrollers in single-sensor dual gas-CO/CH4 detectors

    Science.gov (United States)

    Pietraszek, Stanislaw; Pachole, Aleksander

    2001-08-01

    The main aim of this work is to present an application of PIC16 microcontrollers in single sensor two gas - CO and CH4 detectors, using recently developed by FIS Incorporated, semiconductor sensor SB-95. Microcontroller is used not only for comparison the actual output signal from the sensor with the warning and alarm thresholds, but also provides control and self diagnostic functions. Output logic signals allow user to check the level of gas concentration and detect the improper operation of sensor and electronics circuits.

  19. Understanding and modeling Förster-type resonance energy transfer (FRET) introduction to FRET

    CERN Document Server

    Govorov, Alexander; Demir, Hilmi Volkan

    2016-01-01

    This Brief presents a historical overview of the Förster-type nonradiative energy transfer and a compilation of important progress in FRET research, starting from Förster until today, along with a summary of the current state-of-the-art. Here the objective is to provide the reader with a complete account of important milestones in FRET studies and FRET applications as well as a picture of the current status.

  20. Low-cost vibration sensor based on dual fiber Bragg gratings and light intensity measurement.

    Science.gov (United States)

    Gao, Xueqing; Wang, Yongjiao; Yuan, Bo; Yuan, Yinquan; Dai, Yawen; Xu, Gang

    2013-09-20

    A vibration monitoring system based on light intensity measurement has been constructed, and the designed accelerometer is based on steel cantilever frame and dual fiber Bragg gratings (FBGs). By using numerical simulations for the dual FBGs, the dependence relationship of the area of main lobes on the difference of initial central wavelengths is obtained and the most optimal choice for the initial value and the vibration amplitude of the difference of central wavelengths of two FBGs is suggested. The vibration monitoring experiments are finished, and the measured data are identical to the simulated results.

  1. Dual sensor platforms for UXO/landmine detection using GPR and EMI

    Science.gov (United States)

    Marble, Jay; Hong, Kevin

    2010-04-01

    Metal detectors and ground penetrating radar have become the standard sensors for buried landmine and UXO detection. Joint systems have existed since the late 90s. Recent system development has again led to the placement of MD and GPR sensors on ground vehicles for detection of in-road landmine and UXO objects. In this work, two prominent systems - one GPR and one metal detector - are operated on a test site populated with landmine and deep buried UXO. The strength of the GPR is the ability to detect plastic cased landmines while the strength of the metal detector is to detect deep buried UXO. The sensors' capabilities overlap in regards to metal cased landmines. A simple fusion approach is used to show how these two sensors can be used together to create a platform that carries the strengths of both sensors. The final alarm list averages the confidence values produced by each sensor. ROC curves are used to quantify the performance. Curves are presented for each sensor standing alone and for their fusion performance.

  2. Dual-gate thin-film transistors, integrated circuits and sensors

    NARCIS (Netherlands)

    Spijkman, M.-J.; Myny, K.; Smits, E.C.P.; Heremans, P.; Blom, P.W.M.; Leeuw, D.M. de

    2011-01-01

    The first dual-gate thin-film transistor (DGTFT) was reported in 1981 with CdSe as the semiconductor. Other TFT technologies such as a-Si:H and organic semiconductors have led to additional ways of making DGTFTs. DGTFTs contain a second gate dielectric with a second gate positioned opposite of the f

  3. Dual-Gate Thin-Film Transistors, Integrated Circuits and Sensors

    NARCIS (Netherlands)

    Spijkman, Mark-Jan; Myny, Kris; Smits, Edsger C. P.; Heremans, Paul; Blom, Paul W. M.; de Leeuw, Dago M.

    2011-01-01

    The first dual-gate thin-film transistor (DGTFT) was reported in 1981 with CdSe as the semiconductor. Other TFT technologies such as a-Si:H and organic semiconductors have led to additional ways of making DGTFTs. DGTFTs contain a second gate dielectric with a second gate positioned opposite of the f

  4. A Novel Low-Cost Dual-Wavelength Precipitation Radar Sensor Network Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Remote Sensing Solutions, Inc. (RSS) has developed a novel, practical design that will produce a low-cost precipitation radar / radiometer sensor. Operating in a...

  5. A Dual-Linear Kalman Filter for Real-Time Orientation Determination System Using Low-Cost MEMS Sensors.

    Science.gov (United States)

    Zhang, Shengzhi; Yu, Shuai; Liu, Chaojun; Yuan, Xuebing; Liu, Sheng

    2016-02-20

    To provide a long-time reliable orientation, sensor fusion technologies are widely used to integrate available inertial sensors for the low-cost orientation estimation. In this paper, a novel dual-linear Kalman filter was designed for a multi-sensor system integrating MEMS gyros, an accelerometer, and a magnetometer. The proposed filter precludes the impacts of magnetic disturbances on the pitch and roll which the heading is subjected to. The filter can achieve robust orientation estimation for different statistical models of the sensors. The root mean square errors (RMSE) of the estimated attitude angles are reduced by 30.6% under magnetic disturbances. Owing to the reduction of system complexity achieved by smaller matrix operations, the mean total time consumption is reduced by 23.8%. Meanwhile, the separated filter offers greater flexibility for the system configuration, as it is possible to switch on or off the second stage filter to include or exclude the magnetometer compensation for the heading. Online experiments were performed on the homemade miniature orientation determination system (MODS) with the turntable. The average RMSE of estimated orientation are less than 0.4° and 1° during the static and low-dynamic tests, respectively. More realistic tests on two-wheel self-balancing vehicle driving and indoor pedestrian walking were carried out to evaluate the performance of the designed MODS when high accelerations and angular rates were introduced. Test results demonstrate that the MODS is applicable for the orientation estimation under various dynamic conditions. This paper provides a feasible alternative for low-cost orientation determination.

  6. Electromagnetic soil properties variability in a minefield trial site in Cambodia and its effect on the detection of mine-like targets with a dual-sensor system

    NARCIS (Netherlands)

    Ranada Shaw, A.; Gorriti, A.; Schoolderman, A.J.; Rhebergen, J.B.

    2006-01-01

    In this paper results are presented of a study on the performance of a dual-sensor landmine detector and its dependency on soil moisture. The detector was used on a trial site in the K5 mine belt in Cambodia. Soil samples were taken from the trial lane, as well as GPR measurements. The data obtained

  7. Electromagnetic soil properties variability in a minefield trial site in Cambodia and its effect on the detection of mine-like targets with a dual-sensor system

    NARCIS (Netherlands)

    Ranada Shaw, A.; Gorriti, A.; Schoolderman, A.J.; Rhebergen, J.B.

    2006-01-01

    In this paper results are presented of a study on the performance of a dual-sensor landmine detector and its dependency on soil moisture. The detector was used on a trial site in the K5 mine belt in Cambodia. Soil samples were taken from the trial lane, as well as GPR measurements. The data obtained

  8. A reliable dual-redundant sensor FDI system for the NASA F8C-DFBW aircraft

    Science.gov (United States)

    Deckert, J. C.; Desai, M. N.; Deyst, J. J., Jr.; Willsky, A. S.

    1976-01-01

    In this paper we outline the structure of a sensor failure detection and identification (FDI) system designed for the NASA F8C DFBW aircraft. The system is for use in a dual-redundant environment, and it takes maximal advantage of all functional and kinematic relationships among the sensed variables. The technique is reliable in that we have designed the system to account for a variety of unmodeled effects and have introduced a new concept - the quality sequential probability ratio (QSPR) - that provides a useful measure of our confidence in each test. The methodology used for determining detector parameters and the 'outer loop logic' for combining the various sources of information are described.

  9. A Dynamic Range Expansion Technique for CMOS Image Sensors with Dual Charge Storage in a Pixel and Multiple Sampling

    Science.gov (United States)

    Shafie, Suhaidi; Kawahito, Shoji; Itoh, Shinya

    2008-01-01

    A dynamic range expansion technique for CMOS image sensors with dual charge storage in a pixel and multiple sampling technique is presented. Each pixel contains a photodiode and a storage diode which is connected to the photodiode via a separation gate. The sensitivity of the signal charge in the storage diode can be controlled either by a separation gate which limits the charge to flow into the storage diode or by controlling the accumulation time in the storage diode. The operation of the sensitivity control with separation gate techniques is simulated and it is found that a blocking layer to the storage diode plays an important role for high controllability of sensitivity of the storage diode. A prototype chip for testing multiple short time accumulations is fabricated and measured. PMID:27879802

  10. A Dynamic Range Expansion Technique for CMOS Image Sensors with Dual Charge Storage in a Pixel and Multiple Sampling

    Directory of Open Access Journals (Sweden)

    Shinya Itoh

    2008-03-01

    Full Text Available A dynamic range expansion technique for CMOS image sensors with dual charge storage in a pixel and multiple sampling technique is presented. Each pixel contains a photodiode and a storage diode which is connected to the photodiode via a separation gate. The sensitivity of the signal charge in the storage diode can be controlled either by a separation gate which limits the charge to flow into the storage diode or by controlling the accumulation time in the storage diode. The operation of the sensitivity control with separation gate techniques is simulated and it is found that a blocking layer to the storage diode plays an important role for high controllability of sensitivity of the storage diode. A prototype chip for testing multiple short time accumulations is fabricated and measured.

  11. Nanoscale Electrochemical Sensor Arrays: Redox Cycling Amplification in Dual-Electrode Systems.

    Science.gov (United States)

    Wolfrum, Bernhard; Kätelhön, Enno; Yakushenko, Alexey; Krause, Kay J; Adly, Nouran; Hüske, Martin; Rinklin, Philipp

    2016-09-20

    Micro- and nanofabriation technologies have a tremendous potential for the development of powerful sensor array platforms for electrochemical detection. The ability to integrate electrochemical sensor arrays with microfluidic devices nowadays provides possibilities for advanced lab-on-a-chip technology for the detection or quantification of multiple targets in a high-throughput approach. In particular, this is interesting for applications outside of analytical laboratories, such as point-of-care (POC) or on-site water screening where cost, measurement time, and the size of individual sensor devices are important factors to be considered. In addition, electrochemical sensor arrays can monitor biological processes in emerging cell-analysis platforms. Here, recent progress in the design of disease model systems and organ-on-a-chip technologies still needs to be matched by appropriate functionalities for application of external stimuli and read-out of cellular activity in long-term experiments. Preferably, data can be gathered not only at a singular location but at different spatial scales across a whole cell network, calling for new sensor array technologies. In this Account, we describe the evolution of chip-based nanoscale electrochemical sensor arrays, which have been developed and investigated in our group. Focusing on design and fabrication strategies that facilitate applications for the investigation of cellular networks, we emphasize the sensing of redox-active neurotransmitters on a chip. To this end, we address the impact of the device architecture on sensitivity, selectivity as well as on spatial and temporal resolution. Specifically, we highlight recent work on redox-cycling concepts using nanocavity sensor arrays, which provide an efficient amplification strategy for spatiotemporal detection of redox-active molecules. As redox-cycling electrochemistry critically depends on the ability to miniaturize and integrate closely spaced electrode systems, the

  12. Dual-functional sensor based on switchable plasmonic structure of VO2 nano-crystal films and Ag nanoparticles.

    Science.gov (United States)

    Yi, Mingfang; Lu, Changgui; Gong, Yan; Qi, Zhengqing; Cui, Yiping

    2014-12-01

    Utilizing the insulator-metal phase transition of vanadium dioxide (VO2) crystal films, we develop a dual-functional sensor based on the coupling between VO2 nano-crystal films and Ag nanoparticles, which can probe fluorescence or Raman signals on the same substrate and it is switchable by changing temperature. At room temperature, the VO2 crystal films is insulator phase and the fluorescence signals of probe molecules (R6G) is detectable (Raman is in "off"). At high temperature (such as 85 °C), the VO2 crystal films become metallic phase. Ag nanoparticles interact with the metal phase of VO2 crystal films to produce stronger localized electric field. The stronger electric field can excite the Raman signals of probe molecules (R6G) and the coupled structure can also emit the Raman signals out efficiently (Raman is in "on"). The switchable probe of fluorescence and Raman signals would have potential applications in active photoelectric components, such as intelligent switch and multifunctional active sensor etc.

  13. CO ppb sensors based on monodispersed SnOx:Pd mixed nanoparticle layers: Insight into dual conductance response

    Science.gov (United States)

    Aruna, I.; Kruis, F. E.; Kundu, S.; Muhler, M.; Theissmann, R.; Spasova, M.

    2009-03-01

    This study reports the modifications in CO sensing of SnOx nanoparticle layers by utilizing monodispersed Pd nanoparticles. The distinct advantage of monosized particles and contaminant-free samples with open porosity in addition to size effects resulted in improved CO sensing with decrease in Pd nanoparticle size to 5 nm, decreasing the lowest detection levels of CO using SnOx-based sensor technology down to 10 ppb (parts per billion) in dry synthetic air. The homogeneously mixed nanoparticle layers also exhibit discrimination capability between CO and ethanol in dry air as a manifestation of the dual conductance response. Detailed x-ray photoelectron spectroscopy studies clearly reveal "Mars-van Krevelen" as the key mechanism responsible for the observed sensing in mixed nanoparticle layers. The interfacial/surface PdO formed upon pretreatment in air is continuously "consumed" and "reformed" upon exposure, respectively, to CO and synthetic air. In contrast to the case of ethanol exposure with n-type response, the Pd aided reduction of tin oxide surface in CO ambient leads to p-type response. The sensors of the present study have a wide range of promising applications from air quality control to food and fuel industries.

  14. Full Dynamic-Range Pressure Sensor Matrix Based on Optical and Electrical Dual-Mode Sensing.

    Science.gov (United States)

    Wang, Xiandi; Que, Miaoling; Chen, Mengxiao; Han, Xun; Li, Xiaoyi; Pan, Caofeng; Wang, Zhong Lin

    2017-01-06

    Pressure sensor matrix (PSM) with full dynamic range can accurately detect and spatially map pressure profile. A 100 × 100 large-scale PSM gives both electrical and optical signals by itself without applying an external power. The device represents a major step toward digital imaging and visible display of pressure distribution covers a large dynamic range.

  15. A Novel Dual Traffic/Flash Flood Monitoring System Using Passive Infrared/Ultrasonic Sensors

    KAUST Repository

    Mousa, Mustafa

    2015-10-19

    Floods are the most common type of natural disaster, causing thousands of casualties every year. Among these events, urban flash floods are particularly deadly because of the short timescales on which they occur, and because of the high concentration of population in cities. Since most flash flood casualties are caused by a lack of information, it is critical to generate accurate and detailed warnings of flash floods. However, deploying an infrastructure that solely monitor flash floods makes little economic sense, since the average periodicity of catastrophic flash floods exceeds the lifetime of a typical sensor network. To address this issue, we propose a new sensing device that can simultaneously monitor urban flash floods and another phenomenon of interest (traffic congestion on the present case). This sensing device is based on the combination of an ultrasonic rangefinder with one or multiple remote temperature sensors. We show an implementation of this device, and illustrate its performance in both traffic flow and flash flood sensing. Field data shows that the sensor can detect vehicles with a 99% accuracy, in addition to estimating their speed and classifying them in function of their length. The same sensor can also monitor urban water levels with an accuracy of less than 2 cm. Two of the sensors have been deployed in a flood prone area, where they captured the only (minor) flash flood that occurred over the one-year test period, with no false detection, and an agreement in the estimated water level estimate (during the flash flood event) of about 2 cm.

  16. Corrosion and Fretting of a Modular Hip System: A Retrieval Analysis of 60 Rejuvenate Stems.

    Science.gov (United States)

    De Martino, Ivan; Assini, Joseph B; Elpers, Marcella E; Wright, Timothy M; Westrich, Geoffrey H

    2015-08-01

    Femoral stems with dual-taper modularity were introduced to allow independent control of length, offset, and version. Corrosion and fretting related to micromotion at the neck-stem junction are thought to stimulate an adverse local tissue reaction (ALTR). Analysis of 60 consecutively retrieved modular-neck stem implants (Rejuvenate, Stryker) revised primarily for ALTR was done to determine the variables influencing corrosion and fretting patterns at the neck-stem interface. Taper damage evaluation was performed with stereomicrocopic analysis with two observers. Evidence of fretting and corrosion was seen at the neck-stem taper in all implants, including three implants revised for periprosthetic fractures within four weeks of the index surgery indicating that this process starts early. Femoral stems paired with the long overall neck lengths had significantly higher corrosion scores. Correlation of the corrosion severity at particular locations with the length of implantation suggests that the neck-stem junction experiences cyclic cantilever bending in vivo. The positive correlation between the length of implantation and fretting/corrosion scores bodes poorly for patients who still have this implant. Scanning electron microscopy on a subset of specimens was also performed to evaluate the black corrosion material. We strongly urge frequent follow-up exams for every patient with this particular modular hip stem. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Action-FRET of a Gaseous Protein

    Science.gov (United States)

    Daly, Steven; Knight, Geoffrey; Halim, Mohamed Abdul; Kulesza, Alexander; Choi, Chang Min; Chirot, Fabien; MacAleese, Luke; Antoine, Rodolphe; Dugourd, Philippe

    2017-01-01

    Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question—and thus further develop mass spectrometry as a tool for structural biology—structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state—where a systematic decrease is observed—and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization.

  18. Nanomaterial-based biosensors using dual transducing elements for solution phase detection.

    Science.gov (United States)

    Li, Ning; Su, Xiaodi; Lu, Yi

    2015-05-07

    Biosensors incorporating nanomaterials have demonstrated superior performance compared to their conventional counterparts. Most reported sensors use nanomaterials as a single transducer of signals, while biosensor designs using dual transducing elements have emerged as new approaches to further improve overall sensing performance. This review focuses on recent developments in nanomaterial-based biosensors using dual transducing elements for solution phase detection. The review begins with a brief introduction of the commonly used nanomaterial transducers suitable for designing dual element sensors, including quantum dots, metal nanoparticles, upconversion nanoparticles, graphene, graphene oxide, carbon nanotubes, and carbon nanodots. This is followed by the presentation of the four basic design principles, namely Förster Resonance Energy Transfer (FRET), Amplified Fluorescence Polarization (AFP), Bio-barcode Assay (BCA) and Chemiluminescence (CL), involving either two kinds of nanomaterials, or one nanomaterial and an organic luminescent agent (e.g. organic dyes, luminescent polymers) as dual transducers. Biomolecular and chemical analytes or biological interactions are detected by their control of the assembly and disassembly of the two transducing elements that change the distance between them, the size of the fluorophore-containing composite, or the catalytic properties of the nanomaterial transducers, among other property changes. Comparative discussions on their respective design rules and overall performances are presented afterwards. Compared with the single transducer biosensor design, such a dual-transducer configuration exhibits much enhanced flexibility and design versatility, allowing biosensors to be more specifically devised for various purposes. The review ends by highlighting some of the further development opportunities in this field.

  19. New Low Cost Structure for Dual Axis Mount Solar Tracking System Using Adaptive Solar Sensor

    DEFF Research Database (Denmark)

    Argeseanu, Alin; Ritchie, Ewen; Leban, Krisztina Monika

    2010-01-01

    A solar tracking system is designed to optimize the operation of solar energy receivers. The objective of this paper is proposing a new tracking system structure with two axis. The success strategy of this new project focuses on the economical analysis of solar energy. Therefore it is important...... to determine the most cost effective design, to consider the costs of production and maintenance, and operating. The proposed tracking system uses a new solar sensor position with an adaptive feature....

  20. A Cluster-Based Dual-Adaptive Topology Control Approach in Wireless Sensor Networks.

    Science.gov (United States)

    Gui, Jinsong; Zhou, Kai; Xiong, Naixue

    2016-09-25

    Multi-Input Multi-Output (MIMO) can improve wireless network performance. Sensors are usually single-antenna devices due to the high hardware complexity and cost, so several sensors are used to form virtual MIMO array, which is a desirable approach to efficiently take advantage of MIMO gains. Also, in large Wireless Sensor Networks (WSNs), clustering can improve the network scalability, which is an effective topology control approach. The existing virtual MIMO-based clustering schemes do not either fully explore the benefits of MIMO or adaptively determine the clustering ranges. Also, clustering mechanism needs to be further improved to enhance the cluster structure life. In this paper, we propose an improved clustering scheme for virtual MIMO-based topology construction (ICV-MIMO), which can determine adaptively not only the inter-cluster transmission modes but also the clustering ranges. Through the rational division of cluster head function and the optimization of cluster head selection criteria and information exchange process, the ICV-MIMO scheme effectively reduces the network energy consumption and improves the lifetime of the cluster structure when compared with the existing typical virtual MIMO-based scheme. Moreover, the message overhead and time complexity are still in the same order of magnitude.

  1. FRET in membrane biophysics: an overview

    Directory of Open Access Journals (Sweden)

    Luis MS Loura

    2011-11-01

    Full Text Available Förster resonance energy transfer (FRET, in most applications used as a spectroscopicruler, allows an easy determination of the donor-acceptor intermolecular distance.However, the situation becomes complex in membranes, since around each donor there isan ensemble of acceptors at non-correlated distances. In this review, state-of-the-artmethodologies for this situation are presented, usually involving time-resolved data andmodel fitting. This powerful approach can be used to study the occurrence of phaseseparation (rafts or other type of domains, allowing their detection as well as sizeevaluation. Formalisms for studying lipid-protein and protein-protein interactionsaccording to specific topologies are also addressed. The advantages and addedcomplexity of a specific type of FRET (energy homo-transfer or energy migration aredescribed, as well as applications of FRET under the microscope.

  2. Single Molecule Sensitive FRET in Attoliter Droplets

    CERN Document Server

    Milas, Peker; Gamari, Ben D; Goldner, Lori S

    2013-01-01

    Single molecular-pair fluorescence resonance energy transfer (spFRET) has become an cross-disciplinary tool for understanding molecular folding and interactions. While providing detailed information about the individual members of a molecular ensemble, this technique is always limited by fluorophore brightness and stability. In the case of diffusing molecules, the experiment is further limited by the number of photons that can be collected during the time it takes for a molecule to diffuse across the detection volume. To maximize the number of photons it is common to either increase the detection volume at the expense of increased background, or increase the diffusion time by adding glycerol or sucrose to increase viscosity. Here we demonstrate that FRET from attoliter volume (100 nm radius) aqueous droplets in perfluorinated oil has significantly higher signal-to-noise and a much wider dynamic range than FRET from molecules diffusing in solution. However, our measurements also reveal a droplet environment th...

  3. Highly sensitive liquid-level sensor based on dual-wavelength double-ring fiber laser assisted by beat frequency interrogation.

    Science.gov (United States)

    Dai, Yi; Sun, Qizhen; Tan, Sisi; Wo, Jianghai; Zhang, Jiejun; Liu, Deming

    2012-12-01

    A highly sensitive liquid-level sensor based on dual-wavelength single-longitudinal-mode fiber laser is proposed and demonstrated. The laser is formed by exploiting two parallel arranged phase-shift fiber Bragg gratings (ps-FBGs), acting as ultra-narrow bandwidth filters, into a double-ring resonators. By beating the dual-wavelength lasing output, a stable microwave signal with frequency stability better than 5 MHz is obtained. The generated beat frequency varies with the change of dual-wavelength spacing. Based on this characteristic, with one ps-FBG serving as the sensing element and the other one acting as the reference element, a highly sensitive liquid level sensor is realized by monitoring the beat frequency shift of the laser. The sensor head is directly bonded to a float which can transfer buoyancy into axial strain on the fiber without introducing other elastic elements. The experimental results show that an ultra-high liquid-level sensitivity of 2.12 × 10(7) MHz/m within the measurement range of 1.5 mm is achieved. The sensor presents multiple merits including ultra-high sensitivity, thermal insensitive, good reliability and stability.

  4. Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

    Science.gov (United States)

    Kotera, Ippei; Iwasaki, Takuya; Imamura, Hiromi; Noji, Hiroyuki; Nagai, Takeharu

    2010-02-19

    Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

  5. The measurement of gas-liquid two-phase flows in a small diameter pipe using a dual-sensor multi-electrode conductance probe

    Science.gov (United States)

    Zhai, Lu-Sheng; Bian, Peng; Han, Yun-Feng; Gao, Zhong-Ke; Jin, Ning-De

    2016-04-01

    We design a dual-sensor multi-electrode conductance probe to measure the flow parameters of gas-liquid two-phase flows in a vertical pipe with an inner diameter of 20 mm. The designed conductance probe consists of a phase volume fraction sensor (PVFS) and a cross-correlation velocity sensor (CCVS). Through inserting an insulated flow deflector in the central part of the pipe, the gas-liquid two-phase flows are forced to pass through an annual space. The multiple electrodes of the PVFS and the CCVS are flush-mounted on the inside of the pipe wall and the outside of the flow deflector, respectively. The geometry dimension of the PVFS is optimized based on the distribution characteristics of the sensor sensitivity field. In the flow loop test of vertical upward gas-liquid two-phase flows, the output signals from the dual-sensor multi-electrode conductance probe are collected by a data acquisition device from the National Instruments (NI) Corporation. The information transferring characteristics of local flow structures in the annular space are investigated using the transfer entropy theory. Additionally, the kinematic wave velocity is measured based on the drift velocity model to investigate the propagation behavior of the stable kinematic wave in the annular space. Finally, according to the motion characteristics of the gas-liquid two-phase flows, the drift velocity model based on the flow patterns is constructed to measure the individual phase flow rate with higher accuracy.

  6. FAO-56 Dual Model Combined with Multi-Sensor Remote Sensing for Regional Evapotranspiration Estimations

    Directory of Open Access Journals (Sweden)

    Rim Amri

    2014-06-01

    Full Text Available The main goal of this study is to evaluate the potential of the FAO-56 dual technique for the estimation of regional evapotranspiration (ET and its constituent components (crop transpiration and soil evaporation, for two classes of vegetation (olives trees and cereals in the semi-arid region of the Kairouan plain in central Tunisia. The proposed approach combines the FAO-56 technique with remote sensing (optical and microwave, not only for vegetation characterization, as proposed in other studies but also for the estimation of soil evaporation, through the use of satellite moisture products. Since it is difficult to use ground flux measurements to validate remotely sensed data at regional scales, comparisons were made with the land surface model ISBA-A-gs which is a physical SVAT (Soil–Vegetation–Atmosphere Transfer model, an operational tool developed by Météo-France. It is thus shown that good results can be obtained with this relatively simple approach, based on the FAO-56 technique combined with remote sensing, to retrieve temporal variations of ET. The approach proposed for the daily mapping of evapotranspiration at 1 km resolution is approved in two steps, for the period between 1991 and 2007. In an initial step, the ISBA-A-gs soil moisture outputs are compared with ERS/WSC products. Then, the output of the FAO-56 technique is compared with the output generated by the SVAT ISBA-A-gs model.

  7. CTAB enhancement of FRET in DNA structures.

    Science.gov (United States)

    Oh, Taeseok; Takahashi, Tsukasa; Kim, Sejung; Heller, Michael J

    2016-01-01

    The effect of cetyl-trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye-conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye-conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

  8. Dual Mode Sensing with Low-Profile Piezoelectric Thin Wafer Sensors for Steel Bridge Crack Detection and Diagnosis

    Directory of Open Access Journals (Sweden)

    Lingyu Yu

    2012-01-01

    Full Text Available Monitoring of fatigue cracking in steel bridges is of high interest to many bridge owners and agencies. Due to the variety of deterioration sources and locations of bridge defects, there is currently no single method that can detect and address the potential sources globally. In this paper, we presented a dual mode sensing methodology integrating acoustic emission and ultrasonic wave inspection based on the use of low-profile piezoelectric wafer active sensors (PWAS. After introducing the research background and piezoelectric sensing principles, PWAS crack detection in passive acoustic emission mode is first presented. Their acoustic emission detection capability has been validated through both static and compact tension fatigue tests. With the use of coaxial cable wiring, PWAS AE signal quality has been improved. The active ultrasonic inspection is conducted by the damage index and wave imaging approach. The results in the paper show that such an integration of passive acoustic emission detection with active ultrasonic sensing is a technological leap forward from the current practice of periodic and subjective visual inspection and bridge management based primarily on history of past performance.

  9. Peg-in-Hole Assembly Based on Two-phase Scheme and F/T Sensor for Dual-arm Robot.

    Science.gov (United States)

    Zhang, Xianmin; Zheng, Yanglong; Ota, Jun; Huang, Yanjiang

    2017-09-01

    This paper focuses on peg-in-hole assembly based on a two-phase scheme and force/torque sensor (F/T sensor) for a compliant dual-arm robot, the Baxter robot. The coordinated operations of human beings in assembly applications are applied to the behaviors of the robot. A two-phase assembly scheme is proposed to overcome the inaccurate positioning of the compliant dual-arm robot. The position and orientation of assembly pieces are adjusted respectively in an active compliant manner according to the forces and torques derived by a six degrees-of-freedom (6-DOF) F/T sensor. Experiments are conducted to verify the effectiveness and efficiency of the proposed assembly scheme. The performances of the dual-arm robot are consistent with those of human beings in the peg-in-hole assembly process. The peg and hole with 0.5 mm clearance for round pieces and square pieces can be assembled successfully.

  10. New dual emission fluorescent sensor for pH and Pb(II) based on bis(napfthalimide) derivative

    Energy Technology Data Exchange (ETDEWEB)

    Pina-Luis, Georgina, E-mail: gpinaluis@yahoo.com [Centro de Graduados e Investigacion en Quimica, Instituto Tecnologico de Tijuana, AP 1166, Tijuana 22500, BC (Mexico); Martinez-Quiroz, Marisela; Ochoa-Teran, Adrian [Centro de Graduados e Investigacion en Quimica, Instituto Tecnologico de Tijuana, AP 1166, Tijuana 22500, BC (Mexico); Santacruz-Ortega, Hisila [Departamento de investigacion en Polimeros y Materiales, Universidad de Sonora, Hermosillo, Sonora 83000 (Mexico); Mendez-Valenzuela, Eduardo [Centro de Graduados e Investigacion en Quimica, Instituto Tecnologico de Tijuana, AP 1166, Tijuana 22500, BC (Mexico)

    2013-02-15

    This paper describes a novel dual emission bis-1,8-naphthalimide sensor for selective determination of pH and Pb{sup 2+} ions. The influence of the variability in the backbone that links the two fluorophores (naphthalimides) as a function of pH and metal ions was studied by UV-visible and fluorescence spectroscopy. Compounds 1(a-d) with different length alkyl linkers (CH{sub 2}){sub n} (n=1, 2, 4 and 6) showed no excimer formation in aqueous solution. Fluorescence emission of these derivatives varied in a narrow range of pH (5-8) and was only slightly influenced by the addition of metal ions in CH{sub 3}CN solutions. However, derivative 1e with amino-containing spacer (CH{sub 2}-NH-CH{sub 2}) showed excimer emission in aqueous solution, a wide response to pH (2.5-9.5) and fluorescence enhancement with selective behavior towards metal ions. The pH sensor based in derivative 1e has a sufficient selectivity for practical pH monitoring in the presence of Li{sup +}, Na{sup +}, K{sup +}, Cs{sup +}, Ca{sup 2+}, Mg{sup 2+}, Ba{sup 2+}, Cu{sup 2+}, Pb{sup 2+}, Ni{sup 2+}, Zn{sup 2+} and Cd{sup 2+}. The coordination chemistry of these complexes was studied by UV-Vis, fluorescence and {sup 1}H NMR. This chemosensor displayed high selectivity fluorescence enhancement toward Pb{sup 2+} ions in the presence of the metals ions mentioned in CH{sub 3}CN solutions. Competitive assays show that a 1-fold of metal cations in each case, compared with Pb{sup 2+} ions, results in less than {+-}5% fluorescence intensity changes. Linear calibration up to 1 Multiplication-Sign 10{sup -5} M for Pb(II) ions (R=0.9968) was obtained and detection limit resulted of 5.0 Multiplication-Sign 10{sup -8} M. - Highlights: Black-Right-Pointing-Pointer A novel dual emission bis-1,8-naphthalimide sensor for pH and Pb{sup 2+} ions is synthetized. Black-Right-Pointing-Pointer The excimer formation depends on the spacer that links the two naphthalimide groups. Black-Right-Pointing-Pointer Bis

  11. Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

    Science.gov (United States)

    Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

    2013-07-21

    To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection

  12. Anomalous surplus energy transfer observed with multiple FRET acceptors.

    Directory of Open Access Journals (Sweden)

    Srinagesh V Koushik

    Full Text Available BACKGROUND: Förster resonance energy transfer (FRET is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k(D-->A can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates. METHODOLOGY/PRINCIPAL FINDINGS: The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean and either two or three FRET acceptors (Venus. FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates. CONCLUSIONS/SIGNIFICANCE: We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is

  13. A passive UHF RFID tag chip with a dual-resolution temperature sensor in a 0.18μm standard CMOS process

    Institute of Scientific and Technical Information of China (English)

    Feng Peng; Zhang Qi; Wu Nanjian

    2011-01-01

    This paper presents a passive EPC Gen-2 UHF RFID tag chip with a dual-resolution temperature sensor.The chip tag integrates a temperature sensor,an RF/analog front-end circuit,an NVM memory and a digital baseband in a standard CMOS process.The sensor with a low power sigma-delta (Σ△) ADC is designed to operate in low and high resolution modes.It can not only achieve the target accuracy but also reduce the power consumption and the sensing time.A CMOS-only RF rectifier and a single-poly non-volatile memory (NVM) are designed to realize a low cost tag chip.The 192-bit-NVM tag chip with an area of 1 mm2 is implemented in a 0.18-μm standard CMOS process.The sensitivity of the tag is -10.7 dBm/-8.4 dBm when the sensor is disabled/enabled.It achieves a maximum reading/sensing distance of 4 m/3.1 m at 2 W EIRP.The inaccuracy of the sensor is -0.6 ℃/0.5 ℃ (-1.0 ℃/1.2 ℃) in the operating range from 5 to 15 ℃ in high resolution mode (-30 to 50 ℃ in low resolution mode).The resolution of the sensor achieves 0.02 ℃ (0.18 ℃) in high (low) resolution mode.

  14. Deep Sub-micro mol{\\cdot }mol^{-1} Water-Vapor Measurement by Dual-Ball SAW Sensors for Temperature Compensation

    Science.gov (United States)

    Takeda, N.; Oizumi, T.; Tsuji, T.; Akao, S.; Takayanagi, K.; Nakaso, N.; Yamanaka, K.

    2015-12-01

    A collimated surface acoustic wave (SAW) circles around the equator of a sphere hundreds of times. Because of the long distance travel of the collimated SAW, a small change in the SAW propagation caused by the environment of the sphere can be accumulated as a measurable range in amplitude and/or in delay time. So, a spherical SAW device enables highly sensitive water-vapor measurements. In this paper, deep sub \\upmu mol{\\cdot }mol^{-1} water-vapor detection by 1 mm diameter quartz crystal ball SAW sensors is described. To measure such a low water-vapor concentration in real time, it is necessary to compensate the temperature dependence of the ball SAW sensor, which is about 20 ppm{\\cdot }°C^{-1} in delay time change. A dual-frequency burst analog detector was developed for the temperature compensation in real time. By using a harmonic SAW sensor, which was excited by 80 MHz and 240 MHz at the same time, it was confirmed that the delay time drift for a temperature range of 21.0°C ± 1.0°C became less than 0.05 ppm in delay time change. By using dual-ball SAW sensors (which included a 150 MHz sensor with a water-vapor sensitive layer and a 240 MHz sensor as a reference), water-vapor concentrations from 0.1 \\upmu mol{\\cdot }mol^{-1} to 5 \\upmu mol{\\cdot }mol^{-1} were successfully measured. It appears that the delay time change is proportional to the square root of the water-vapor concentration. The detection limit determined by the electrical noise of the system was estimated at 0.01 \\upmu mol{\\cdot }mol^{-1}.

  15. Sensors

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, H. [PBI-Dansensor A/S (Denmark); Toft Soerensen, O. [Risoe National Lab., Materials Research Dept. (Denmark)

    1999-10-01

    A new type of ceramic oxygen sensors based on semiconducting oxides was developed in this project. The advantage of these sensors compared to standard ZrO{sub 2} sensors is that they do not require a reference gas and that they can be produced in small sizes. The sensor design and the techniques developed for production of these sensors are judged suitable by the participating industry for a niche production of a new generation of oxygen sensors. Materials research on new oxygen ion conducting conductors both for applications in oxygen sensors and in fuel was also performed in this project and finally a new process was developed for fabrication of ceramic tubes by dip-coating. (EHS)

  16. Sensors

    CERN Document Server

    Pigorsch, Enrico

    1997-01-01

    This is the 5th edition of the Metra Martech Directory "EUROPEAN CENTRES OF EXPERTISE - SENSORS." The entries represent a survey of European sensors development. The new edition contains 425 detailed profiles of companies and research institutions in 22 countries. This is reflected in the diversity of sensors development programmes described, from sensors for physical parameters to biosensors and intelligent sensor systems. We do not claim that all European organisations developing sensors are included, but this is a good cross section from an invited list of participants. If you see gaps or omissions, or would like your organisation to be included, please send details. The data base invites the formation of effective joint ventures by identifying and providing access to specific areas in which organisations offer collaboration. This issue is recognised to be of great importance and most entrants include details of collaboration offered and sought. We hope the directory on Sensors will help you to find the ri...

  17. RUNNING CONDITION FRETTING MAPS OF POLYMER MATERIALS

    Directory of Open Access Journals (Sweden)

    Szilárd Tamás Vezér

    2009-04-01

    Full Text Available Due to the industrial demand, the determination of the wear behaviour of polymeric materials is an important research task. Rubbers and elastomers are used widely in contacts, where wear is the dominant failure mechanisms. Furthermore, only the material properties under large displacements were investigated in the majority of existing studies. Depending on the input physical parameters of the tribological systems small oscillations are also observed on the measured signals (due to stick-slip like effect in the contact zone of the elastomers. To describe the failure behaviour under this special condition, a novel fretting fatigue test system was developed and built on a electro-dynamical shaker in this study. The contact area were defined with some additional test, like 2D full field strain analysis, compression and creep tests. Based on the methodology developed and applied for steels and polymer composites, Running Condition Fretting Maps for two elastomer grades (HNBR and TPU were determined.

  18. The role of oxidation in the fretting wear process

    Science.gov (United States)

    Bill, R. C.

    1980-01-01

    Fretting experiments were conducted on titanium, a series of Ni-Cr-Al alloys and on some high temperature turbine alloys at room temperature and at elevated temperatures in air and in various inert environments. It was found that, depending on temperature and environment, the fretting behavior of the materials examined could be classified according to four general types of behavior. Briefly, these types of behavior were: (1) the complete absence of oxidation, as in inert environments, generally leading to low rates of fretting wear but high fretting friction; (2) gradual attrition of surface oxide with each fretting stroke, found in these experiments to operate in concert with other dominating mechanisms; (3) rapid oxidation at surface fatigue damage sites, resulting in undermining and rapid disintegration of the load bearing surface; and (4) the formation of coherent, protective oxide film, resulting in low rates of fretting wear. An analytical model predicting conditions favorable to the fourth type of behavior was outlined.

  19. Fretting wear behavior of AZ91D magnesium alloy

    Institute of Scientific and Technical Information of China (English)

    CHEN An-hua; HUANG Wei-jiu; LI Zhao-feng

    2006-01-01

    The fretting behaviour of the AZ91D magnesium alloy was investigated. The influence of the number of cycles, normal load (contact pressure) and the amplitude of slip on the fretting behavior of the material were focused. Fretting tests were performed under various running conditions with regard to normal load levels and slip amplitudes. The friction coefficient between the surfaces at the fretting junction was continuously recorded. The fretting damage on the magnesium specimens was studied by SEM. The results show that the wear volume increases with the increase of slip amplitude, and linearly increases with the increase of normal load in the mixed and gross slip regime, but the normal load has no obvious effect on the wear volume in the partial slip regime. The predominant fretting wear mechanism of magnesium alloy in the slip regime is the oxidation wear, delaminated wear and abrasive wear.

  20. FRET and Non-FRET processes in quantum dot - dye nanoassemblies

    Energy Technology Data Exchange (ETDEWEB)

    Kowerko, Danny; Schuster, Joerg; Borczyskowski, Christian von [Center of Nanostructured Materials and Analytics, TU-Chemnitz, 09107 Chemnitz (Germany)

    2009-07-01

    Assemblies of semiconductor quantum dots (QDs - donor) and organic (dye - acceptor) molecules are of increasing interest for numerous applications in science and technology. Recent publications on such systems report on charge and fluorescence resonant energy transfer (FRET), prevalently neglecting an exact quantitative spectroscopic analysis of the acceptor. Recently, calculations of Seifert et al. and Blaudeck and coworkers demonstrated the crucial dependence of trap state emission from the surface coverage. In our contribution we emphasize the role of dye induces surface trap states as one mechanism - besides FRET - for QD quenching. Thus detailed quantitative analysis of titration experiments of differently functionalized perylenebismide molecules and CdSe/ZnS quantum dots will be discussed. By means of single particle spectroscopy we may even compare spectroscopic properties of the free and bound species on one and the same sample under identical conditions. Furthermore, analysis of FRET allows for a distinction between different geometries of the dye-QD assemblies.

  1. The Design of Dual-Emissive Composite Material [Zn2(HL)3](+)@MOF-5 as Self-Calibrating Luminescent Sensors of Al(3+) Ions and Monoethanolamine.

    Science.gov (United States)

    Wu, Meng-Meng; Wang, Jiao-Yang; Sun, Rui; Zhao, Cui; Zhao, Jiong-Peng; Che, Guang-Bo; Liu, Fu-Chen

    2017-08-21

    Introducing another chromophore into a luminescent MOF is a potential way to assembling novel dual-emissive luminescent materials. Putting the chromophore, for which luminescence can be enhanced by Zn(2+) ion, into MOF-5 by the "bottle around ship" strategy is a simple but efficient synthesis method to realize such dual-emissive materials. According to this strategy, a novel dual-emissive luminescent composite material [Zn2(HL)3](+)@MOF-5 was constructed by loading the [La3(HL)2L2(NO3)3H2O] (1) (H2L = 7,7'-(ethane-1,1'-diyl)8-hydro-quinoline) into MOF-5, in which the [Zn2(HL)3](+) anions were transformed from 1 with the existence of Zn(2+). The dual-emissive composite materials show excellent luminescence with two emissions of MOF-5 at 410 nm and [Zn2(HL)3](+) at 524 nm. Furthermore, by combining characteristics of MOF-5 and the guest chromophore, the composite material is highly selectively sensitive toward Al(3+) and monoethanolamine, which makes [Zn2(HL)3](+)@MOF-5 a potential self-calibrated fluorescence sensor.

  2. A general strategy to facilely design ratiometric electrochemical sensors in electrolyte solution by directly using a bare electrode for dual-signal sensing of analytes.

    Science.gov (United States)

    Yu, Jianbo; Jin, Hui; Gui, Rijun; Wang, Zonghua; Ge, Feng

    2017-01-01

    In this paper, we have described a general strategy to facilely design ratiometric electrochemical sensors in electrolyte solutions, directly using a bare electrode for dual-signal sensing of analytes. Two types of substances (methylene blue/MB, doxorubicin/DOX) with different electrochemical signal peaks were added into electrolyte solutions (phosphate buffered saline, NaCl), where one was the analyte (DOX) and the other was used as a reference (MB). A linear plotting of DOX concentration [DOX] versus ratiometric electrochemical signal peak intensity (IDOX/IMB) was achieved, with a good linear coefficient and low detection limit of DOX (0.4nM). Experimental results implied that this ratiometric electrochemical sensor (ECS) of DOX enabled highly selective and sensitive detection of DOX in real samples, with high detection recoveries. In comparison with previous reports about ratiometric ECS, this as-proposed strategy can directly fabricate a ratiometric ECS in electrolyte solution (not on electrode), only using a bare electrode for dual- signal sensing of analytes. This strategy is not only novel and facile, but also flexible and general, as adequately confirmed in experiments, which would facilitate a further development in the facile fabrication and efficient applications of electrochemical sensors.

  3. A Dual Conductance Sensor for Simultaneous Measurement of Void Fraction and Structure Velocity of Downward Two-Phase Flow in a Slightly Inclined Pipe

    Directory of Open Access Journals (Sweden)

    Yeon-Gun Lee

    2017-05-01

    Full Text Available In this study, a new and improved electrical conductance sensor is proposed for application not only to a horizontal pipe, but also an inclined one. The conductance sensor was designed to have a dual layer, each consisting of a three-electrode set to obtain two instantaneous conductance signals in turns, so that the area-averaged void fraction and structure velocity could be measured simultaneously. The optimum configuration of the electrodes was determined through numerical analysis, and the calibration curves for stratified and annular flow were obtained through a series of static experiments. The fabricated conductance sensor was applied to a 45 mm inner diameter U-shaped downward inclined pipe with an inclination angle of 3° under adiabatic air-water flow conditions. In the tests, the superficial velocities ranged from 0.1 to 3.0 m/s for water and from 0.1 to 18 m/s for air. The obtained mean void fraction and the structure velocity from the conductance sensor were validated against the measurement by the wire-mesh sensor and the cross-correlation technique for the visualized images, respectively. The results of the flow regime classification and the corresponding time series of the void fraction at a variety of flow velocities were also discussed.

  4. A Dual Conductance Sensor for Simultaneous Measurement of Void Fraction and Structure Velocity of Downward Two-Phase Flow in a Slightly Inclined Pipe.

    Science.gov (United States)

    Lee, Yeon-Gun; Won, Woo-Youn; Lee, Bo-An; Kim, Sin

    2017-05-08

    In this study, a new and improved electrical conductance sensor is proposed for application not only to a horizontal pipe, but also an inclined one. The conductance sensor was designed to have a dual layer, each consisting of a three-electrode set to obtain two instantaneous conductance signals in turns, so that the area-averaged void fraction and structure velocity could be measured simultaneously. The optimum configuration of the electrodes was determined through numerical analysis, and the calibration curves for stratified and annular flow were obtained through a series of static experiments. The fabricated conductance sensor was applied to a 45 mm inner diameter U-shaped downward inclined pipe with an inclination angle of 3° under adiabatic air-water flow conditions. In the tests, the superficial velocities ranged from 0.1 to 3.0 m/s for water and from 0.1 to 18 m/s for air. The obtained mean void fraction and the structure velocity from the conductance sensor were validated against the measurement by the wire-mesh sensor and the cross-correlation technique for the visualized images, respectively. The results of the flow regime classification and the corresponding time series of the void fraction at a variety of flow velocities were also discussed.

  5. Discrimination between FRET and non-FRET quenching in a photochromic CdSe quantum dot/dithienylethene dye system

    Science.gov (United States)

    Dworak, Lars; Reuss, Andreas J.; Zastrow, Marc; Rück-Braun, Karola; Wachtveitl, Josef

    2014-11-01

    A photochromic Förster resonance energy transfer (FRET) system was employed to disentangle the fluorescence quenching mechanisms in quantum dot/photochromic dye hybrids. In the off-state of the dye the main quenching mechanism is FRET whereas the moderate quenching in the on-state is due to non-FRET pathways opened up upon assembly.A photochromic Förster resonance energy transfer (FRET) system was employed to disentangle the fluorescence quenching mechanisms in quantum dot/photochromic dye hybrids. In the off-state of the dye the main quenching mechanism is FRET whereas the moderate quenching in the on-state is due to non-FRET pathways opened up upon assembly. Electronic supplementary information (ESI) available: QD and DTE synthesis, preparation of the DTE/QD coupled system, TEM image of the nanocrystals and experimental details. See DOI: 10.1039/c4nr05144k

  6. Performance of a Distributed Simultaneous Strain and Temperature Sensor Based on a Fabry-Perot Laser Diode and a Dual-Stage FBG Optical Demultiplexer

    Directory of Open Access Journals (Sweden)

    Shinwon Kang

    2013-11-01

    Full Text Available A simultaneous strain and temperature measurement method using a Fabry-Perot laser diode (FP-LD and a dual-stage fiber Bragg grating (FBG optical demultiplexer was applied to a distributed sensor system based on Brillouin optical time domain reflectometry (BOTDR. By using a Kalman filter, we improved the performance of the FP-LD based OTDR, and decreased the noise using the dual-stage FBG optical demultiplexer. Applying the two developed components to the BOTDR system and using a temperature compensating algorithm, we successfully demonstrated the simultaneous measurement of strain and temperature distributions under various experimental conditions. The observed errors in the temperature and strain measured using the developed sensing system were 0.6 °C and 50 με, and the spatial resolution was 1 m, respectively.

  7. A dual-emitting 4d-4f nanocrystalline metal-organic framework as a self-calibrating luminescent sensor for indoor formaldehyde pollution.

    Science.gov (United States)

    Hao, Ji-Na; Yan, Bing

    2016-06-09

    A dual-emissive 4d-4f Ag(i)-Eu(iii) functionalized MOF nanocomposite was fabricated and utilized as a self-calibrating luminescent nanoprobe for detecting indoor formaldehyde (FA). The implantation of Ag(+) ions can tune the dual-emissive characters of the material. FA can interact with the Ag(+) ions and induce opposite luminescence behaviors of the two emitters in the singular molecular material, thus realizing its recognition. This nanoprobe for FA exhibits many merits, such as excellent selectivity, high sensitivity with a detection limit of 51 ppb, fast response, room-temperature testing ability, easy preparation and low cost. This is the first example of a MOF-implicated self-calibrated sensor for indoor FA detection.

  8. Förster Resonance Energy Transfer (FRET as a Tool for Dissecting the Molecular Mechanisms for Maturation of the Shigella Type III Secretion Needle Tip Complex

    Directory of Open Access Journals (Sweden)

    William D. Picking

    2012-11-01

    Full Text Available Förster resonance energy transfer (FRET provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying Å-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB’s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system.

  9. A dual-emitting 4d-4f nanocrystalline metal-organic framework as a self-calibrating luminescent sensor for indoor formaldehyde pollution

    Science.gov (United States)

    Hao, Ji-Na; Yan, Bing

    2016-06-01

    A dual-emissive 4d-4f Ag(i)-Eu(iii) functionalized MOF nanocomposite was fabricated and utilized as a self-calibrating luminescent nanoprobe for detecting indoor formaldehyde (FA). The implantation of Ag+ ions can tune the dual-emissive characters of the material. FA can interact with the Ag+ ions and induce opposite luminescence behaviors of the two emitters in the singular molecular material, thus realizing its recognition. This nanoprobe for FA exhibits many merits, such as excellent selectivity, high sensitivity with a detection limit of 51 ppb, fast response, room-temperature testing ability, easy preparation and low cost. This is the first example of a MOF-implicated self-calibrated sensor for indoor FA detection.A dual-emissive 4d-4f Ag(i)-Eu(iii) functionalized MOF nanocomposite was fabricated and utilized as a self-calibrating luminescent nanoprobe for detecting indoor formaldehyde (FA). The implantation of Ag+ ions can tune the dual-emissive characters of the material. FA can interact with the Ag+ ions and induce opposite luminescence behaviors of the two emitters in the singular molecular material, thus realizing its recognition. This nanoprobe for FA exhibits many merits, such as excellent selectivity, high sensitivity with a detection limit of 51 ppb, fast response, room-temperature testing ability, easy preparation and low cost. This is the first example of a MOF-implicated self-calibrated sensor for indoor FA detection. Electronic supplementary information (ESI) available: Experimental procedure, N2 adsorption-desorption isotherms, IR spectra, EDX mappings, ICP data, luminescence data, PXRD patterns, UV-Vis spectra, and XPS spectra. See DOI: 10.1039/c6nr02446g

  10. Single molecule FRET data analysis procedures for FRET efficiency determination: probing the conformations of nucleic acid structures.

    Science.gov (United States)

    Krüger, Asger Christian; Birkedal, Victoria

    2013-11-01

    Single molecule FRET microscopy is an attractive technique for studying structural dynamics and conformational diversity of nucleic acid structures. Some of its strengths are that it can follow structural changes on a fast time scale and identify conformation distributions arising from dynamic or static population heterogeneity. Here, we give a description of the experiment and data analysis procedures of this method and detail what parameters are needed for FRET efficiency calculation. Using single molecule FRET data obtained on G-quadruplex DNA structures that exhibit large conformation diversity, we illustrate that the shape of the FRET distribution changes depending on what parameters are included in the data analysis procedure.

  11. In silico FRET from simulated dye dynamics

    Science.gov (United States)

    Hoefling, Martin; Grubmüller, Helmut

    2013-03-01

    Single molecule fluorescence resonance energy transfer (smFRET) experiments probe molecular distances on the nanometer scale. In such experiments, distances are recorded from FRET transfer efficiencies via the Förster formula, E=1/(1+(). The energy transfer however also depends on the mutual orientation of the two dyes used as distance reporter. Since this information is typically inaccessible in FRET experiments, one has to rely on approximations, which reduce the accuracy of these distance measurements. A common approximation is an isotropic and uncorrelated dye orientation distribution. To assess the impact of such approximations, we present the algorithms and implementation of a computational toolkit for the simulation of smFRET on the basis of molecular dynamics (MD) trajectory ensembles. In this study, the dye orientation dynamics, which are used to determine dynamic FRET efficiencies, are extracted from MD simulations. In a subsequent step, photons and bursts are generated using a Monte Carlo algorithm. The application of the developed toolkit on a poly-proline system demonstrated good agreement between smFRET simulations and experimental results and therefore confirms our computational method. Furthermore, it enabled the identification of the structural basis of measured heterogeneity. The presented computational toolkit is written in Python, available as open-source, applicable to arbitrary systems and can easily be extended and adapted to further problems. Catalogue identifier: AENV_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENV_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GPLv3, the bundled SIMD friendly Mersenne twister implementation [1] is provided under the SFMT-License. No. of lines in distributed program, including test data, etc.: 317880 No. of bytes in distributed program, including test data, etc.: 54774217 Distribution format: tar.gz Programming language

  12. Fret Replica Inspection Laser Scanner (FRILS)

    Energy Technology Data Exchange (ETDEWEB)

    Kretz, S.; Hanley, K., E-mail: steve.kretz@opg.com, E-mail: kelly.hanley@opg.com [Ontario Power Generation, Inspection Maintenance and Commercial Services, Pickering, Ontario (Canada)

    2008-07-01

    In the stress analysis of flaws and artifacts found in pressure tubes, it is crucial to have detailed knowledge of the flaw geometry. Fuel channel inspections by ultrasonic or eddy current inspection methods alone cannot provide the complete required geometry information. Replicas, which are a negative impression of surface pressure tube indications, are scanned with a laser system which will provide the additional detail required. FRILS was initially developed in 1993 to establish in-house capability of profiling indications on the inside diameter surface of pressure tubes. The need of this profiling was initially a response to the discovery of fuel bundle bearing pad fretting (FBBPF) caused by flow induced fuel bundle vibration. The benefits of the system were soon realized as a tool for profiling debris type indications. Although the primary use of FRILS is to profile FBBBF and Debris Fretting, since its inception the FRILS inspection system has become an instrumental tool in flaw assessment for: Fuel Bundle Bearing Pad Frets (FBBPF); Debris Frets; Scratches; Crevice Corrosion; Oxide Jacking; Areas of surface roughness; and, Weld Profiling. Replicas are collected via acquisition from tooling on both the Channel and Gauging Apparatus for Reactors (CIGAR) and the Advanced Non-Destructive Examination (ANDE) systems. The ANDE system is a high speed data acquisition system which includes both an ultrasonic inspection tool and a replication tool. Although both of these tools were designed to be delivered with the UDM, the platform for these tools was built with flexibility allowing for adoption to other delivery systems. These tools were based on the experience of the CIGAR inspection system. The CIGAR system has also undergone many system upgrades resulting in reduced inspection times. The FRILS system - Fret Replication Inspection Laser Scanner system was developed and has been upgraded to meet the demands of the improved inspection and replication systems. FRILS

  13. Influence of Fretting Wear on Lifetime of Tin Plated Connectors

    Science.gov (United States)

    Ikeda, Hirosaka; Ito, Tetsuya; Sawada, Shigeru; Hattori, Yasuhiro; Saitoh, Yasushi; Tamai, Terutaka; Iida, Kazuo

    Due to the recent increase in electronic devices mounted on automobiles, a large number of connectors, especially low-cost tin plated connectors are being used. As a result, their contact reliability has become problematic. Furthermore, for the connectors which are subjected to fretting wear caused by heat cycle and vibrations, the contact resistance increases because of wear of tin and deposition of oxides, which generates problems of poor contact. This study is intended to analyze the change in contact resistance of tin plated connectors from the start of fretting wear to the end of their lifetime from the viewpoint of practical reliability, and to observe the trace and the characteristics of fretting wear microscopically. This study found that wear and oxidation of tin plated connectors start immediately with fretting wear, and thus accumulation of abrasion powder on fretting areas causes connectors to reach to the end of their useful lifetime quickly. Especially, it was demonstrated that amplitude of fretting has a considerable influence on a connector's lifetime. It is made clear that air-tightness, so-called “gas-tight” of tin in a fretting area influences fretting wear considerably.

  14. CONSIDERATIONS REGARDING THE FRETTING PHENOMENON USING LEAF SPRINGS

    Directory of Open Access Journals (Sweden)

    Stefan GHIMIȘI

    2015-05-01

    Full Text Available The fretting phenomenon represents particulary and complex form of wear who is; generaly, and/or weary of fretting who is produced on the load contact in a relative oscialatory movement lay small amplitude.A simultaneoustly applied tangential force and normal into contact appears a adhesion force

  15. BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

    Directory of Open Access Journals (Sweden)

    Sebastian L B König

    Full Text Available Time-binned single-molecule Förster resonance energy transfer (smFRET experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc. are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1 is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET, as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/.

  16. A passive UHF RFID tag chip with a dual-resolution temperature sensor in a 0.18 μm standard CMOS process

    Science.gov (United States)

    Peng, Feng; Qi, Zhang; Nanjian, Wu

    2011-11-01

    This paper presents a passive EPC Gen-2 UHF RFID tag chip with a dual-resolution temperature sensor. The chip tag integrates a temperature sensor, an RF/analog front-end circuit, an NVM memory and a digital baseband in a standard CMOS process. The sensor with a low power sigma—delta (ΣΔ) ADC is designed to operate in low and high resolution modes. It can not only achieve the target accuracy but also reduce the power consumption and the sensing time. A CMOS-only RF rectifier and a single-poly non-volatile memory (NVM) are designed to realize a low cost tag chip. The 192-bit-NVM tag chip with an area of 1 mm2 is implemented in a 0.18-μm standard CMOS process. The sensitivity of the tag is -10.7 dBm/-8.4 dBm when the sensor is disabled/enabled. It achieves a maximum reading/sensing distance of 4 m/3.1 m at 2 W EIRP. The inaccuracy of the sensor is -0.6 °C/0.5 °C (-1.0 °C/1.2 °C) in the operating range from 5 to 15 °C in high resolution mode (-30 to 50 °C in low resolution mode). The resolution of the sensor achieves 0.02 °C (0.18 °C) in high (low) resolution mode.

  17. Ice and debris in the fretted terrain, Mars

    Science.gov (United States)

    Lucchitta, B. K.

    1984-01-01

    Viking moderate and high resolution images along the northern highland margin have been monoscopically and stereoscopically examined in order to study the development of fretted terrain. Young debris aprons around mesas and debris in tributary channels create typical fretted morphologies identical to ancient fretted morphologies. This suggests that the debris-apron process operating relatively recently also shaped the fretted terrain of the past. The debris aprons were lubricated by interstitial ice derived from ground ice. Abundant collapse features suggest that ground ice existed and may have flowed in places. The fretting process has been active for a long period and may be active today. The location of debris aprons in two latitudinal belts may be controlled by atmospheric conditions that permit ice in the region to remain in the ground below depths of about one meter and temperatures warm enough for ice to flow.

  18. Near surface stress analysis strategies for axisymmetric fretting

    Indian Academy of Sciences (India)

    M Ramesh; Satish V Kailas; K R Y Simha

    2008-06-01

    Fretting is essentially a surface phenomenon, but bulk stresses and material properties contribute to subsequent failure. This feature of fretting demands a thorough understanding of near surface stresses under the joint action of normal, shear and thermal loading. Axisymmetric fretting is of great concern in piping and coupling design. In this paper, we develop design tools for Near Surface Analysis (NSA) for understanding axisymmetric fretting. Axisymmetric Fretting Analysis (AFA) becomes formidable owing to localised tractions that call for Fourier transform techniques. We develop two different NSA strategies based on two-dimensional plane strain models: 2D strip model (2DS) and half-plane Flamant model (2DF). We compare the results of 2DS and 2DF with the exact results for AFA obtained using Love’s stress function in conjunction with Fourier transform. There is a good correspondence between stress components obtained from 2D-models.

  19. Sensor Fusion Based on an Integrated Neural Network and Probability Density Function (PDF) Dual Kalman Filter for On-Line Estimation of Vehicle Parameters and States.

    Science.gov (United States)

    Vargas-Melendez, Leandro; Boada, Beatriz L; Boada, Maria Jesus L; Gauchia, Antonio; Diaz, Vicente

    2017-04-29

    Vehicles with a high center of gravity (COG), such as light trucks and heavy vehicles, are prone to rollover. This kind of accident causes nearly 33 % of all deaths from passenger vehicle crashes. Nowadays, these vehicles are incorporating roll stability control (RSC) systems to improve their safety. Most of the RSC systems require the vehicle roll angle as a known input variable to predict the lateral load transfer. The vehicle roll angle can be directly measured by a dual antenna global positioning system (GPS), but it is expensive. For this reason, it is important to estimate the vehicle roll angle from sensors installed onboard in current vehicles. On the other hand, the knowledge of the vehicle's parameters values is essential to obtain an accurate vehicle response. Some of vehicle parameters cannot be easily obtained and they can vary over time. In this paper, an algorithm for the simultaneous on-line estimation of vehicle's roll angle and parameters is proposed. This algorithm uses a probability density function (PDF)-based truncation method in combination with a dual Kalman filter (DKF), to guarantee that both vehicle's states and parameters are within bounds that have a physical meaning, using the information obtained from sensors mounted on vehicles. Experimental results show the effectiveness of the proposed algorithm.

  20. Design and implementation of a Sun tracker with a dual-axis single motor for an optical sensor-based photovoltaic system.

    Science.gov (United States)

    Wang, Jing-Min; Lu, Chia-Liang

    2013-03-06

    The dual threats of energy depletion and global warming place the development of methods for harnessing renewable energy resources at the center of public interest. Solar energy is one of the most promising renewable energy resources. Sun trackers can substantially improve the electricity production of a photovoltaic (PV) system. This paper proposes a novel design of a dual-axis solar tracking PV system which utilizes the feedback control theory along with a four-quadrant light dependent resistor (LDR) sensor and simple electronic circuits to provide robust system performance. The proposed system uses a unique dual-axis AC motor and a stand-alone PV inverter to accomplish solar tracking. The control implementation is a technical innovation that is a simple and effective design. In addition, a scaled-down laboratory prototype is constructed to verify the feasibility of the scheme. The effectiveness of the Sun tracker is confirmed experimentally. To conclude, the results of this study may serve as valuable references for future solar energy applications.

  1. Design and Implementation of a Sun Tracker with a Dual-Axis Single Motor for an Optical Sensor-Based Photovoltaic System

    Directory of Open Access Journals (Sweden)

    Chia-Liang Lu

    2013-03-01

    Full Text Available The dual threats of energy depletion and global warming place the development of methods for harnessing renewable energy resources at the center of public interest. Solar energy is one of the most promising renewable energy resources. Sun trackers can substantially improve the electricity production of a photovoltaic (PV system. This paper proposes a novel design of a dual-axis solar tracking PV system which utilizes the feedback control theory along with a four-quadrant light dependent resistor (LDR sensor and simple electronic circuits to provide robust system performance. The proposed system uses a unique dual-axis AC motor and a stand-alone PV inverter to accomplish solar tracking. The control implementation is a technical innovation that is a simple and effective design. In addition, a scaled-down laboratory prototype is constructed to verify the feasibility of the scheme. The effectiveness of the Sun tracker is confirmed experimentally. To conclude, the results of this study may serve as valuable references for future solar energy applications.

  2. Recent Advances in Development of Genetically Encoded Fluorescent Sensors.

    Science.gov (United States)

    Sanford, Lynn; Palmer, Amy

    2017-01-01

    Genetically encoded fluorescent sensors are essential tools in modern biological research, and recent advances in fluorescent proteins (FPs) have expanded the scope of sensor design and implementation. In this review we compare different sensor platforms, including Förster resonance energy transfer (FRET) sensors, fluorescence-modulated single FP-based sensors, translocation sensors, complementation sensors, and dimerization-based sensors. We discuss elements of sensor design and engineering for each platform, including the incorporation of new types of FPs and sensor screening techniques. Finally, we summarize the wide range of sensors in the literature, exploring creative new sensor architectures suitable for different applications.

  3. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

    Directory of Open Access Journals (Sweden)

    Alsu I. Zamaleeva

    2015-09-01

    Full Text Available Semiconductor nanocrystals (NCs or quantum dots (QDs are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxyethane-N,N,N′,N′-tetra-acetic acid for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging.

  4. Handheld FRET-Aptamer Sensor for Water Safety Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Operational Technologies Corporation (OpTech) proposes to expand its current NASA Phase 2 SBIR handheld fluorometer and bone marker fluorescence resonance energy...

  5. Handheld FRET-Aptamer Sensor for Bone Markers Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Astronauts lose approximately 1-1.5% of their bone mass per month during space travel due to a lack of physical stress in the microgravity environment. Although, no...

  6. Handheld FRET-Aptamer Sensor for Bone Markers Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Astronauts lose significant bone mass during lengthy spaceflights. Although, no effective treatments or prophylactics have yet been defined, it is important to...

  7. Understanding FRET as a Research Tool for Cellular Studies

    Directory of Open Access Journals (Sweden)

    Dilip Shrestha

    2015-03-01

    Full Text Available Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET. FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

  8. Dual-wavelength speckle-based SI-POF sensor for frequency detection and localization of remote vibrations

    Science.gov (United States)

    Pinzón, P. J.; Montero, D. S.; Tapetado, A.; Torres, J. C.; Vázquez, C.

    2016-05-01

    A novel speckle-based method for sensing frequency vibration is demonstrated in a reflective configuration. By employing a visible dual-wavelength approach it is also possible to determine the relative spatial location of the vibrations along a plastic optical fiber lead of 8 m in a distributed scheme.

  9. Intonation and Compensation of Fretted String Instruments

    CERN Document Server

    Varieschi, Gabriele U

    2009-01-01

    In this paper we present mathematical models and we analyze the physics related to the problem of intonation of musical instruments such as guitars, mandolins and similar, i.e., we study how to produce perfectly in tune notes on these instruments. This analysis begins with the correct fret placement on the instrument fingerboard, following precise mathematical laws, but then it becomes increasingly complicated due to the geometrical deformation of the strings when these instruments are played, and also due to the inharmonic characteristics of the same strings. As a consequence of these factors, perfect intonation of all the notes on the instrument can never be achieved, but complex compensation procedures are introduced and studied to minimize the problem. To test the validity of these compensation procedures, we have performed extensive measurements using standard monochord sonometers and other basic acoustical devices, which confirm the correctness of our theoretical models. In particular, these experimenta...

  10. Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast.

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Takanaga, Hitomi; Chermak, Diane; Frommer, Wolf B

    2011-10-27

    Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (K(d)) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.

  11. An Experimental Study of Fretting of Gear Teeth

    Science.gov (United States)

    Krantz, Timothy L.

    2008-01-01

    Experiments were conducted to study fretting of gears. The gears were made from case-carburized AISI 9310 alloy to match the material of a flight actuator gearbox of interest. The objective of the testing was to produce damage representative of that observed on flight hardware. The following correlations and observations were noted. The amplitude of dithering motion very strongly influenced the type and magnitude of damage. Sliding amounts on the order of 30% of the width of the line contact were judged to most readily produce fretting damage. There was observed an incubation period on the order of tens-of-thousands of cycles, and the incubation period was influenced by surface roughness, torque, and the motion extent. Fretting damage could be produced for any of the torques tested, and the severity of damage increased slightly with torque. Gear teeth having surface roughness of 0.7-0.8 micrometer were somewhat more resistant to fretting than were smoother surfaces.

  12. Pulse-shaping based two-photon FRET stoichiometry.

    Science.gov (United States)

    Flynn, Daniel C; Bhagwat, Amar R; Brenner, Meredith H; Núñez, Marcos F; Mork, Briana E; Cai, Dawen; Swanson, Joel A; Ogilvie, Jennifer P

    2015-02-09

    Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

  13. Frequency-domain fluorescence lifetime imaging system (pco.flim) based on a in-pixel dual tap control CMOS image sensor

    Science.gov (United States)

    Franke, Robert; Holst, Gerhard A.

    2015-03-01

    The luminescence lifetime as a beneficial analytical parameter is known for many years and is well described by a large variety of publications. Many instruments including 2D measuring systems with cameras have been developed and applied in the past years. However, since the current instrumentation to perform either time- or frequency-domain lifetime measurements is rather complex, new developments in CMOS image sensor technology have achieved to create new image sensors, which can efficiently be integrated into easier-to-handle luminescence lifetime measuring systems. The principle of these modulatable CMOS image sensors, while initially being designed for distance measurements, shows a clear analogy to frequency-domain FLIM measurements, which was proven by researchers [1, 2]. Based on this principle a new CMOS image sensor has been developed, integrated into a camera system and has been investigated within a research project. The image sensor has a resolution of 1024 × 1024 pixels with a 5.6 μm pitch and can be modulated up to 50 MHz. First measurements show an effective dynamic range of larger than 1:1024 (corresponding to 10 bit dynamic). The maximum frame rate is in the range of 90 frames/s in dual-tap mode, resulting in an effective lifetime image frame rate for realistic measurements of approximately 22 frames/s. The camera system pco.flim, featuring that image sensor, generates all required modulation signals from 5 kHz to 50 MHz (sinusoidal and rectangular). It performs advanced pixel correction to generate linear and high-quality images, while the basic lifetime image processing is done in the computer. The modulation frequency can be freely adjusted within the specified range. The characteristics of the camera systems are presented, and first results are discussed using different representations of the data like for example the phasor approach [3], which has been established to provide a more global view to pixelwise fluorescence lifetime data and

  14. A Dual Sensor for pH and Hydrogen Peroxide Using Polymer-Coated Optical Fibre Tips

    Directory of Open Access Journals (Sweden)

    Malcolm S. Purdey

    2015-12-01

    Full Text Available This paper demonstrates the first single optical fibre tip probe for concurrent detection of both hydrogen peroxide (H2O2 concentration and pH of a solution. The sensor is constructed by embedding two fluorophores: carboxyperoxyfluor-1 (CPF1 and seminaphtharhodafluor-2 (SNARF2 within a polymer matrix located on the tip of the optical fibre. The functionalised fibre probe reproducibly measures pH, and is able to accurately detect H2O2 over a biologically relevant concentration range. This sensor offers potential for non-invasive detection of pH and H2O2 in biological environments using a single optical fibre.

  15. a Study on the Fretting Fatigue Life of Zircaloy Alloys

    Science.gov (United States)

    Kwon, Jae-Do; Park, Dae-Kyu; Woo, Seung-Wan; Chai, Young-Suck

    Studies on the strength and fatigue life of machines and structures have been conducted in accordance with the development of modern industries. In particular, fine and repetitive cyclic damage occurring in contact regions has been known to have an impact on fretting fatigue fractures. The main component of zircaloy alloy is Zr, and it possesses good mechanical characteristics at high temperatures. This alloy is used in the fuel rod material of nuclear power plants because of its excellent resistance. In this paper, the effect of the fretting damage on the fatigue behavior of the zircaloy alloy is studied. Further, various types of mechanical tests such as tension and plain fatigue tests are performed. Fretting fatigue tests are performed with a flat-flat contact configuration using a bridge-type contact pad and plate-type specimen. Through these experiments, it is found that the fretting fatigue strength decreases by about 80% as compared to the plain fatigue strength. Oblique cracks are observed in the initial stage of the fretting fatigue, in which damaged areas are found. These results can be used as the basic data for the structural integrity evaluation of corrosion-resisting alloys considering the fretting damages.

  16. Indicators based on fluorescence resonance energy transfer (FRET).

    Science.gov (United States)

    Tsien, Roger Y

    2009-07-01

    One of the major new trends in the design of indicators for optically imaging biochemical and physiological functions of living cells has been the exploitation of fluorescence resonance energy transfer (FRET). FRET is a well-known spectroscopic technique for monitoring changes in the proximity and mutual orientation of pairs of chromophores. It has long been used in biochemistry and cell biology to assess distances and orientations between specific labeling sites within a single macromolecule or between two separate molecules. More recently, macromolecules or molecular pairs have been engineered to change their FRET in response to biochemical and physiological signals such as membrane potential, cyclic AMP (cAMP), protease activity, free Ca(2+) and Ca(2+)-calmodulin (CaM) concentrations, protein-protein heterodimerization, phosphorylation, and reporter-gene expression. Because FRET is general, nondestructive, and easily imaged, it has proven to be one of the most versatile spectroscopic readouts available to the designer of new probes. FRET is particularly amenable to emission ratioing, which is more reliably quantifiable than single-wavelength monitoring and better suited than excitation ratioing to high-speed and laser-excited imaging. This article summarizes the photophysical principles of FRET and the types of indicators used.

  17. FRET-based identification of mRNAs undergoing translation.

    Directory of Open Access Journals (Sweden)

    Benjamin Stevens

    Full Text Available We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET. With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

  18. FRET-based identification of mRNAs undergoing translation.

    Science.gov (United States)

    Stevens, Benjamin; Chen, Chunlai; Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

  19. Nanoblinker: Brownian Motion Powered Bio-Nanomachine for FRET Detection of Phagocytic Phase of Apoptosis

    Science.gov (United States)

    Minchew, Candace L.; Didenko, Vladimir V.

    2014-01-01

    We describe a new type of bio-nanomachine which runs on thermal noise. The machine is solely powered by the random motion of water molecules in its environment and does not ever require re-fuelling. The construct, which is made of DNA and vaccinia virus topoisomerase protein, can detect DNA damage by employing fluorescence. It uses Brownian motion as a cyclic motor to continually separate and bring together two types of fluorescent hairpins participating in FRET. This bio-molecular oscillator is a fast and specific sensor of 5′OH double-strand DNA breaks present in phagocytic phase of apoptosis. The detection takes 30 s in solution and 3 min in cell suspensions. The phagocytic phase is critical for the effective execution of apoptosis as it ensures complete degradation of the dying cells’ DNA, preventing release of pathological, viral and tumor DNA and self-immunization. The construct can be used as a smart FRET probe in studies of cell death and phagocytosis. PMID:25268504

  20. Wave propagation in coated cylinders with reference to fretting fatigue

    Indian Academy of Sciences (India)

    M Ramesh; Satish V Kailas; K R Y Simha

    2008-06-01

    Fretting fatigue is the phenomenon of crack initiation due to dynamic contact loading, a situation which is commonly encountered in mechanical couplings subjected to vibration. The study of fretting fatigue in high frequency regime has gained importance in recent years. However the stress wave effects at high frequency y loading is scanty in the literature. The objective of present investigation is to study stress wave propagation in cylinders with reference to high frequency fretting. The case of a coated cylinder is considered since coating is often provided to improve tribological properties of the component. Rule of mixtures is proposed to understand the dispersion phenomenon in coated or layered cylinder knowing the dispersion relation for the cases of homogeneous cylinders made of coating and substrate materials separately. The possibility of stress wave propagation at the interface with a particular phase velocity without dispersion is also discussed. Results are given for two different thicknesses of coating.

  1. Fretting wear behavior of nitrogen implanted Zircaloy-4 alloy

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Zircaloy-4 was implanted with nitrogen at 120keV with various ion doses between 1 × 1013 and 1 × 1014 ions/m2. Fretting wear tests were performed at various cycles and loads under water immersion condition by the fretting simulator, The implanted surfaces were analyzed by Auger electron spectroscopy (AES) and transmission electron microscope(TEM). Micro-hardnees tester measured surface hardness. It is shown that nitrogen imphantation produced Zirconium nitride oxide and high density dislocations in implanted ltyer, surface hardness was enhanced from HK280 for unimplantedspecimen to HK1800 for a total ion dose of 1 × 1014 ions/m2. The nitrogen ion implantation treatment provided significantimprovements in the resistance of fretting damage.

  2. Borromean three-body FRET in frozen Rydberg gases

    Science.gov (United States)

    Faoro, R.; Pelle, B.; Zuliani, A.; Cheinet, P.; Arimondo, E.; Pillet, P.

    2015-09-01

    Controlling the interactions between ultracold atoms is crucial for quantum simulation and computation purposes. Highly excited Rydberg atoms are considered in this prospect for their strong and controllable interactions known in the dipole-dipole case to induce non-radiative energy transfers between atom pairs, similarly to fluorescence resonance energy transfer (FRET) in biological systems. Here we predict few-body FRET processes in Rydberg atoms and observe the first three-body resonance energy transfer in cold Rydberg atoms using cold caesium atoms. In these resonances, additional relay atoms carry away an energy excess preventing the two-body resonance, leading thus to a Borromean type of energy transfer. These few-body processes present strong similarities with multistep FRET between chromophores sometimes called donor-bridge-acceptor or superexchange. Most importantly, they generalize to any Rydberg atom and could lead to new implementations of few-body quantum gates or entanglement.

  3. PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs.

    Science.gov (United States)

    Feige, Jérôme N; Sage, Daniel; Wahli, Walter; Desvergne, Béatrice; Gelman, Laurent

    2005-09-01

    Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed-throughs (SBTs), which requires to calculate the ratios between the intensities in the FRET and in the donor or acceptor settings, when only the donor or acceptor are present. Theoretically, SBT ratios should be constant. However, experimentally, these ratios can vary as a function of fluorophore intensity, and assuming constant values may hinder precise FRET calculation. One possible cause for such a variation is the use of a microscope set-up with different photomultipliers for the donor and FRET channels, a set-up allowing higher speed acquisitions on very dynamic fluorescent molecules in living cells. Herein, we show that the bias introduced by the differential response of the two PMTs can be circumvented by a simple modeling of the SBT ratios as a function of fluorophore intensity. Another important issue when performing FRET is the localization of FRET within the cell or a population of cells. We hence developed a freely available ImageJ plug-in, called PixFRET, that allows a simple and rapid determination of SBT parameters and the display of normalized FRET images. The usefulness of this modeling and of the plug-in are exemplified by the study of FRET in a system where two interacting nuclear receptors labeled with ECFP and EYFP are coexpressed in living cells.

  4. 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.

  5. A colorimetric and fluorometric dual-signal sensor for arginine detection by inhibiting the growth of gold nanoparticles/carbon quantum dots composite.

    Science.gov (United States)

    Liu, Ting; Li, Na; Dong, Jiang Xue; Zhang, Ying; Fan, Yu Zhu; Lin, Shu Min; Luo, Hong Qun; Li, Nian Bing

    2017-01-15

    A bidimensional optical sensing platform which combines the advantages of fluorescence and colorimetry has been designed for arginine (Arg) detection. The system was established by monitoring the influence of Arg on the growth of gold nanoparticles/carbon quantum dots (Au/CQDs) composite, and the CQDs synthesized by ethylene glycol were used as the reducing and stabilizing agent in this paper. Considering that Arg is the only amino acid with guanidine group and has the highest isoelectric point (pI) value at 10.76, Arg would carry positive charges at pH 7.4. Consequently, the positively charged guanidine group of Arg could attract AuCl4(-) and CQDs through electrostatic interaction, which inhibited the growth of Au/CQDs composite. Thereby, the color of the system almost did not change and the fluorescence quenching of CQDs was prevented in the presence of Arg. Based on the color change a low detection limit for Arg was 37nM, and a detection limit of 450nM was obtained by fluorescence spectroscopy. Moreover, this dual-signal sensor also revealed excellent selectivity toward Arg over other amino acids. Besides, Arg can be detected in urine samples with satisfactory results, which demonstrate the potential applications for real analysis.

  6. Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

    Science.gov (United States)

    Oscar, Breland G.; Liu, Weimin; Zhao, Yongxin; Tang, Longteng; Wang, Yanli; Campbell, Robert E.; Fang, Chong

    2014-01-01

    Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca2+) sensing. This study reveals that, in the absence of Ca2+, the dominant skeletal motion is a ∼170 cm−1 phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca2+ in physiologically relevant environments. PMID:24987121

  7. A BTT-Based Colorimetric Dual Sensor for Hg(II and Selected Anions with Molecular Logic Operations

    Directory of Open Access Journals (Sweden)

    Veikko Uahengo

    2016-01-01

    Full Text Available A benzotrithiophenes (BTT based colorimetric sensor (2 was synthesized via a simple one-step reaction process, and its recognition properties towards several biologically important anions and cations were investigated by naked eye observation and spectroscopic methods of UV-vis and 1H NMR analysis in DMSO. The molar addition of anions such as F−, AcO−, and H2PO4- induced a marked red shift of the charge transfer band (Δλ=94 nm, from 440 nm to 534 nm concomitant with a clearly visible “naked eye” detectable colorimetric activities (from orange to pink. However, the molar addition of H2PO4- did not induce significant spectral changes, compared to F− and AcO−. Furthermore, the molar addition of Hg2+ to 2 resulted in hypochromic shift at 440 nm and the eventual disappearance of a low energy band at 534 nm, concomitant with the colorimetric activities (from orange to yellow. Moreover, both spectral and color changes induced by the addition of anions (F− and AcO− were all reversible by the molar addition of metal cations of 3d5–10. The reversibility properties triggered the logic operation studies, and, subsequently, the complementary IMP/INH logic functions based on color and spectral switching (ON/OFF were confirmed. Conclusively, 2 can thus be utilized as a colorimetric molecular switch modulated by AcO−/MII.

  8. Fretting Fatigue of Gamma TiAl Studied

    Science.gov (United States)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.

    2003-01-01

    Gamma titanium-aluminum alloy (g-TiAl) is an attractive new material for aerospace applications because of its low density and high specific strength in comparison to currently used titanium and nickel-base alloys. Potential applications for this material are compressor and low-pressure turbine blades. These blades are fitted into either the compressor or turbine disks via a dovetail connection. The dovetail region experiences a complex stress state due to the alternating centrifugal force and the natural high-frequency vibration of the blade. Because of the dovetail configuration and the complex stress state, fretting is often a problem in this area. Furthermore, the local stress state becomes more complex when the influence of the metal-metal contact and the edge of the contact is evaluated. Titanium and titanium-based alloys in the clean state exhibit strong adhesive bonds when in contact with themselves and other materials (refs. 1 and 2). This adhesion causes heavy surface damage and high friction in practical cases. Although the wear produced by fretting may be mild, the reduction in fatigue life can be substantial. Thus, there is the potential for fretting problems with these TiAl applications. Since TiAl is an emerging material, there has been limited information about its fretting behavior.

  9. Alternating-laser excitation : single-molecule FRET and beyond

    NARCIS (Netherlands)

    Hohlbein, Johannes; Craggs, Timothy D.; Cordes, Thorben

    2014-01-01

    The alternating-laser excitation (ALEX) scheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Forster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables

  10. Application of FRET probes in the analysis of neuronal plasticity

    Directory of Open Access Journals (Sweden)

    Yoshibumi eUeda

    2013-10-01

    Full Text Available Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since GFP was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET, which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo.

  11. Plant-based FRET biosensor discriminates enviornmental zinc levels

    Science.gov (United States)

    Heavy metal accumulation in the environment poses great risks to flora and fauna. However, monitoring sites prone to accumulation poses scale and economic challenges. In this study, we present and test a method for monitoring these sites using fluorescent resonance energy transfer (FRET) change in r...

  12. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee

    2016-03-01

    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  13. Fretting Fatigue Behavior of Shot-Peened IN 100

    Science.gov (United States)

    2006-03-01

    117 - x - List of Figures Figure Page Figure 1. Blade/ Disc ...Blade/ Disc Dovetail Joint in a Turbine Engine. - 7 - Figure 2. Simplified Fretting Configuration Contact Load (P) Fatigue Specimen (P...steam turbine power plants, medical applications, nuclear power systems, and in the chemical petrochemical industries 28. Nickel superalloys are

  14. Color control through FRET efficiency modulation using CDI (Conference Presentation)

    Science.gov (United States)

    Wolowelsky, Karni; Guyes, Eric; Rubin, Shimon; Suss, Matthew; Bercovici, Moran; Rotschild, Carmel

    2017-02-01

    Although much progress was made in light emitting devices, the ability to electrically control their spectral emission remains limited. We will present a novel approach and experimental results for dynamic color control, by electrically modulating the non-radiative Forster resonance energy transfer (FRET) efficiency between donor and acceptor dyes in a solution. FRET efficiency depends on the 6th power of the distance between donor and acceptor dye molecules, and thus, it is sensitive to variations in acceptor's concentration. Controlled acceptor concentrations could be achieved by attracting or repelling ionic dyes from the electrodes using a capacitive deionization (CDI) cell, with high surface area porous electrodes. This approach to dynamic color control may open new directions in 100% fill-factor displays, and can be expanded to energy saving applications such as controlling building's external wall emissivity. We studied the modulation of a single dye emission using a CDI cell with negatively charged Fluorescein Sodium Salt in aquatic solution. Photoluminescence was measured along few charging-discharging CDI cycles and showed the ability to control extensive optical response through CDI. We experimented with two types of FRET-pair dyes: a) anion-cation, where the acceptor and the donor ions are oppositely charged, and b) zwitterion and ion, where the donor is neutral. We found that electrical control on FRET in aquatic solution is weak, due to hydrophobic attractive interaction between the acceptor and the donor. In order to avoid this effect, we are experimenting FRET control in organic solvents. These results will be presented in the talk.

  15. Single-Molecule Pull-down FRET (SiMPull-FRET) to dissect the mechanisms of biomolecular machines

    Science.gov (United States)

    Kahlscheuer, Matthew L.; Widom, Julia; Walter, Nils G.

    2016-01-01

    Spliceosomes are multi-megadalton RNA-protein complexes responsible for the faithful removal of non-coding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition and versatile structural dynamics. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues towards studying the mechanisms of biomolecular machines isolated directly from complex biological specimens such as cell extracts. Here we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy in exemplary single molecule pull-down FRET (SiMPull-FRET) studies of the yeast spliceosome and discuss the broad application potential of this technique. PMID:26068753

  16. 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.

  17. Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.

    Science.gov (United States)

    Afsari, Hamid Samareh; Cardoso Dos Santos, Marcelina; Lindén, Stina; Chen, Ting; Qiu, Xue; van Bergen En Henegouwen, Paul M P; Jennings, Travis L; Susumu, Kimihiro; Medintz, Igor L; Hildebrandt, Niko; Miller, Lawrence W

    2016-06-01

    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide-mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

  18. Specific survivin dual fluorescence resonance energy transfer molecular beacons for detection of human bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-qiang WANG; Jun ZHAO; Jin ZENG; Kai-jie WU; Yu-le CHEN; Xin-ya ng WANG; Luke S CHANG; Da-lin HE

    2011-01-01

    Survivin molecular beacons can be used to detectbladder cancer cells in urine samples non-invasively.The aim of this study is to improve the specificity of detection of bladder cancer cells using survivin dual fluorescence resonance energy transfer molecular beacons (FRET MBs) that have fluorophores forming one donor-acceptor pair.Methods:Survivin-targeting dual fluorescence resonance energy transfer molecular beacons with unique target sequences were designed,which had no overlap with the other genes in the apoptosis inhibitor protein family.Human bladder cancer cell lines 5637,253J and T24,as well as the exfoliated cells in the urine of healthy adults and patients with bladder cancer were examined.Images of cells were taken using a laser scanning confocal fluorescence microscope.For assays using dual FRET MBs,the excitation wavelength was 488 nm,and the emission detection wavelengths were 520+20 nm and 560+20 nm,respectively.Results:The human bladder cancer cell lines and exfoliated cells in the urine of patients with bladder cancer incubated with the survivin dual FRET MBs exhibited strong fluorescence signals.In contrast,no fluorescence was detected in the survivin-negative human dermal fibroblasts-adult (HDF-a) cells or exfoliated cells in the urine of healthy adults incubated with the survivin dual FRET MBs.Conclusion:The results suggest that the survivin dual FRET MBs may be used as a specific and non-invasive method for early detection and follow-up of patients with bladder cancer.

  19. Detecting stoichiometry of macromolecular complexes in live cells using FRET

    Science.gov (United States)

    Ben-Johny, Manu; Yue, Daniel N.; Yue, David T.

    2016-01-01

    The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca2+-induced switch in calmodulin stoichiometry with Ca2+ channels—one calmodulin binds at basal cytosolic Ca2+ levels while two calmodulins interact following Ca2+ elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells. PMID:27922011

  20. Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

    Directory of Open Access Journals (Sweden)

    Ruud G J Detert Oude Weme

    Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

  1. Interference Assembly and Fretting Wear Analysis of Hollow Shaft

    OpenAIRE

    Chuanjun Han; Jie Zhang

    2014-01-01

    Fretting damage phenomenon often appears in the interference fit assembly. The finite element model of hollow shaft and shaft sleeve was established, and the equivalent stress and contact stress were computed after interference assembly. The assembly body of hollow shaft and shaft sleeve was in whirling bending load, and the contact status (sticking, sliding, and opening) and the distribution of stress along one typical contact line were computed under different loads, interferences, hollow d...

  2. Fretting Fatigue Behavior of Nickel Alloy IN-100

    Science.gov (United States)

    2006-03-01

    K., B. F. Dyson, and M. McLean. “Tension-Compression Creep Asym- metry In a Turbine Disc Superalloy : Roles of Internal Stress and Thermal Age- ing...understand the fretting phenomenon and its mechanisms, but only a few have been performed on nickel-based alloys. This superalloy is considered one...excellent magnetic properties, but only at low magnetic induction. Nowadays, the need for the use of high-strength nickel-based superalloys in

  3. Fretting corrosion in power contacts: Electrical and thermal analysis

    OpenAIRE

    El Mossouess, S; Benjemâa, N; Carvou, E; El Abdi, R; Benmamas, L; Doublet, L

    2014-01-01

    International audience; -Fretting corrosion phenomenon is known as the main cause of contact resistance increasing in signal contact. But for power connectors this undesirable phenomenon in embarked systems is more complex because high current induces high voltages and subsequent Joule overheating can be expected during vibration. Our study deals with contact similar of power connectors ones submitted to vibration am-plitudes up to 25 micrometres at frequencies of a few 10Hz under a current r...

  4. Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells.

    Science.gov (United States)

    Hou, Bi-Huei; Takanaga, Hitomi; Grossmann, Guido; Chen, Li-Qing; Qu, Xiao-Qing; Jones, Alexander M; Lalonde, Sylvie; Schweissgut, Oliver; Wiechert, Wolfgang; Frommer, Wolf B

    2011-10-27

    Knowledge of the in vivo levels, distribution and flux of ions and metabolites is crucial to our understanding of physiology in both healthy and diseased states. The quantitative analysis of the dynamics of ions and metabolites with subcellular resolution in vivo poses a major challenge for the analysis of metabolic processes. Genetically encoded Förster resonance energy transfer (FRET) sensors can be used for real-time in vivo detection of metabolites. FRET sensor proteins, for example, for glucose, can be targeted genetically to any cellular compartment, or even to subdomains (e.g., a membrane surface), by adding signal sequences or fusing the sensors to specific proteins. The sensors can be used for analyses in individual mammalian cells in culture, in tissue slices and in intact organisms. Applications include gene discovery, high-throughput drug screens or systematic analysis of regulatory networks affecting uptake, efflux and metabolism. Quantitative analyses obtained with the help of FRET sensors for glucose or other ions and metabolites provide valuable data for modeling of flux. Here we provide a detailed protocol for monitoring glucose levels in the cytosol of mammalian cell cultures through the use of FRET glucose sensors; moreover, the protocol can be used for other ions and metabolites and for analyses in other organisms, as has been successfully demonstrated in bacteria, yeast and even intact plants. The whole procedure typically takes ∼4 d including seeding and transfection of mammalian cells; the FRET-based analysis of transfected cells takes ∼5 h.

  5. Distinguishing the cross-beta spine arrangements in amyloid fibrils using FRET analysis.

    Science.gov (United States)

    Deng, Wei; Cao, Aoneng; Lai, Luhua

    2008-06-01

    The recently published microcrystal structures of amyloid fibrils from small peptides greatly enhanced our understanding of the atomic-level structure of the amyloid fibril. However, only a few amyloid fibrils can form microcrystals. The dansyl-tryptophan fluorescence resonance energy transfer (FRET) pair was shown to be able to detect the inter-peptide arrangement of the Transthyretin (105-115) amyloid fibril. In this study, we combined the known microcrystal structures with the corresponding FRET efficiencies to build a model for amyloid fibril structure classification. We found that fibrils with an antiparallel structural arrangement gave the largest FRET signal, those with a parallel arrangement gave the lowest FRET signal, and those with a mixed arrangement gave a moderate FRET signal. This confirms that the amyloid fibril structure patterns can be classified based on the FRET efficiency.

  6. Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

    Science.gov (United States)

    Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

  7. Fretting fatigue crack propagation rate under variable loading conditions

    Directory of Open Access Journals (Sweden)

    C. Gandiolle

    2016-01-01

    Full Text Available Fretting fatigue experiments aim to represent industrial problems and most of them endure variable loading. Being able to assess lifetime of assemblies, especially for low propagation rate conditions, is essential as experimental validation is often too expensive. Both experimental and numerical approaches are proposed to follow the crack propagation rate of steel on steel cylinder/plane fretting fatigue contact submitted to variable loading conditions. An original experimental monitoring has been implemented on the fretting-fatigue test device to observe crack propagation using a potential drop technique. A calibration curve relating crack length and electrical potential was established for the studied contact. It allows direct knowledge of the crack length and crack propagation rate. It was applied to mixed load test showing crack arrest for the last loading condition. To explain this behavior, a 2-dimensional FE modeling was implemented to simulate the complexes multi-axial contact stressing. The crack propagation rate was formalized using an effective stress intensity factor amplitude ΔKeff coupled with Paris law of the material. The crack arrest condition for a given loading was related to ΔKeff along the expected crack path crossing the material crack arrest threshold ΔK0. The failure was related to ΔKeff reaching the critical stress intensity factor KIC. A good correlation with experiments was observed allowing to predict the crack arrest condition although the model tends to overestimate the final crack length extension.

  8. pyFRET: A Python Library for Single Molecule Fluorescence Data Analysis

    OpenAIRE

    Murphy, Rebecca R.; Jackson, Sophie E.; Klenerman, David

    2014-01-01

    Single molecule F\\"orster resonance energy transfer (smFRET) is a powerful experimental technique for studying the properties of individual biological molecules in solution. However, as adoption of smFRET techniques becomes more widespread, the lack of available software, whether open source or commercial, for data analysis, is becoming a significant issue. Here, we present pyFRET, an open source Python package for the analysis of data from single-molecule fluorescence experiments from freely...

  9. Considerations for sensitivity analysis, uncertainty quantification, and data assimilation for grid-to-rod fretting

    Energy Technology Data Exchange (ETDEWEB)

    Michael Pernice

    2012-10-01

    Grid-to-rod fretting is the leading cause of fuel failures in pressurized water reactors, and is one of the challenge problems being addressed by the Consortium for Advanced Simulation of Light Water Reactors to guide its efforts to develop a virtual reactor environment. Prior and current efforts in modeling and simulation of grid-to-rod fretting are discussed. Sources of uncertainty in grid-to-rod fretting are also described.

  10. Rise-time of FRET-acceptor fluorescence tracks protein folding

    OpenAIRE

    Simon Lindhoud; Adrie H. Westphal; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Jan Willem Borst

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based F...

  11. Assessment of Adaptive Rate Response Provided by Accelerometer, Minute Ventilation and Dual Sensor Compared with Normal Sinus Rhythm During Exercise: A Self-controlled Study in Chronotropically Competent Subjects

    Institute of Scientific and Technical Information of China (English)

    Yuanyuan Cao; Yiqun Zhang; Yangang Su; Jin Bai; Wei Wang; Junbo Ge

    2015-01-01

    Background:Dual sensor (DS) for rate adaption was supposed to be more physiological.To evaluate its superiority,the DS (accelerometer [ACC] and minute ventilation [MV]) and normal sinus rate response were compared in a self-controlled way during exercise treadmill testing.Methods:This self-controlled study was performed in atrioventricular block patients with normal sinus function who met the indications of pacemaker implant.Twenty-one patients came to the 1-month follow-up visit.Patients performed a treadmill test 1-month post implant while programmed in DDDR and sensor passive mode.For these patients,sensor response factors were left at default settings (ACC =8,MV =3) and sensor indicated rates (SIRs) for DS,ACC and MV sensor were retrieved from the pacemaker memories,along with measured sinus node (SN) rates from the beginning to 1-minute after the end of the treadmill test,and compared among study groups.Repeated measures analysis of variance and profile analysis,as well as variance analysis of randomized block designs,were used for statistical analysis.Results:Fifteen patients (15/2 l) were determined to be chronotropically competent.The mean differences between DS SIRs and intrinsic sinus rates during treadmill testing were smaller than those for ACC and MV sensor (mean difference between SIR and SN rate:ACC vs.SN,MV vs.SN,DS vs.SN,respectively,34.84,17.60,16.15 beats/min),though no sensors could mimic sinus rates under the default settings for sensor response factor (ACC vs.SN P-adjusted < 0.001; MV vs.SN P-adjusted =0.002; DS vs.SN P-adjusted =0.005).However,both in the range of 1st minute and first 3 minutes of exercise,only the DS SIR profile did not differ from sinus rates (P-adjusted =0.09,0.90,respectively).Conclusions:The DS under default settings provides more physiological rate response during physical activity than the corresponding single sensors (ACC or MV sensor).Further study is needed to determine if individual optimization would further

  12. Assessment of Adaptive Rate Response Provided by Accelerometer, Minute Ventilation and Dual Sensor Compared with Normal Sinus Rhythm During Exercise: A Self-controlled Study in Chronotropically Competent Subjects

    Directory of Open Access Journals (Sweden)

    Yuanyuan Cao

    2015-01-01

    Full Text Available Background: Dual sensor (DS for rate adaption was supposed to be more physiological. To evaluate its superiority, the DS (accelerometer [ACC] and minute ventilation [MV] and normal sinus rate response were compared in a self-controlled way during exercise treadmill testing. Methods: This self-controlled study was performed in atrioventricular block patients with normal sinus function who met the indications of pacemaker implant. Twenty-one patients came to the 1-month follow-up visit. Patients performed a treadmill test 1-month post implant while programmed in DDDR and sensor passive mode. For these patients, sensor response factors were left at default settings (ACC = 8, MV = 3 and sensor indicated rates (SIRs for DS, ACC and MV sensor were retrieved from the pacemaker memories, along with measured sinus node (SN rates from the beginning to 1-minute after the end of the treadmill test, and compared among study groups. Repeated measures analysis of variance and profile analysis, as well as variance analysis of randomized block designs, were used for statistical analysis. Results: Fifteen patients (15/21 were determined to be chronotropically competent. The mean differences between DS SIRs and intrinsic sinus rates during treadmill testing were smaller than those for ACC and MV sensor (mean difference between SIR and SN rate: ACC vs. SN, MV vs. SN, DS vs. SN, respectively, 34.84, 17.60, 16.15 beats/min, though no sensors could mimic sinus rates under the default settings for sensor response factor (ACC vs. SN P-adjusted < 0.001; MV vs. SN P-adjusted = 0.002; DS vs. SN P-adjusted = 0.005. However, both in the range of 1 st minute and first 3 minutes of exercise, only the DS SIR profile did not differ from sinus rates (P-adjusted = 0.09, 0.90, respectively. Conclusions: The DS under default settings provides more physiological rate response during physical activity than the corresponding single sensors (ACC or MV sensor. Further study is needed to

  13. Fretting Wear Behavior of Tin Plated Contacts:. Influence on Contact Resistance

    Science.gov (United States)

    Park, Young Woo; Sankara Narayanan, T. S. N.; Lee, Kang Yong

    The fretting wear behavior of tin plated copper alloy contacts and its influence on the contact resistance are addressed in this paper. Based on the change in the area of contact zone as well as the wear depth as a function of fretting cycles, a model was proposed to explain the observed low and stable contact resistance. The extent of wear of tin coating and the formation of wear debris as a function of fretting cycles were assessed by scanning electron microscopy (SEM). Energy dispersive X-ray line scanning (EDX), X-ray mapping, and EDX spot analysis were employed to characterize the nature of changes that occur at the contact zone. The study reveals that the fretted area increases linearly up to 8000 cycles due to the continuous removal of the tin coating and attains saturation when the fretting path length reaches a maximum. The observed low and stable contact resistance observed up to 8000 cycles is due to the common area of contact which provides an electrically conducting area. Surface analysis by SEM, EDX, and X-ray elemental mapping elucidate the nature of changes that occurred at the contact zone. Based on the change in contact resistance as a function of fretting cycles, the fretting wear and fretting corrosion dominant regimes are proposed. The interdependence of extent of wear and oxidation increases the complexity of the fretting corrosion behavior of tin plated contacts.

  14. A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously.

    Science.gov (United States)

    Götz, M; Wortmann, P; Schmid, S; Hugel, T

    2016-01-01

    Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other. Multicolor smFRET experiments enable the direct observation of such correlated dynamics and interactions. Here we demonstrate the power and limitations of multicolor smFRET experiments including the description of a multicolor smFRET setup and data analysis. A general analytical procedure for multicolor smFRET data is presented and applied to the multicomponent heat shock protein 90 system. This allows us to identify microscopic states in transient complexes. Conformational dynamics and nucleotide binding are simultaneously detected, which is impossible using two-color smFRET. Additionally, their correlation is quantified using 3D ensemble hidden Markov analysis, in and out of equilibrium. This method is perfectly suited for protein systems that are much more sophisticated than previously studied DNA-based systems. By extending the application to biologically relevant systems, multicolor smFRET comes of age and provides a unique mechanistic insight into protein machines.

  15. Fretting Fatigue Experiment and Analysis of AlSi9Cu2Mg Alloy

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2016-12-01

    Full Text Available An investigation was carried out in order to study the fretting fatigue behavior of an AlSi9Cu2Mg aluminum alloy. The fretting fatigue tests of AlSi9Cu2Mg were performed using a specially designed testing machine. The failure mechanism of fretting fatigue was explored by studying the fracture surfaces, fretting scars, fretting debris, and micro-hardness of fretting fatigue specimens using scanning electron microscopy (SEM, energy-dispersive X-ray spectroscopy (EDX, and micro Vickers hardness test techniques. The experimental results show that the fretting fatigue limit (42 MPa is significantly reduced to approximately 47% of the plain fatigue limit (89 MPa under 62.5 MPa contact pressure. Furthermore, the fretting fatigue life decreases with increasing alternating stress and increasing contact pressure. The examination results suggest that the stress concentrates induced by oxidation-assisted wear on the contact interface led to the earlier initiation and propagation of crack under the fretting condition.

  16. Understanding and modeling Förster-type resonance energy transfer (FRET)

    CERN Document Server

    Hernández Martínez, Pedro Ludwig; Demir, Hilmi Volkan

    2017-01-01

    This Brief presents a complete study of the generalized theory of Förster-type energy transfer in nanostructures with mixed dimensionality. Here the aim is to obtain a generalized theory of FRET including a comprehensive set of analytical equations for all combinations and configurations of nanostructures and deriving generic expressions for the dimensionality involved. In this brief, the modification of FRET mechanism with respect to the nanostructure serving as the donor vs. the acceptor will be included, focusing on the rate’s distance dependency and the role of the effective dielectric function in FRET, which will be a unique, useful source for those who study and model FRET.

  17. AccPbFRET: An ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images

    Directory of Open Access Journals (Sweden)

    Vereb György

    2008-08-01

    Full Text Available Abstract Background The acceptor photobleaching fluorescence resonance energy transfer (FRET method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios. Results AccPbFRET is a user-friendly, efficient ImageJ plugin which allows fully corrected, pixel-wise calculation and detailed, ROI (region of interest-based analysis of FRET efficiencies in microscopic images. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing FRET evaluation software. Conclusion Despite of the widespread applicability of the acceptor photobleaching FRET technique, this is the first paper where all possible sources of major errors of the measurement and analysis are considered, and AccPbFRET is the only program which provides the complete suite of corrections – for registering image pairs, for unwanted photobleaching of the donor, for cross-talk of the acceptor and/or its photoproduct to the donor channel and for partial photobleaching of the acceptor. The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.

  18. Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, Evgenia G., E-mail: evgenia_matveeva@hotmail.co [Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States); Gryczynski, Zygmunt [Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States); Stewart, Donald R. [Omm Scientific, Inc., 2600 N. Stemmons Freeway, Suite 129, Dallas, TX 75207 (United States); Gryczynski, Ignacy [Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States)

    2010-04-15

    A new FRET-based method for the ratiometric detection of DNA oligomers on a surface using TIRF detection mode is reported. The dual-labeled system consisting of two hybridized oligomers, Cy3oligoY:Cy5oligoX was immobilized on the surface, and the total internal reflection fluorescence (TIRF) was used to detect emission signals from the surface. Two signals, green and red, which originated from the green donor Cy3 and the red acceptor Cy5, have been simultaneously detected. When the target single-stranded complimentary oligomer was present in the solution, this oligomer replaced the Cy3oligoY in the donor:acceptor complex on the surface and the ratio of red-to-green signal was dramatically changed. This detection scheme is generally applicable to the detection of DNA or RNA on a surface.

  19. FRET and colocalization analyzer--a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug-in.

    Science.gov (United States)

    Hachet-Haas, Muriel; Converset, Noël; Marchal, Olivier; Matthes, Hans; Gioria, Sophie; Galzi, Jean-Luc; Lecat, Sandra

    2006-12-01

    Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.

  20. Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols.

    Directory of Open Access Journals (Sweden)

    Alexandre Bourdès

    Full Text Available Förster resonance energy transfer (FRET biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens. To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.Two new vectors were developed for cloning genes for solute-binding proteins (SBPs between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2 and red fluorescent protein (mKate2 FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose, D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate. To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP transport systems.FRET based on orange (mOrange2 and red fluorescent protein (mKate2 partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i cyclic polyols, (ii L-deoxy sugars, (iii β-linked disaccharides and (iv C4-dicarboxylates could be developed to study metabolism in vivo.

  1. Bullet Design and Fabrication of Dual Mode Pyroelectric Sensor: High Sensitive Energymeter for Nd: YAG Laser and Detector for Chopped He-Ne Laser

    Directory of Open Access Journals (Sweden)

    S. SATAPATHY

    2008-05-01

    Full Text Available Pyroelectric sensor using TGS has been designed and fabricated which can be operated in laser energy meter mode as well as pyroelectric detector mode. The amplifying circuit configuration has very good signal to noise ratio, very high input impedance and low drift. The pyroelectric sensor has been tested using Q-switched Nd: YAG laser and chopped He-Ne laser. The sensitivity of pyroelectric sensor in energymeter mode is 421.7V/J and the voltage responsivity of the pyroelectric sensor is 3.27 V/W in detector mode.

  2. Disentangling picosecond events that complicate the quantative use of the calcium sensor YC3.60

    NARCIS (Netherlands)

    Laptenok, S.; Stokkum, van I.H.M.; Borst, J.W.; Oort, van B.F.; Visser, A.J.W.G.; Amerongen, van H.

    2012-01-01

    Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein

  3. Disentangling picosecond events that complicate the quantitative use of the calcium sensor YC3.60

    NARCIS (Netherlands)

    Laptenok, S.; Stokkum, van I.H.M.; Borst, J.W.; Oort, van B.F.; Visser, A.J.W.G.; Amerongen, van H.

    2012-01-01

    Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein

  4. A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex.

    Science.gov (United States)

    Arsenovic, Paul T; Bathula, Kranthidhar; Conway, Daniel E

    2017-04-11

    The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

  5. 双层预紧式六维力传感器及其静态标定%Dual Layers Pre-stressed Six-axis Force Sensor and Its Static Calibration

    Institute of Scientific and Technical Information of China (English)

    王志军; 姚建涛; 吴遥; 叶冬; 赵永生

    2013-01-01

    针对基于传统Stewart结构六维力传感器在解耦、刚度等方面性能的不足,设计并研制一种双层预紧式六维力传感器,并对其进行静态标定试验研究.所设计的六维力传感器采用球窝锥头式球面副代替传统球副,有效地减小了球副的摩擦力矩,另外测量分支只承受压力,消除了力在换向时对接触球面的冲击,提高了整体刚度.基于螺旋理论推导出传感器所施加外力与测量分支轴向力之间的映射关系,进而说明预紧式六维力传感器的测量原理.采用最小二乘线性化标定方法对传感器进行静态解耦和测量误差分析,推导出传感器的标定矩阵和误差矩阵.研制出双层预紧式六维力传感器样机,并进行静态标定试验.经过标定试验,该传感器各方向最大非线性度误差低于满量程的1.17%,最大耦合误差低于满量程的1.82%.试验结果表明双层预紧式六维力传感器具有很高的测量精度,可用于工业生产中对六维力测量要求较高的场合.%According to the deficiency of performance of the traditional six-axis force sensor based on Stewart platform structure on decoupling and stiffness, a pre-stressed six-axis force sensor with dual layers is designed and developed, and the static calibration of the sensor is carried out. The proposed six-axis force sensor uses cone-socket type spherical joint instead of the traditional one, which reduces the friction torque of spherical pair effectively and eliminates the commutation impact force for the measuring limbs compressed all the time, so the overall stiffness is improved. Based on screw theory, the mapping of the applied external force and the axial force of measuring limbs of the sensor is derived, and then the measuring principle of the pre-stressed six-axis force sensor is described. Analysis of static decoupling and measurement error of the sensor is conducted by using linear least squares calibration method, and

  6. Evaluating the Relationship between FRET Changes and Distance Changes Using DNA Length and Restriction Enzyme Specificity

    Science.gov (United States)

    Pazhani, Yogitha; Horn, Abigail E.; Grado, Lizbeth; Kugel, Jennifer F.

    2016-01-01

    FRET (Fo¨rster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students…

  7. FRET Imaging Trackable Long-Circulating Biodegradable Nanomedicines for Ovarian Cancer Therapy

    Science.gov (United States)

    2014-09-01

    Award Number: W81XWH-13-1-0160 TITLE: FRET Imaging Trackable Long-Circulating Biodegradable ...TITLE AND SUBTITLE FRET Imaging Trackable Long-Circulating Biodegradable Nanomedicines for Ovarian Cancer Therapy 5a. CONTRACT NUMBER...strategy, which permits visualizing the biodegradation of copolymer-drug conjugates at the body, tissue and cell levels in real time. The information

  8. Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A., E-mail: nelambert@gru.edu

    2015-08-14

    Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells. - Highlights: • Diffusion enhances TR-FRET from membrane proteins labeled with lanthanide donors. • Diffusion-dependent FRET can overshadow FRET due to oligomerization or clustering. • FRET studies using lanthanide-tagged membrane proteins should consider diffusion. • FRET from lanthanide donors can be used to monitor membrane protein diffusion.

  9. Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity.

    Science.gov (United States)

    Massey, Melissa; Li, Jia Jun; Algar, W Russ

    2017-01-01

    Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolytic activity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolytic activity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.

  10. Engineering of weak helper interactions for high-efficiency FRET probes.

    Science.gov (United States)

    Grünberg, Raik; Burnier, Julia V; Ferrar, Tony; Beltran-Sastre, Violeta; Stricher, François; van der Sloot, Almer M; Garcia-Olivas, Raquel; Mallabiabarrena, Arrate; Sanjuan, Xavier; Zimmermann, Timo; Serrano, Luis

    2013-10-01

    Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

  11. Development of a DNA sensor using molecular logic gate

    CERN Document Server

    Bhattacharjee, D; Chakraborty, S; Hussain, Syed Arshad

    2014-01-01

    This communication reports the increase in fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in presence of Deoxyribonucleic acid (DNA). Two types of molecular logic gates have been designed where DNA acts as input signal and fluorescence intensity of different bands are taken as output signal. Use of these logic gates as DNA sensor has been demonstrated

  12. A Versatile Multiple Target Detection System Based on DNA Nano-assembled Linear FRET Arrays.

    Science.gov (United States)

    Li, Yansheng; Du, Hongwu; Wang, Wenqian; Zhang, Peixun; Xu, Liping; Wen, Yongqiang; Zhang, Xueji

    2016-05-27

    DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as inconstant programmable templates for assembly of biosensors. In this paper, a versatile, scalable and multiplex detection system is reported based on an extending fluorescent resonance energy transfer (FRET) cascades on a linear DNA assemblies. Seven combinations of three kinds of targets are successfully detected through the changes of fluorescence spectra because of the three-steps FRET or non-FRET continuity mechanisms. This nano-assembled FRET-based nanowire is extremely significant for the development of rapid, simple and sensitive detection system. The method used here could be extended to a general platform for multiplex detection through more-step FRET process.

  13. pyFRET: A Python Library for Single Molecule Fluorescence Data Analysis

    CERN Document Server

    Murphy, Rebecca R; Klenerman, David

    2014-01-01

    Single molecule F\\"orster resonance energy transfer (smFRET) is a powerful experimental technique for studying the properties of individual biological molecules in solution. However, as adoption of smFRET techniques becomes more widespread, the lack of available software, whether open source or commercial, for data analysis, is becoming a significant issue. Here, we present pyFRET, an open source Python package for the analysis of data from single-molecule fluorescence experiments from freely diffusing biomolecules. The package provides methods for the complete analysis of a smFRET dataset, from burst selection and denoising, through data visualisation and model fitting. We provide support for both continuous excitation and alternating laser excitation (ALEX) data analysis. pyFRET is available as a package downloadable from the Python Package Index (PyPI) under the open source three-clause BSD licence, together with links to extensive documentation and tutorials, including example usage and test data. Additio...

  14. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

    Directory of Open Access Journals (Sweden)

    George Chennell

    2016-08-01

    Full Text Available We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP Activated Protein Kinase (AMPK FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR, for fluorescence lifetime imaging (FLIM readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP, in the original FRET biosensor, AMPK activity reporter (AMPKAR, with mTurquoise2 (mTq2FP, increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

  15. Fretting damage behavior and mechanism of tin coated zircaloy-4 tube

    Institute of Scientific and Technical Information of China (English)

    Tae-HyungKim; Ji-HyunSung; Seock-SamKim

    2001-01-01

    The fretting characteristics of TiN coated Zircaloy-4 tube were investigated experimen-tally. The fretting experiment was performed using TIN coated Zircaloy-4 tube as the fuel rod clad-ding material and uncoated Zircaloy-4 tube as one of grids. TIN coating is probably one of the mostfrequently and successfully used PVD coatings for the mitigation of fretting. In this study, TiN coat-ing by PVD was employed for improvement of Zircaloy-4 tube fretting characteristics. The frettingtester was designed and manufactured for this experiment. The number of cycles, slip amplitudeand normal load were selected as main factors of fretting. The results of this research showed thatthe wear volume of TiN coated Zircaloy-4 tube increased as number of cycles, normal load andslip amplitude increase but the quantity of volume was lower than the case of uncoated Zircaloy-4tube pairs.

  16. DESIGN OF A DUAL KEYBOARD

    OpenAIRE

    V. Ragavi; G. Geetha

    2013-01-01

    The design of a computer keyboard with dual function is proposed. This computer keyboard called Dual Keyboard can function both as a normal keyboard and as a pressure sensitive keyboard. The proposed device has a switch that decides the function. The keyboard makes use of sensors placed beneath the keys to measure the pressure applied on the key by the user. This device has many applications. In this study, it is applied to mitigate Denial of Service (DoS) attack.

  17. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging

    Science.gov (United States)

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger

    2016-03-01

    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02-04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity.

  18. Crack path simulation for cylindrical contact under fretting conditions

    Directory of Open Access Journals (Sweden)

    R.A. Cardoso

    2016-02-01

    Full Text Available In this work different strategies to estimate crack path for cylindrical contacts under fretting conditions are carried out. The main goal is to propose and to evaluate methodologies not only to estimate the direction of crack initiation but also the subsequent propagation in its earlier stages, where the stress field is multiaxial, non-proportional and decays very fast due to the proximity with the contact interface. Such complex conditions pose a substantial challenge to the modelling of crack path. The numerical simulations are provided by a 2D Finite Element Analysis taking into account interactions between the crack faces. The results show that, under fretting conditions, models based on the critical plane method are not effective to estimate the crack initiation orientation, while models based on a so called “critical direction” applied along a critical distance provide better results. Regarding the subsequent crack propagation orientation, it was possible to see that stress intensity factor based models where one considers an infinitesimal virtual crack emerging from an original preexistent crack are powerful mechanisms of crack orientation estimation.

  19. Paths to Förster's resonance energy transfer (FRET) theory

    Science.gov (United States)

    Masters, B. R.

    2014-02-01

    Theodor Förster (1910-1974) developed a phenomenological theory of nonradiative resonance energy transfer which proved to be transformative in the fields of chemistry, biochemistry, and biology. This paper explores the experimental and the theoretical antecedents of Förster's theory of resonance energy transfer (FRET). Early studies of sensitized fluorescence, fluorescence depolarization, and photosynthesis demonstrated the phenomena of long-range energy transfer. At the same time physicists developed theoretical models which contained common physical mechanisms and parameters: oscillating dipoles as models for the atoms or molecules, dipole-dipole coupling for the interaction, and a distance R0 that is optimal for resonance energy transfer. Early theories predicted R0 that was too large as compared to experiments. Finally, in 1946 Förster developed a classical theory and in 1948 he developed a quantum mechanical theory; both theories predicted an inverse sixth power dependence of the rate of energy transfer and a R0 that agreed with experiments. This paper attempts to determine why Förster succeeded when the other theoreticians failed to develop the correct theory. The putative roles of interdisciplinary education and collaborative research are discussed. Furthermore, I explore the role of science journals and their specific audiences in the popularization of FRET to a broad interdisciplinary community.

  20. DYNAMIC ANALYSIS OF CYTOSOLIC GLUCOSE AND ATP LEVELS IN YEAST WITH OPTICAL SENSORS

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Takanaga, Hitomi; Chermak, Diane; Frommer, Wolf B.

    2010-01-01

    Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. Here, genetically encoded Förster Resonance Energy Transfer (FRET)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP at varying external supply and in glucose transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes in intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in an hxt5Δ hexose transporter mutant compared to wild type and to other hxtΔ strains. PMID:20854260

  1. Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.

    Science.gov (United States)

    Bermejo, Clara; Haerizadeh, Farzad; Takanaga, Hitomi; Chermak, Diane; Frommer, Wolf B

    2010-12-01

    Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. In the present study, genetically encoded FRET (Förster resonance energy transfer)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP with varying external supply and in glucose-transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes of intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in a hxt5Δ hexose-transporter mutant compared with wild-type and other hxtΔ strains.

  2. Dissecting the effect of anions on Hg2+ detection using a FRET based DNA probe.

    Science.gov (United States)

    Kiy, Mehmet Murat; Zaki, Ahmed; Menhaj, Arsalsan Beg; Samadi, Azadeh; Liu, Juewen

    2012-08-07

    Many biosensors have been developed to detect Hg(2+) using thymine-rich DNA. While sensor response to various cations is often studied to demonstrate selectivity, the effect of anions has been largely overlooked. Anions may compete with DNA for metal binding and thus produce a false negative result. Anions cannot be added alone; the cation part of a salt may cause DNA compaction and other effects, obscuring the role of anions. We find that the sensitivity of a FRET-based Hg(2+) probe is independent of Na(+) concentration. Therefore, by using various sodium salts, any change in sensitivity can be attributed solely to the effect of anions. Halide salts, sulfides, and amines are strong inhibitors; anions containing oxo or hydroxyl groups (e.g. nitrate, sulfate, phosphate, carbonate, acetate, and citrate) do not interfere with Hg(2+) detection even at 100 mM concentration. Mercury hydrolysis and its diffusion into polypropylene containers can also strongly affect the detection results. We conclude that thymine-rich DNA should be useful for Hg(2+) detection in many environmental water samples.

  3. Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.

    Science.gov (United States)

    Nakanishi, Yoichi; Iida, Syuntaro; Ueoka-Nakanishi, Hanayo; Niimi, Tomoaki; Tomioka, Rie; Maeshima, Masayoshi

    2013-01-01

    Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

  4. Fluorescence resonance energy transfer (FRET) in chemistry and biology: Non-Förster distance dependence of the FRET rate

    Indian Academy of Sciences (India)

    Sangeeta Saini; Harjinder Singh; Biman Bagchi

    2006-01-01

    Fluorescence resonance energy transfer (FRET) is a popular tool to study equilibrium and dynamical properties of polymers and biopolymers in condensed phases and is now widely used in conjunction with single molecule spectroscopy. In the data analysis, one usually employs the Förster expression which predicts (1/6) distance dependence of the energy transfer rate. However, critical analysis shows that this expression can be of rather limited validity in many cases. We demonstrate this by explicitly considering a donor-acceptor system, polyfluorene (PF6)-tetraphenylporphyrin (TPP), where the size of both donor and acceptor is comparable to the distance separating them. In such cases, one may expect much weaker distance (as 1/2 or even weaker) dependence. We have also considered the case of energy transfer from a dye to a nanoparticle. Here we find 1/4 distance dependence at large separations, completely different from Förster. We also discuss recent application of FRET to study polymer conformational dynamics.

  5. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  6. Impact Fretting Wear Behavior of Alloy 690 Tubes in Dry and Deionized Water Conditions

    Science.gov (United States)

    Cai, Zhen-Bing; Peng, Jin-Fang; Qian, Hao; Tang, Li-Chen; Zhu, Min-Hao

    2017-07-01

    The impact fretting wear has largely occurred at nuclear power device induced by the flow-induced vibration, and it will take potential hazards to the service of the equipment. However, the present study focuses on the tangential fretting wear of alloy 690 tubes. Research on impact fretting wear of alloy 690 tubes is limited and the related research is imminent. Therefore, impact fretting wear behavior of alloy 690 tubes against 304 stainless steels is investigated. Deionized water is used to simulate the flow environment of the equipment, and the dry environment is used for comparison. Varied analytical techniques are employed to characterize the wear and tribochemical behavior during impact fretting wear. Characterization results indicate that cracks occur at high impact load in both water and dry equipment; however, the water as a medium can significantly delay the cracking time. The crack propagation behavior shows a jagged shape in the water, but crack extended disorderly in dry equipment because the water changed the stress distribution and retarded the friction heat during the wear process. The SEM and XPS analysis shows that the main failure mechanisms of the tube under impact fretting are fatigue wear and friction oxidation. The effect of medium(water) on fretting wear is revealed, which plays a potential and promising role in the service of nuclear power device and other flow equipments.

  7. Cognitive Workload and Fatigue in a Vigilance Dual Task: Miss Errors, False Alarms, and the Effect of Wearing Biometric Sensors While Working.

    Science.gov (United States)

    Guastello, Stephen J; Reiter, Katherine E; Malon, Matthew

    2016-10-01

    The effects of workload, fatigue, and practice on the performance of cognitive tasks are often intertwined. Previous research has shown that these influences can be separated with the two cusp catastrophe models. This study expanded an earlier investigation of the two models for workload and fatigue in a vigilance task to include a wider range of bifurcation variables that could affect the elasticity versus rigidity of the operator in response to workload and added performance variability resulting from fatigue. The study also responded to a concern in the literature that performance on cognitive tasks can be complicated by adaptive responses to artificial task situations and thus distort underlying cognitive events. Therefore, we also explored whether wearing biometric sensors, frequently used in workload studies, can affect performance dynamics. Participants were 279 undergraduates who responded to target stimuli that appeared on a simulated security camera display at three rates of speed while completing a secondary task. Participants worked alone, in pairs, or in pairs wearing GSR sensors. Results supported the efficacy of the two models and isolated the impact of wearing sensors on the fatigue process. The strongest control variables across both the workload and fatigue models were field independence, anxiety, indecisiveness, inflexibility, secondary task completion, working in pairs, and wearing the sensors. The contributing effect of wearing sensors could possibly extend to other types of wearable technologies.

  8. Design and proposal of dual line-of-defense perimeter watchdog incorporating optimally designed FBG based accelerometers and strain sensors using single optical fiber

    Science.gov (United States)

    Khan, Mohd. Mansoor; Sonkar, Ramesh Kumar

    2015-06-01

    Paper presents Opto-Mechanical intrusion sensor fence with FBGs attached to mechanical accelerometers and strain sensors, optimized on SolidWorks 2013 for desired frequency to 35 Hz, picking up accelerations/ strains and its deployment for perimeter security. The accelerometer structure consists of inertial mass supported by an L-shaped modified cantilever beam having non-uniform cross section area connected to base by a thin neck element which acts as strain concentrated centre hence an optimum zone for FBG sensors placement. Bragg wavelength shifts were obtained on Optigrating software for the obtained strain values on mechanical assembly of fence. CFD wind analysis is performed on the assembly to obtain the spot for accelerometer's placement to avoid false alarms up to wind velocities of 20 m/s.

  9. Fretting wear damage of HexTOOL{sup TM} composite depending on the different fibre orientations

    Energy Technology Data Exchange (ETDEWEB)

    Terekhina, S; Salvia, M; Fouvry, S [Laboratoire de Tribologie et Dynamique des Systemes, UMR CNRS ECL ENISE ENSMSE 5513, Ecole Centrale de Lyon, 69134 Ecully cedex (France); Malysheva, G; Tarasova, T, E-mail: svetlana.terekhina@ec-lyon.fr, E-mail: svetlanaterekhina@yandex.ru [Bauman Moscow State Technical University, 105005 Moscow, 5, 2nd Baumanskaya str (Russian Federation)

    2009-09-15

    The composites have drawn considerable interest in the mould processes. The vibrations and fatigue stresses induced in the moulds made evident to characterize the composite HexTOOL{sup TM} under fretting conditions. Fretting is a small-amplitude oscillatory motion between contacting surfaces. The running conditions fretting maps (RCFM) of composite at ambient conditions were established. The influence of different fiber orientations of HexTOOL{sup TM} composite on the wear kinetics was shown. An energy wear approach was developed. According to results of dynamic mechanical analysis (DMA), the viscoelastic properties of composite material were obtained.

  10. Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET.

    Science.gov (United States)

    Kudryashova, Kseniya S; Chertkov, Oleg V; Nikitin, Dmitry V; Pestov, Nikolai A; Kulaeva, Olga I; Efremenko, Anastasija V; Solonin, Alexander S; Kirpichnikov, Mikhail P; Studitsky, Vasily M; Feofanov, Alexey V

    2015-01-01

    Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.

  11. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    OpenAIRE

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unrav...

  12. Fretting Fatigue Improvement of Ti6Al4V by Coating and Shot Peening

    Institute of Scientific and Technical Information of China (English)

    Daoxin LIU; Xiaodong ZHU; Bin TANG; Jiawen HE

    2005-01-01

    Ion beam enhanced deposition (IBED) was employed to increase the fretting fatigue resistance of Ti6AI4V. CrN and TiN hard coatings were applied on the base material and shot peening was combined with the hard coatings to study the duplex effect on fretting fatigue resistance, The IBED coatings exhibited a good bonding strength. They did not spall off even after shot peening. However, an optimum composition of CrN showed better fretting fatigue resistance than that of TiN with the same processing parameters.

  13. The Effect of Elevated Temperature on the Fretting Fatigue Behavior of Nickel Alloy IN-100

    Science.gov (United States)

    2008-04-01

    Saladin S-N data points for plain fatigue 86 4.16 Comparison of Ownby and Saladin S-N data points for fretting fatigue 87 4.17 Comparison of Ownby...and Saladin S-N data for all tests 88 4.18 Plot comparing ΔQ vs Nf from Ownby, Saladin & Madhi data points 89 4.19 Plot from Kawagoishi et al. [21...in this study 93 4.3 Madhi fretting and plain fatigue testing data 94 4.4 Saladin fretting and plain fatigue testing data 94 4.5 Comparison of

  14. Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins.

    Science.gov (United States)

    Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A

    2015-08-14

    Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells.

  15. A new unsymmetrical azine derivative based on coumarin group as dual-modal sensor for CN- and fluorescent "OFF-ON" for Zn2 +

    Science.gov (United States)

    Hu, Jing-Han; Sun, You; Qi, Jing; Li, Qiao; Wei, Tai-Bao

    2017-03-01

    A simple chemosensor YS based on coumarin group was designed and synthesized. Its structure was determined by single crystal X-ray diffraction analysis. In the presence of cyanide the sensor showed an immediate visible change in color from colorless to pale yellow by naked-eyes and produced a strong blue fluorescence rapidly in DMSO/H2O (3:7, v/v). The detection limit on fluorescence response of the sensor to CN- is down to 1.69 × 10- 7 M. In addition YS could show fluorescence turn-on response rapidly for Zn2 + over other metals ions such as Fe3 +, Hg2 +, Ag+, Ca2 +, Cu2 +, Co2 +, Ni2 +, Cd2 +, Pb2 +, Cr3 + and Mg2 + in aqueous solutions. Notably, this sensor served as a molecular switch, which was controlled by Zn2 + and EDTA cyclically. Test strips based on YS were fabricated, which could act as a convenient and efficient CN- and Zn2 + test kit. This chemosensor could achieve the detection of two ions, which need not depend on two different sensors.

  16. Methodological considerations for global analysis of cellular FLIM/FRET measurements

    Science.gov (United States)

    Adbul Rahim, Nur Aida; Pelet, Serge; Kamm, Roger D.; So, Peter T. C.

    2012-02-01

    Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

  17. Nucleic Acid Base Analog FRET-Pair Facilitating Detailed Structural Measurements in Nucleic Acid Containing Systems

    DEFF Research Database (Denmark)

    Börjesson, Karl; Preus, Søren; El-Sagheer, Afaf

    2009-01-01

    distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes...... as the number of bases separating tCO and tC(nitro) is varied. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. In combination with the good nucleobase analog properties, this points...... toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid...

  18. Combined effects of fretting and pollutant particles on the contact resistance of the electrical connectors

    Directory of Open Access Journals (Sweden)

    Zhigang Kong

    2017-06-01

    Full Text Available Usually, when electrical connectors operate in vibration environments, fretting will be produced at the contact interfaces. In addition, serious environmental pollution particles will affect contact resistance of the connectors. The fretting will worsen the reliability of connectors with the pollutant particles. The combined effects of fretting and quartz particles on the contact resistance of the gold plating connectors are studied with a fretting test system. The results show that the frequencies have obvious effect on the contact resistance. The higher the frequency, the higher the contact resistance is. The quartz particles cause serious wear of gold plating, which make the nickel and copper layer exposed quickly to increase the contact resistance. Especially in high humidity environments, water supply certain adhesion function and make quartz particles easy to insert or cover the contact surfaces, and even cause opening resistance.

  19. Fretting Behavior of SPR Joining Dissimilar Sheets of Titanium and Copper Alloys

    Directory of Open Access Journals (Sweden)

    Xiaocong He

    2016-12-01

    Full Text Available The fretting performance of self-piercing riveting joining dissimilar sheets in TA1 titanium alloy and H62 copper alloy was studied in this paper. Load-controlled cyclic fatigue tests were carried out using a sine waveform and in tension-tension mode. Scanning electron microscopy and energy-dispersive X-ray techniques were employed to analyze the fretting failure mechanisms of the joints. The experimental results showed that there was extremely severe fretting at the contact interfaces of rivet and sheet materials for the joints at relatively high loads levels. Moreover, the severe fretting in the region on the locked sheet in contact with the rivet was the major cause of the broken locked sheet for the joints at low load level.

  20. Fretting wear in titanium, Monel-400, and cobalt 25-percent-molybdenum using scanning electron microscopy

    Science.gov (United States)

    Bill, R. C.

    1972-01-01

    Damage scar volume measurements taken from like metal fretting pairs combined with scanning electron microscopy observations showed that three sequentially operating mechanisms result in the fretting of titanium, Monel-400, and cobalt - 25-percent molybdenum. Initially, adhesion and plastic deformation of the surface played an important role. This was followed after a few hundred cycles by a fatigue mechanism which produced spall-like pits in the damage scar. Finally, a combination of oxidation and abrasion by debris particles became most significant. Damage scar measurements made on several elemental metals after 600,000 fretting cycles suggested that the ratio of oxide hardness to metal hardness was a measure of the susceptibility of a metal to progressive damage by fretting.

  1. Influence of fretting on the fatigue strength at the vise clamp–specimen interface

    Indian Academy of Sciences (India)

    Abdulhaqq A Hamid; Rafi K Yahya

    2003-12-01

    Fretting fatigue is one of the most important phenomena for inducing a significant reduction of fatigue strength and consequently, leading to unexpected failure accidents of the engineering structures even at very low stresses. In the present study, both plain and fretting fatigue tests with zero mean stress were carried out on two different types of steel, low-carbon steel and martensitic stainless steel, by means of a reversed bending fatigue testing machine. The drop in the fatigue strengths through fretting at vise clamp–specimen interface were significant for both tested steels. The fretting processes produced a reduction in fatigue strength of about 27% for low-carbon steel and 16% for martensitic stainless steel.

  2. Single-molecule three-color FRET with both negligible spectral overlap and long observation time.

    Directory of Open Access Journals (Sweden)

    Sanghwa Lee

    Full Text Available Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF microscopy.

  3. Effects of Contact Load on the Fretting Fatigue Behavior of IN-100 at Elevated Temperature

    Science.gov (United States)

    2009-03-01

    experimental setup which did not accurately model the turbine engine environment. Kawagoishi et al. [23] studied the nickel-based alloy Inconel 718 ...In their study with Inconel 718 [48, 49], it was found that the beneficial glaze oxide had formed and did affect the fretting fatigue life. Their...Waterhouse [52] investigated the fretting fatigue behaviors of austenitic steel type 321 and nickel-based alloy Inconel 718 at elevated temperatures. He

  4. Quantitative thermoelastic measurement on a helicopter glass fibre component underneath a surface anti-fretting coating.

    Directory of Open Access Journals (Sweden)

    Costa A.

    2010-06-01

    Full Text Available Thermoelastic stress analysis (TSA has been applied to measure the first stress invariant on a composite helicopter component under load. The component comprised inner mono-directional glass fiber layers with an outer central plate in glass fiber cloth, covered by an anti-fretting surface coating. In order to obtain quantitative results, a previous calibration of the thermoelastic constant obtained on a composite sample with a similar anti-fretting coating has been necessary.

  5. EXPERIMENTAL INVESTIGTION OF THE FRETTING PHENOMENON-DEPENDENCE OF NUMBERS CYCLES

    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI

    2014-12-01

    Full Text Available The present paper argues that adhesion forces and elastic deformation in the contact zone may contribute significantly to the relative displacement during fretting of metals. A simultaneously applied tangential force and normal into contact appears a adhesion force. A tangential force whose magnitude is less equal on greater than the force of limiting friction will not give rise on give rise to a sliding motion.It is determined the energy loss dissipated per fretting cycle.

  6. Switch on or switch off: an optical DNA sensor based on poly(p-phenylenevinylene) grafted magnetic beads.

    Science.gov (United States)

    Srinivas, Anupama R Gulur; Peng, Hui; Barker, David; Travas-Sejdic, Jadranka

    2012-05-15

    There has been an enormous demand for commercial label-free DNA sensors in a diverse range of fields including pre-emptive medicine, diagnostics, environmental monitoring, and food industry. Addressing the need for sensitive, selective and facile DNA sensors, we demonstrate a novel switch on/off sensor design that utilizes sandwich hybridization between photoluminescent anionic conjugated polyelectrolyte (CPE) bound captureprobe coated onto magnetic beads, target and the signaling probe. The hybridization-readout in our sensor was monitored by either fluorescence resonance energy transfer (FRET, switch-on) or superquenching (switch-off) depending on the type of signaling probe used. Moreover recent designs that utilize beads for sensing DNA have been limited towards using electrostatic interactions or intercalation of dyes to observe FRET. To our knowledge this is the first report of a switch on/off sensor utilizing either FRET or superquenching thus providing flexibility for future development of such rapid, facile and sensitive DNA sensors. The FRET-based sensor was investigated by optimizing the reaction parameters and selectivity. A low detection limit of 240 fmol in 2 mL of SSC buffer was achieved.

  7. Large-area, uniform and low-cost dual-mode plasmonic naked-eye colorimetry and SERS sensor with handheld Raman spectrometer.

    Science.gov (United States)

    Xu, Zhida; Jiang, Jing; Wang, Xinhao; Han, Kevin; Ameen, Abid; Khan, Ibrahim; Chang, Te-Wei; Liu, Gang Logan

    2016-03-21

    We demonstrated a highly-sensitive, wafer-scale, highly-uniform plasmonic nano-mushroom substrate based on plastic for naked-eye plasmonic colorimetry and surface-enhanced Raman spectroscopy (SERS). We gave it the name FlexBrite. The dual-mode functionality of FlexBrite allows for label-free qualitative analysis by SERS with an enhancement factor (EF) of 10(8) and label-free quantitative analysis by naked-eye colorimetry with a sensitivity of 611 nm RIU(-1). The SERS EF of FlexBrite in the wet state was found to be 4.81 × 10(8), 7 times stronger than in the dry state, making FlexBrite suitable for aqueous environments such as microfluid systems. The label-free detection of biotin-streptavidin interaction by both SERS and colorimetry was demonstrated with FlexBrite. The detection of trace amounts of the narcotic drug methamphetamine in drinking water by SERS was implemented with a handheld Raman spectrometer and FlexBrite. This plastic-based dual-mode nano-mushroom substrate has the potential to be used as a sensing platform for easy and fast analysis in chemical and biological assays.

  8. Fretting properties of biodegradable Mg-Nd-Zn-Zr alloy in air and in Hank's solution.

    Science.gov (United States)

    Li, Wenting; Li, Nan; Zheng, Yufeng; Yuan, Guangyin

    2016-11-04

    Fretting is a significant cause for the failure of orthopedic implants. Currently, since magnesium and its alloys have been developed as promising biodegradable implant materials, the fretting behavior of the Mg alloys is of great research significance. In this study, a Mg-Nd-Zn-Zr alloy (hereafter, denoted as JDBM alloy) was selected as experimental material, and its fretting behaviors were evaluated under 5 N, 10 N and 20 N normal loads with a displacement of 200 μm under the frequency of 10 Hz at 37 °C in air and in Hank's solution, respectively. The results indicated that while the friction coefficient decreased with the increment of the normal load, the wear volume of the alloy increased with the increment of the normal load both in air and in Hank's solution. Both the friction coefficients and the wear volume of the fretting in Hank's solution were much lower than those in air environment. The evolution trend of friction coefficients with time had different performance in air environment and the Hank's solution group. Although oxidation occurred during the fretting tests in Hank's solution, the damage of JDBM alloy was still reduced due to the lubrication effects of Hank's solution. Moreover, the addition of Fetal bovine serum (FBS) could act as lubrication and result in the reduction of the fretting damage.

  9. Test Methodology of Reproducing Fuel Rod Failure by Debris Fretting Wear

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Oh Joon; Park, Nam Gyu; Kim, Jae Ik [KEPCO NF, Daejeon (Korea, Republic of)

    2015-10-15

    A test was conducted with simple debris to reproduce debris fretting wear. 68% of fuel rod cladding thickness is worn out by Inconel debris in 75 hours. The test result shows that a simple link system is useful to accommodate debris oscillation, and mid grid mixing vanes could be a source of debris forcing. Additional tests will be conducted with various debris such as wire brush, metal chip, etc which are suspected to generate actual debris fretting wear in future works. Debris fretting is one of the most common cause of the nuclear fuel rod failure. Even the most of the nuclear fuels has debris protection system, debris still cause fuel rod failure. From 1994 to 2006, debris fretting failure is around 11% of the total fuel failure. In 2006-2010, the portion of debris rises to over 13%. The total number of fuel rods failure is decreasing, but the portion of the debris fretting wear is growing with time. Therefore reproducing and identifying the mechanism of fuel rod failure by debris fretting wear is needed to improve reliability of the nuclear fuel.

  10. Non-conjugated small molecule FRET for differentiating monomers from higher molecular weight amyloid beta species.

    Directory of Open Access Journals (Sweden)

    Chongzhao Ran

    Full Text Available BACKGROUND: Systematic differentiation of amyloid (Aβ species could be important for diagnosis of Alzheimer's disease (AD. In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathology, and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher molecular weight species (HrMW would be beneficial for drug screening, diagnosis, and molecular mechanism studies. However, fast and cheap methods for these specific aims are still lacking. PRINCIPAL FINDINGS: We demonstrated the feasibility of a non-conjugated FRET (Förster resonance energy transfer technique that utilized amyloid beta (Aβ species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivatives that served as the small molecule FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer solution. Lastly, this FRET technique enabled us to quantify the concentrations of Aβ monomers and high molecular weight species in solution. SIGNIFICANCE: We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.

  11. A 5.2GHz, 0.5mW RF powered wireless sensor with dual on-chip antennas for implantable intraocular pressure monitoring

    KAUST Repository

    Arsalan, Muhammad

    2013-06-01

    For the first time a single chip implantable wireless sensor system for Intraocular Pressure Monitoring (IOPM) is presented. This system-on-chip (SoC) is battery-free and harvests energy from incoming RF signals. The chip is self-contained and does not require external components or bond wires to function. This 1.4mm3 SoC has separate 2.4GHz-transmit and 5.2GHz-receive antennas, an energy harvesting module, a temperature sensor, a 7-bit TIQ Flash ADC, a 4-bit RFID, a power management and control unit, and a VCO transmitter. The chip is fabricated in a standard 6-metal 0.18μm CMOS process and is designed to work with a post-processed MEMS pressure sensor. It consumes 513μW of peak power and when implanted inside the eye, it is designed to communicate with an external reader using on-off keying (OOK). © 2013 IEEE.

  12. Dual-MWCNT Probe Thermal Sensor Assembly and Evaluation Based on Nanorobotic Manipulation inside a Field-Emission-Scanning Electron Microscope

    Directory of Open Access Journals (Sweden)

    Zhan Yang

    2015-03-01

    Full Text Available We report a thermal sensor composed of two multiwalled carbon nano-tubes (MWCNTs inside a field-emission-scanning electron microscope. The sensor was assembled using a nanorobotic manipulation system, which was used to construct a probe tip in order to detect the local environment of a single cell. An atomic force microscopy (AFM cantilever was used as a substrate; the cantilever was composed of Si3N4 and both sides were covered with a gold layer. MWCNTs were individually assembled on both sides of the AFM cantilever by employing nanorobotic manipulation. Another AFM cantilever was subsequently used as an end effector to manipulate the MWCNTs to touch each other. Electron-beam-induced deposition (EBID was then used to bond the two MWCNTs. The MWCNT probe thermal sensor was evaluated inside a thermostated container in the temperature range from 25°C to 60°C. The experimental results show the positive characteristics of the temperature coefficient of resistance (TCR.

  13. Protonophore- and pH-insensitive glucose and sucrose accumulation detected by FRET nanosensors in Arabidopsis root tips.

    Science.gov (United States)

    Chaudhuri, Bhavna; Hörmann, Friederike; Lalonde, Sylvie; Brady, Siobhan M; Orlando, David A; Benfey, Philip; Frommer, Wolf B

    2008-12-01

    Although soil contains only traces of soluble carbohydrates, plant roots take up glucose and sucrose efficiently when supplied in artificial media. Soluble carbohydrates and other small metabolites found in soil are in part products from exudation from plant roots. The molecular nature of the transporters for uptake and exudation is unknown. Here, fluorescence resonance energy transfer (FRET) glucose and sucrose sensors were used to characterize accumulation and elimination of glucose and sucrose in Arabidopsis roots tips. Using an improved image acquisition set-up, FRET responses to perfusion with carbohydrates were detectable in roots within less than 10 sec and over a wide concentration range. Accumulation was fully reversible within 10-180 sec after glucose or sucrose had been withdrawn; elimination may be caused by metabolism and/or efflux. The rate of elimination was unaffected by pre-incubation with high concentrations of glucose, suggesting that elimination is not due to accumulation in a short-term buffer such as the vacuole. Glucose and sucrose accumulation was insensitive to protonophores, was comparable in media differing in potassium levels, and was similar at pH 5.8, 6.8 and 7.8, suggesting that both influx and efflux may be mediated by proton-independent transport systems. High-resolution expression mapping in root tips showed that only a few proton-dependent transport of the STP (Sugar Transport Protein) and SUT/SUC (Sucrose Transporter/Carrier) families are expressed in the external cell layers of root tips. The root expression maps may help to pinpoint candidate genes for uptake and release of carbohydrates from roots.

  14. Rational design of a thermalresponsive-polymer-switchable FRET system for enhancing the temperature sensitivity of upconversion nanophosphors

    Science.gov (United States)

    Xiao, Qingbo; Li, Yanfang; Li, Fujin; Zhang, Mengxin; Zhang, Zhijun; Lin, Hongzhen

    2014-08-01

    Here we propose a thermoresponsive polymer PNIPAM modulated fluorescence resonance energy transfer (FRET) system to enhance the temperature sensitivity of upconversion nanophosphors (UCNPs). By utilizing red/near-infrared dual emitting NaLuF4:Mn2+,Ln3+ (Ln3+ = Yb3+, Er3+, Tm3+) UCNPs as the energy donor and Au nanoparticles as the acceptor, the temperature resolution of the UCNPs is significantly increased from 3.1 °C to 0.9 °C in the physiological temperature range. Conjugating the UCNPs and acceptors into discrete nanocomposites in our samples facilitates reversible regulation of the emission intensity of UCNPs, which thus would extend their application range in biosensing, especially for probing the dynamic changes of local micro-environments in biological tissues. As there are a broad variety of stimuli to which smart polymers can reversibly respond, our experiments are also extendable to various external conditions in local micro-environments, such as pH values, metal ions, glucose, and tissue-specific enzymes.Here we propose a thermoresponsive polymer PNIPAM modulated fluorescence resonance energy transfer (FRET) system to enhance the temperature sensitivity of upconversion nanophosphors (UCNPs). By utilizing red/near-infrared dual emitting NaLuF4:Mn2+,Ln3+ (Ln3+ = Yb3+, Er3+, Tm3+) UCNPs as the energy donor and Au nanoparticles as the acceptor, the temperature resolution of the UCNPs is significantly increased from 3.1 °C to 0.9 °C in the physiological temperature range. Conjugating the UCNPs and acceptors into discrete nanocomposites in our samples facilitates reversible regulation of the emission intensity of UCNPs, which thus would extend their application range in biosensing, especially for probing the dynamic changes of local micro-environments in biological tissues. As there are a broad variety of stimuli to which smart polymers can reversibly respond, our experiments are also extendable to various external conditions in local micro

  15. Spectral studies on anthracene based dual sensor for Hg2 + and Al3 + ions with two distinct output modes of detection

    Science.gov (United States)

    Kaur, Navneet; Kaur, Baljeet

    2017-06-01

    A simple and easily synthesized colorimetric and fluorescent chemosensor 1, based on aromatic sbnd OH and imine moieties as a binding and anthracene as signaling unit, has been synthesized in a one-step procedure. The chemosensor 1 is developed as a dual chemosensor for detection of Hg2 + and Al3 + ions in CH3OH, which exhibited a color change from light yellow to dark yellow with Hg2 + ions, enabling 1 a suitable ;bare eye; indicator for Hg2 + ions. On the other hand, fluorescent enhancement with blue shift along with brilliant cyan fluorescence was observed upon binding with Al3 + ions. A possible sensing mechanism has been proposed by means of Job's plot and 1H NMR titration.

  16. Effects of temperature, slip amplitude, contact pressure on fretting fatigue behavior of Ti811 alloys at elevated temperatures

    Institute of Scientific and Technical Information of China (English)

    Xiaohua ZHANG; Daoxin LIU

    2009-01-01

    Effects of the temperature, slip amplitude, and contact pressure on fretting fatigue (FF) behavior of the Ti811 titanium alloy were investigated using a high frequency fatigue machine and a home-made high temperature apparatus. The fretting fatigue failure mechanism was studied by observing the fretting surface morphology features. The results show that the sensitivity to fretting fatigue is high at both 350 and 500 ℃. The higher the temperature, the more sensitive to the fretting fatigue failure is. Creep is an important factor that influences the fretting fatigue failure process at elevated temperatures. The fretting fatigue life of the Ti811 alloy does not change in a monotonic way as the slip amplitude and contact pressure increase. This is owing to the fact that the slip amplitude affects the action of fatigue and wear in the fretting process, and the nominal contact pressure affects the distribution and concentration of the stress and the amplitude of fretting slip at the contact surface, and thus further influences the crack initiation probability and the driving force for propagation.

  17. Interference Assembly and Fretting Wear Analysis of Hollow Shaft

    Directory of Open Access Journals (Sweden)

    Chuanjun Han

    2014-01-01

    Full Text Available Fretting damage phenomenon often appears in the interference fit assembly. The finite element model of hollow shaft and shaft sleeve was established, and the equivalent stress and contact stress were computed after interference assembly. The assembly body of hollow shaft and shaft sleeve was in whirling bending load, and the contact status (sticking, sliding, and opening and the distribution of stress along one typical contact line were computed under different loads, interferences, hollow degrees, friction coefficient, and wear quantity. Judgment formula of contact state was fixed by introducing the corrected coefficient k. The computation results showed that the “edge effect” appears in the contact surface after interference fit. The size of slip zone is unchanged along with the increase of bending load. The greater the interference value, the bigger the wear range. The hollow degree does not influence the size of stick zone but controls the position of the junction point of slip-open. Tangential contact stress increases with the friction coefficient, which has a little effect on normal contact stress. The relationship between open size and wear capacity is approximately linear.

  18. Analysis of initial crack path in fretting fatigue

    Directory of Open Access Journals (Sweden)

    J. Vázquez

    2016-07-01

    Full Text Available The initial crack path is analysed in a fretting fatigue test with cylindrical contact, where there is a stress gradient and a multiaxial and non-proportional stress state. For this, a cylindrical pad is pressed, with a constant normal load, N, against a dog-bone type fatigue test specimen. Then, the test specimen is subjected to a cyclic axial stress, σ. Due to the cyclical axial stress, the assembly used and the friction between the contact pair, a tangential cyclic load Q is generated. In these tests, both components are made of Al7075-T651 alloy. The crack initiation path along the fracture surface is optically measured using a focus variation technique. The contact stress/strain fields obtained analytically, in junction with the Fatemi-Socie (FS and Smith-Watson- Topper (SWT multiaxial fatigue parameters, allow us to determine the controlling parameters of the crack initiation process observed in the tests and to estimate the crack path during the early stage of the crack growth.

  19. Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

    Science.gov (United States)

    Gahl, Robert F; Tekle, Ephrem; Tjandra, Nico

    2014-03-15

    Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented. Published by Elsevier Inc.

  20. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application. Copyright

  1. APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

    Directory of Open Access Journals (Sweden)

    Heidi J Chial

    Full Text Available BACKGROUND: Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported. METHODOLOGY: Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species. CONCLUSIONS: All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes

  2. Large-area, uniform and low-cost dual-mode plasmonic naked-eye colorimetry and SERS sensor with handheld Raman spectrometer

    CERN Document Server

    Xu, Zhida; Wang, Xinhao; Han, Kevin; Ameen, Abid; Khan, Ibrahim; Chang, Te-Wei; Liu, Gang Logan

    2016-01-01

    We demonstrated a highly sensitive, waferscale, highly uniform plasmonic nanomushroom substrate based on plastic for nakedeye plasmonic colorimetry and surface enhanced Raman spectroscopy (SERS). We gave it the name FlexBrite. The dualmode functionality of FlexBrite allows for label-free qualitative analysis by SERS with an enhancement factor (EF) of 10^8 and labelfree quantitative analysis by naked eye colorimetry with a sensitivity of 611 nm RIU-1. The SERS EF of FlexBrite in the wet state was found to be 4.81 X 10^8, 7 times stronger than in the dry state, making FlexBrite suitable for aqueous environments such as microfluid systems. The labelfree detection of biotin streptavidin interaction by both SERS and colorimetry was demonstrated with FlexBrite. The detection of trace amounts of the narcotic drug methamphetamine in drinking water by SERS was implemented with a handheld Raman spectrometer and FlexBrite. This plastic based dual-mode nano-mushroom substrate has the potential to be used as a sensing pla...

  3. A Supramolecular Sensor Array Using Lanthanide-Doped Nanoparticles for Sensitive Detection of Glyphosate and Proteins.

    Science.gov (United States)

    Wang, Meng; Ye, Hebo; You, Lei; Chen, Xueyuan

    2016-01-13

    Lanthanide (Ln(3+))-doped nanoparticles (NPs) are an intensive area of research in chemical and materials sciences. Herein a sensor array of Ln(3+)-doped NPs was developed for the first time toward sensitive molecular sensing based on a novel strategy of the hybridized time-resolved Förster resonance energy transfer (TR-FRET) with the indicator displacement assay (IDA) concept (TR-FRET-IDA). The sensor platform was generated in situ by binding a series of negatively charged indicators on the surface of ligand-free LiYF4:Ce/Tb NPs. The TR-FRET between NPs and dyes resulted in indicator emission and was employed as a means of removing undesired short-lived background luminescence from the indicator effectively. Displacement of indicators from the NP/indicator ensembles by glyphosate, a common herbicide, led to turn-off of the indicator emission. The sensor array was able to successfully discriminate 11 biologically relevant anions with high accuracy and sensitivity in pure aqueous buffer both qualitatively and quantitatively. Furthermore, the differentiation of six model proteins in the nM range was achieved with 100% accuracy for the classification, thereby demonstrating the versatility of this simple sensor platform. The study of the mechanism of binding and signal modulation further verified TR-FRET-IDA as a reliable sensing paradigm.

  4. DESIGN OF A DUAL KEYBOARD

    Directory of Open Access Journals (Sweden)

    V. Ragavi

    2013-01-01

    Full Text Available The design of a computer keyboard with dual function is proposed. This computer keyboard called Dual Keyboard can function both as a normal keyboard and as a pressure sensitive keyboard. The proposed device has a switch that decides the function. The keyboard makes use of sensors placed beneath the keys to measure the pressure applied on the key by the user. This device has many applications. In this study, it is applied to mitigate Denial of Service (DoS attack.

  5. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples.

    Directory of Open Access Journals (Sweden)

    Joachim Goedhart

    Full Text Available BACKGROUND: Fluorescence Resonance Energy Transfer (FRET between the green fluorescent protein (GFP variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells.

  6. A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology.

    Science.gov (United States)

    Li, Chunjie; Imanishi, Ayako; Komatsu, Naoki; Terai, Kenta; Amano, Mutsuki; Kaibuchi, Kozo; Matsuda, Michiyuki

    2017-01-11

    Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.

  7. pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins.

    Science.gov (United States)

    Betolngar, Dahdjim-Benoît; Erard, Marie; Pasquier, Hélène; Bousmah, Yasmina; Diop-Sy, Awa; Guiot, Elvire; Vincent, Pierre; Mérola, Fabienne

    2015-05-01

    It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.

  8. Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen

    Directory of Open Access Journals (Sweden)

    Shashi Bhuckory

    2016-02-01

    Full Text Available The integration of semiconductor quantum dots (QDs into homogeneous Förster resonance energy transfer (FRET immunoassay kits for clinical diagnostics can provide significant advantages concerning multiplexing and sensitivity. Here we present a facile and functional QD-antibody conjugation method using three commercially available QDs with different photoluminescence (PL maxima (605 nm, 655 nm, and 705 nm. The QD-antibody conjugates were successfully applied for FRET immunoassays against prostate specific antigen (PSA in 50 µL serum samples using Lumi4-Tb (Tb antibody conjugates as FRET donors and time-gated PL detection on a KRYPTOR clinical plate reader. Förster distance and Tb donor background PL were directly related to the analytical sensitivity for PSA, ...which resulted in the lowest limits of detection for Tb-QD705 (2 ng/mL, followed by Tb-QD655 (4 ng/mL, and Tb-QD605 (23 ng/mL. Duplexed PSA detection using the Tb-QD655 and Tb-QD705 FRET-pairs demonstrated the multiplexing ability of our immunoassays. Our results show that FRET based on QD acceptors is suitable for multiplexed and sensitive biomarker detection in clinical diagnostics.

  9. Significant FRET between SWNT/DNA and rare earth ions: a signature of their spatial correlations.

    Science.gov (United States)

    Ignatova, Tetyana; Najafov, Hikmat; Ryasnyanskiy, Aleksandr; Biaggio, Ivan; Zheng, Ming; Rotkin, Slava V

    2011-07-26

    Significant acceleration of the photoluminescence (PL) decay rate was observed in water solutions of two rare earth ions (REIs), Tb and Eu. We propose that the time-resolved PL spectroscopy data are explained by a fluorescence resonance energy transfer (FRET) between the REIs. FRET was directly confirmed by detecting the induced PL of the energy acceptor, Eu ion, under the PL excitation of the donor ion, Tb, with FRET efficiency reaching 7% in the most saturated solution, where the distance between the unlike REIs is the shortest. Using this as a calibration experiment, a comparable FRET was measured in the mixed solution of REIs with single-wall nanotubes (SWNTs) wrapped with DNA. From the FRET efficiency of 10% and 7% for Tb and Eu, respectively, the characteristic distance between the REI and SWNT/DNA was obtained as 15.9 ± 1.3 Å, suggesting that the complexes are formed because of Coulomb attraction between the REI and the ionized phosphate groups of the DNA.

  10. A Study on Surface Modification of Al7075-T6 Alloy against Fretting Fatigue Phenomenon

    Directory of Open Access Journals (Sweden)

    E. Mohseni

    2014-01-01

    Full Text Available Aircraft engines, fuselage, automobile parts, and energy saving strategies in general have promoted the interest and research in the field of lightweight materials, typically on alloys based on aluminum. Aluminum alloy itself does not have suitable wear resistance; therefore, it is necessary to enhance surface properties for practical applications, particularly when aluminum is in contact with other parts. Fretting fatigue phenomenon occurs when two surfaces are in contact with each other and one or both parts are subjected to cyclic load. Fretting drastically decreases the fatigue life of materials. Therefore, investigating the fretting fatigue life of materials is an important subject. Applying surface modification methods is anticipated to be a supreme solution to gradually decreasing fretting damage. In this paper, the authors would like to review methods employed so far to diminish the effect of fretting on the fatigue life of Al7075-T6 alloy. The methods include deep rolling, shot peening, laser shock peening, and thin film hard coatings. The surface coatings techniques are comprising physical vapor deposition (PVD, hard anodizing, ion-beam-enhanced deposition (IBED, and nitriding.

  11. Role of surface roughness on corrosion and fretting corrosion behaviour of commercially pure titanium in Ringer's solution for bio-implant application

    Science.gov (United States)

    Sivakumar, Bose; Pathak, Lokesh Chandra; Singh, Raghuvir

    2017-04-01

    Influence of roughness (ra) from 43 to 474 nm on corrosion and fretting corrosion of commercially pure titanium (CpTi) was studied in the Ringer's solution. The anodic polarization and electrochemical impedance spectroscopy (EIS) revealed the highest corrosion resistance of CpTi with ra 43 nm and correlated well with the surface energy (SE). The highest potential drop associated with the fretting corrosion is observed for CpTi with ra 43 nm followed by 474 nm; this is found to correspond with the worn out area. The fretting current density (ifretting) is several order higher than obtained during the potentiodynamic polarization (without fretting) study. Fretting corrosion manifested by the drop in electrochemical potential is simulated with high accuracy using fretting current density and an initial contact area. Fretting corrosion at an applied potential (+250 mV(SCE)) is produced much larger fretting corrosion current density than during the open circuit potential (OCP).

  12. Branched DNA nanostructures efficiently stabilised and monitored by novel pyrene-perylene 2'-α-l-amino-LNA FRET pairs

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Santhosh Kumar, T; Campbell, Meghan A;

    2013-01-01

    Novel pyrene-perylene α-l-LNA FRET pairs described herein effectively detect assembly of 2- and 3-way branched DNA nanostructures prepared by postsynthetic microwave-assisted CuAAC click chemistry. The fluorescent signalling of assembly by internally positioned FRET pairs is achieved with low to ...

  13. Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid

    Directory of Open Access Journals (Sweden)

    Norio Maruyama, Sachiko Hiromoto, Eiji Akiyama and Morihiko Nakamura

    2013-01-01

    Full Text Available Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-. For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR both in air and in PBS(-. A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR. The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.

  14. The Contact Ageing Effect on Fretting Damage of an Electro-Deposited Coating against an AISI52100 Steel Ball

    Directory of Open Access Journals (Sweden)

    Kyungmok Kim

    2016-09-01

    Full Text Available This article investigates the effect of contact ageing on fretting damage of an epoxy-based cathodic electro-deposited coating for use on automotive seat slide tracks (made of cold-rolled high strength steel. Static normal load was induced at the contact between the coating and an AISI52100 ball for a certain duration. It was identified that plastically deformed contact area increased logarithmically as a function of time when the contact was under static normal load. Fretting tests after various durations of static contact were conducted using a ball-on-flat plate apparatus. All fretting tests were halted when the friction coefficient reached a critical value of 0.5, indicating complete coating failure. The total number of fretting cycles to the critical friction coefficient was found to vary with the duration of static contact before fretting. It was identified that the number of cycles to the critical friction coefficient decreased with the increased duration of static contact. Meanwhile, the friction coefficient at steady-state sliding was not greatly affected by the duration of static contact before fretting. Finally, the relation between coating thickness after indentation creep and the number of cycles to the critical friction coefficient was found to be linear. Obtained results show that the duration of static contact before fretting has an influence on the fretting lifetime of an electro-deposited coating.

  15. Phanta: a non-fluorescent photochromic acceptor for pcFRET.

    Directory of Open Access Journals (Sweden)

    Craig Don Paul

    Full Text Available We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003 we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.

  16. A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

    Science.gov (United States)

    Chen, WeiYue; Avezov, Edward; Schlachter, Simon C; Gielen, Fabrice; Laine, Romain F; Harding, Heather P; Hollfelder, Florian; Ron, David; Kaminski, Clemens F

    2015-03-10

    FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.

  17. On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies.

    Science.gov (United States)

    Tahtaoui, Chouaib; Guillier, Fabrice; Klotz, Philippe; Galzi, Jean-Luc; Hibert, Marcel; Ilien, Brigitte

    2005-12-01

    The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.

  18. Analysis of Fretting Fatigue Strength of Integral Shroud Blade for Steam Turbine

    Science.gov (United States)

    Kaneko, Yasutomo; Tomii, Masayuki; Ohyama, Hiroharu; Kurimura, Takayuki

    To improve the reliability and the thermal efficiency of LP (Low Pressure) end blades of steam turbine, new standard series of LP end blades have been developed. The new LP end blades are characterized by the ISB (Integral Shroud Blade) structure. In the ISB structure, blades are continuously coupled by blade untwist due to centrifugal force when the blades rotate at high speed. One of the probable failure modes of the ISB structure seems to be fretting fatigue, because the ISB utilizes friction damping between adjacent shrouds and stubs. Therefore, in order to design a blade with high reliability, the design procedure for evaluating the fretting fatigue strength was established by the model test and the nonlinear contact analysis. This paper presents the practical design method for predicting the fretting fatigue strength of the ISB structure, and the some applications are explained.

  19. Low-Frequency Reciprocating Fretting Wear Testing System Design and Experiment Research

    Institute of Scientific and Technical Information of China (English)

    Zhongnan Wang∗,Wuyi Wang; Guangyu Zhang

    2015-01-01

    The fretting wear is resulted from different or same sample’ s surfaces by the small variationand leads to mechanism failures. The main factors consist of the variation of normal load and oscillation frequencies, among which surface topography of different materials are the main factors to the problems of the fretting wear. Therefore, a novel low⁃frequency reciprocating fretting wear test system is designed upon the principle of Friction coefficient measurement. Four metal and non⁃metallic samples are measured under various normal load and oscillation frequencies to obtain the instantaneous friction coefficient in the repeat experiments. In fact, the experimental results show that CoF curves of different samples with the increase of the normal load are the similar exponential decay or parabolic shapes, which are consistent with the literatures to verify the rational design and reliable⁃operation of the system under the conditions of different frequencies.

  20. Fretting corrosion of CoCr alloy: Effect of load and displacement on the degradation mechanisms.

    Science.gov (United States)

    Bryant, Michael; Neville, Anne

    2017-02-01

    Fretting corrosion of medical devices is of growing concern, yet, the interactions between tribological and electrochemical parameters are not fully understood. Fretting corrosion of CoCr alloy was simulated, and the components of damage were monitored as a function of displacement and contact pressure. Free corrosion potential (Ecorr), intermittent linear polarisation resistance and cathodic potentiostatic methods were used to characterise the system. Interferometry was used to estimate material loss post rubbing. The fretting regime influenced the total material lost and the dominant degradation mechanism. At high contact pressures and low displacements, pure corrosion was dominant with wear and its synergies becoming more important as the contact pressure and displacement decreased and increased, respectively. In some cases, an antagonistic effect from the corrosion-enhanced wear contributor was observed suggesting that film formation and removal may be present. The relationship between slip mechanism and the contributors to tribocorrosion degradation is presented.

  1. Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

    Science.gov (United States)

    Biener, Eva; Charlier, Madia; Ramanujan, V Krishnan; Daniel, Nathalie; Eisenberg, Avital; Bjørbaek, Christian; Herman, Brian; Gertler, Arieh; Djiane, Jean

    2005-12-01

    Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

  2. Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers.

    Science.gov (United States)

    Patowary, Suparna; Pisterzi, Luca F; Biener, Gabriel; Holz, Jessica D; Oliver, Julie A; Wells, James W; Raicu, Valerică

    2015-04-07

    Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

  3. Dual diagnosis

    OpenAIRE

    2013-01-01

    Dual diagnosis denotes intertwining of intellectual disabilities with mental disorders. With the help of systematic examination of literature, intellectual disabilities are determined (they are characterized by subaverage intellectual activity and difficulties in adaptive skills), along side mental disorders. Their influence is seen in changes of thinking, perception, emotionality, behaviour and cognition. Mental disorders often occur with people with intellectual disabilities (data differs f...

  4. FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET.

    Science.gov (United States)

    Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon; Weiss, Shimon; Michalet, Xavier

    2016-01-01

    Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to

  5. Fretting fatigue behavior of high-strength steel monostrands under bending load

    DEFF Research Database (Denmark)

    Winkler, Jan; Georgakis, Christos T.; Fischer, Gregor

    2015-01-01

    In this paper, the fretting fatigue behavior of pretensioned high-strength steel monostrands is investigated. To measure the local deformations on the strands, a novel method based on the digital image correlation (DIC) technique was used to quantify the relative movement between individual wires...... along the length of the monostrand. Information about the monostrand bending stiffness and the extent of relative displacement between core and outer wires of a monostrand undergoing flexural deformations is provided. From the series of dynamic fatigue tests, a fretting fatigue spectrum is derived...

  6. Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopy

    Science.gov (United States)

    Sun, Yuansheng; Booker, Cynthia F.; Day, Richard N.; Periasamy, Ammasi

    2009-02-01

    Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However, the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy. Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor photobleaching FRET microscopy.

  7. Aircraft Engine Sensor Fault Diagnostics Through Dual-Channel Sensor Measurements Based on a Bank of Hybrid Kalman Filters%基于混合卡尔曼滤波器组的航空发动机双通道传感器故障检测

    Institute of Scientific and Technical Information of China (English)

    张书刚; 郭迎清; 陆军

    2012-01-01

    Based on a bank of hybrid Kalman filters which are hybrids of a nonlinear on-board engine model (NOBEM) and piecewise linear Kalman fillers, a civil aircraft engine sensor fault diagnostics system which utilizes dual -channel sensor measurements is developed. Principles and algorithms of sensor fault detection, isolation and accommodation are given. By this system applied to some typical civil turbo-fan engine sensor faults, simulation results show that the diagnostic effectiveness of the system is maintained to avoid false alarms as the health of the engine degrades over time through a simple process: by feeding the health degradation values into the NUBEM and not changing the parameters of the linear Kalman filters. The update process, which can be completed automatically online to save time and effort, is feasible in the real application environment.%基于机载非线性模型与分段线性卡尔曼滤波器混合组成的混合卡尔曼滤波器组,结合双通道传感器的特点,建立了民用航空发动机传感器故障诊断系统;给出故障诊断原理及算法的同时,将该系统应用于民用涡扇发动机传感器常见典型故障进行了仿真;仿真结果表明,诊断系统可以在发动机发生健康蜕化后,通过只简单更新机载模型的蜕化因子,而保持线性卡尔曼滤波器的参数不变,便能准确地检测和隔离各类传感器故障而不发生误报;该更新过程可以在线自动完成,省时省力,易于工程实现.

  8. Reliability Analysis and Design of the Shell Based on Portable Dual Sensor Night Vision Goggles%便携式双传感器夜视镜壳体可靠性分析与设计

    Institute of Scientific and Technical Information of China (English)

    孟瑞; 邱亚峰

    2014-01-01

    便携式双传感器夜视镜是一种应用于军事作战中的重要仪器,而作为其核心零部件--壳体,必须保证在复杂作战环境下不会发生过量变形及应力集中,以免造成仪器失效。从选择该仪器壳体材料和建立模型着手,考虑夜视镜壳体上所受其他零部件给予的等效压力及弯矩,对其在2种极端的环境下进行仿真分析,得到-30℃及70℃两种情况下壳体所受最大应力及应变,并分析是否能够满足使用要求。%Portable dual sensor night vision goggles are important instruments in military operations. As its core component, the shell must avoid excessive deformation and stress concentration in complex operational environment in order to prevent equipment failures. This article introduced how to select the instrument shell material and build the modeling .Taking into account the equivalent pressure on night vision goggles the shell suffered from other components, the paper gives simulation analysis under two extreme conditions, simulating its biggest stress and strain at-30℃ and 70℃, andanalyzing whether to meet the requirements.

  9. QCD Dual

    DEFF Research Database (Denmark)

    Sannino, Francesco

    2009-01-01

    We uncover a novel solution of the 't Hooft anomaly matching conditions for QCD. Interestingly in the perturbative regime the new gauge theory, if interpreted as a possible QCD dual, predicts the critical number of flavors above which QCD in the nonperturbative regime, develops an infrared stable...... fixed point. Remarkably this value is identical to the maximum bound predicted in the nonpertubative regime via the all-orders conjectured beta function for nonsupersymmetric gauge theories.......We uncover a novel solution of the 't Hooft anomaly matching conditions for QCD. Interestingly in the perturbative regime the new gauge theory, if interpreted as a possible QCD dual, predicts the critical number of flavors above which QCD in the nonperturbative regime, develops an infrared stable...

  10. Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging

    NARCIS (Netherlands)

    Afsari, Hamid Samareh; Cardoso Dos Santos, Marcelina; Lindén, Stina; Chen, Ting; Qiu, Xue; van Bergen En Henegouwen, Paul M P; Jennings, Travis L; Susumu, Kimihiro; Medintz, Igor L; Hildebrandt, Niko; Miller, Lawrence W

    2016-01-01

    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress samp

  11. Imaging real-time HIV-1 virion fusion with FRET-based biosensors

    Science.gov (United States)

    Jones, Daniel M.; Padilla-Parra, Sergi

    2015-01-01

    We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously. PMID:26300212

  12. 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.

  13. Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions.

    Science.gov (United States)

    Kempe, Daryan; Cerminara, Michele; Poblete, Simón; Schöne, Antonie; Gabba, Matteo; Fitter, Jörg

    2017-01-03

    The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye-dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil-globule transition.

  14. Rise-time of FRET-acceptor fluorescence tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Van Mierlo, C.P.M.; Visser, A.J.W.G.; Borst, J.W.

    2014-01-01

    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no correspon

  15. Tracing the conformational changes in BSA using FRET with environmentally-sensitive squaraine probes

    Science.gov (United States)

    Govor, Iryna V.; Tatarets, Anatoliy L.; Obukhova, Olena M.; Terpetschnig, Ewald A.; Gellerman, Gary; Patsenker, Leonid D.

    2016-06-01

    A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.

  16. Metal ion induced FRET OFF-ON in tren/dansyl-appended rhodamine.

    Science.gov (United States)

    Lee, Min Hee; Kim, Hyun Jung; Yoon, Sangwoon; Park, Noejung; Kim, Jong Seung

    2008-01-17

    A series of new fluorescent probes bearing tren-spaced rhodamine B and dansyl groups have been synthesized. Compound 1 exhibits selective changes in the absorption and the emission spectra toward Cu2+ ion over miscellaneous metal cations. Among 1-3, 1 shows the best FRET efficiency through dansyl emission to rhodamine absorption for the Cu2+ ion.

  17. Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells.

    Science.gov (United States)

    Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D; Thomas, David D; Gillispie, Gregory D

    2017-03-01

    We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

  18. An FITC-BODIPY FRET couple: application to selective, ratiometric detection and bioimaging of cysteine.

    Science.gov (United States)

    Ma, Dong Hee; Kim, Dokyoung; Akisawa, Takuya; Lee, Kyung-Ha; Kim, Kyong-Tai; Ahn, Kyo Han

    2015-04-01

    A novel FRET couple of fluorescein is disclosed, and it was readily constructed by conjugating an amino-BODIPY dye, a new FRET donor, with fluorescein isocyanate. Its potential was demonstrated by a fluorescence sensing system for cysteine, which was prepared by introducing acryloyl groups to the fluorescein moiety. The FRET probe exhibited promising ratiometric response to cysteine with high selectivity and sensitivity in a buffer solution containing acetonitrile at a physiological pH of 7.4, but showed slow reactivity. This slow response was solved by addition of a surfactant, thus allowing ratiometric imaging and determination of the endogenous level of cysteine in cells in HEPES buffer, by confocal fluorescence microscopy. Imaging experiments toward various cells suggested that such aryl acrylate type probes are vulnerable to the ubiquitous esterase activity. For the selected C6 cell line, in which the esterase activity was minimal, the ratiometric quantification of cysteine level was demonstrated. The FRET probe was also applied to determine the level of cysteine in human blood plasma.

  19. Titanium carbide nanoparticles reinforcing nickel matrix for improving nanohardness and fretting wear properties in wet conditions

    Science.gov (United States)

    Dănăilă, Eliza; Benea, Lidia; Caron, Nadège; Raquet, Olivier

    2016-09-01

    In this study Ni/nano-TiC functional composite coatings were produced by electro-codeposition of TiC nanoparticles (50 nm mean diameter) with nickel on 304L stainless steel support. Coatings were obtained from a Watts classical solution in which TiC nanoparticles were added. The surface morphology, chemical composition, structure, roughness and thickness, were evaluated in relation to the effect of TiC nanoparticles incorporation into Ni matrix. It was found that incorporation of TiC nanoparticles into the nickel matrix produces morphological changes in the deposit and increases the roughness. The fretting wear behavior in wet conditions of the obtained coatings was evaluated on a ball-on-plate configuration. To evaluate the wet fretting wear (tribocorrosion) behavior the open circuit potential was measured before, during and after the fretting tests at room temperature in the solution that simulates the primary water circuit of Pressurized Water Reactors. The results show that Ni/nano-TiC composite coatings exhibited a low friction coefficient, high nanohardness and fretting wear resistance in wet conditions compared with pure Ni coatings.

  20. Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy

    NARCIS (Netherlands)

    Borst, J.W.; Laptenok, S.; Westphal, A.H.; Kühnemuth, R.; Hornen, H.; Visser, N.V.; Kalinin, S.; Aker, J.C.M.; Hoek, van A.; Seidel, C.A.M.; Visser, A.J.W.G.

    2008-01-01

    Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of

  1. Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution

    NARCIS (Netherlands)

    Bader, A.N.; Hofman, E.G.; Voortman, J.; Henegouwen, P.; Gerritsen, H.C.

    2009-01-01

    Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and

  2. Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET

    NARCIS (Netherlands)

    Hohlbein, J.; Kapanidis, A.N.

    2016-01-01

    Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open

  3. FRET-based modified graphene quantum dots for direct trypsin quantification in urine

    Energy Technology Data Exchange (ETDEWEB)

    Poon, Chung-Yan; Li, Qinghua [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Zhang, Jiali; Li, Zhongping [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Dong, Chuan [Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Lee, Albert Wai-Ming; Chan, Wing-Hong [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Li, Hung-Wing, E-mail: hwli@hkbu.edu.hk [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong)

    2016-04-21

    A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening. - Highlights: • A FRET-based biosensor was developed for direct quantification of trypsin. • Fast and sensitive screening of pancreatic disease was facilitated. • The direct quantification of trypsin in urine samples was demonstrated.

  4. Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

    Science.gov (United States)

    Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

    2016-08-01

    This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.

  5. Influence of oil and water media on fretting behaviour of AISI 52100 steel rubbing against AISI 1045 steel

    Institute of Scientific and Technical Information of China (English)

    REN Ping-di; CHEN Guang-xiong; ZHU Min-hao; ZHOU Zhong-rong

    2004-01-01

    A series of fretting test were carried out using a DELTA PLINT testing system to study the influence of hydraulic oil and water on fretting behaviour of AISI 52100 steel rubbing against AISI 1045 steel. The test result shows that media hydraulic oil and water have a distinct influence on fretting behaviour of the tested materials. Medium water can lead to shifting of the partial slip regime in the fretting map from a larger displacement amplitude toward a smaller one and enlargement of the mixed slip regime, in comparison with that in ambient atmosphere. While medium hydraulic oil can result in shifting of the partial slip regime from a smaller displacement amplitude toward a larger one. In the gross slip regime, hydraulic oil and water play a positive role as lubrication media. They can clearly decrease the fretting friction coefficient between AISI 52100 and AISI 1045. The test result also demonstrates that this lubrication effect will get better with increasing displacement amplitude and that hydraulic oil is better than water for lubrication. SEM observation of the wear scars displays that the fretting wear mainly results from abrasive wear and delamination of the fretted materials when using these two kinds of substances as lubrication media.

  6. Intracellular localization and interaction of mRNA binding proteins as detected by FRET

    Directory of Open Access Journals (Sweden)

    Port J

    2010-09-01

    Full Text Available Abstract Background A number of RNA binding proteins (BPs bind to A+U rich elements (AREs, commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. Results All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general, or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. Conclusions Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as

  7. Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging.

    Science.gov (United States)

    Omer, Travis; Intes, Xavier; Hahn, Juergen

    2015-01-01

    Fluorescence lifetime imaging (FLIM) when paired with Förster resonance energy transfer (FLIM-FRET) enables the monitoring of nanoscale interactions in living biological samples. FLIM-FRET model-based estimation methods allow the quantitative retrieval of parameters such as the quenched (interacting) and unquenched (non-interacting) fractional populations of the donor fluorophore and/or the distance of the interactions. The quantitative accuracy of such model-based approaches is dependent on multiple factors such as signal-to-noise ratio and number of temporal points acquired when sampling the fluorescence decays. For high-throughput or in vivo applications of FLIM-FRET, it is desirable to acquire a limited number of temporal points for fast acquisition times. Yet, it is critical to acquire temporal data sets with sufficient information content to allow for accurate FLIM-FRET parameter estimation. Herein, an optimal experimental design approach based upon sensitivity analysis is presented in order to identify the time points that provide the best quantitative estimates of the parameters for a determined number of temporal sampling points. More specifically, the D-optimality criterion is employed to identify, within a sparse temporal data set, the set of time points leading to optimal estimations of the quenched fractional population of the donor fluorophore. Overall, a reduced set of 10 time points (compared to a typical complete set of 90 time points) was identified to have minimal impact on parameter estimation accuracy (≈5%), with in silico and in vivo experiment validations. This reduction of the number of needed time points by almost an order of magnitude allows the use of FLIM-FRET for certain high-throughput applications which would be infeasible if the entire number of time sampling points were used.

  8. FRET-based trilateration of probes bound within functional ryanodine receptors.

    Science.gov (United States)

    Svensson, Bengt; Oda, Tetsuro; Nitu, Florentin R; Yang, Yi; Cornea, Iustin; Chen-Izu, Ye; Fessenden, James D; Bers, Donald M; Thomas, David D; Cornea, Razvan L

    2014-11-04

    To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

  9. Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements.

    Directory of Open Access Journals (Sweden)

    Rosemary A Bamford

    Full Text Available Förster resonance energy transfer (FRET technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5 complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

  10. Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

    Science.gov (United States)

    Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

    2015-10-01

    DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR

  11. A FRET enzyme-based probe for monitoring hydrogen sulfide.

    Science.gov (United States)

    Strianese, Maria; Palm, Gottfried J; Milione, Stefano; Kühl, Olaf; Hinrichs, Winfried; Pellecchia, Claudio

    2012-11-05

    Fluorescently labeled cobalt peptide deformylase (Co-PDF) can be efficiently used as a fluorescence-resonance-energy-transfer-based sensing device for hydrogen sulfide (H(2)S). The proof of concept of our sensor system is substantiated by spectroscopic, structural, and theoretical results. Monohydrogen sulfide coordination to Co-PDF and Ni-PDF was verified by X-ray crystallography. Density functional theory calculations were performed to gain insight into the characteristics of the coordination adduct between H(2)S and the cobalt cofactor in Co-PDF.

  12. Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

    Science.gov (United States)

    Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

    2015-01-01

    Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

  13. Study of fretting wear in titanium, Monel-400, and cobalt-25 percent molybdenum using scanning electron microscopy.

    Science.gov (United States)

    Bill, R. C.

    1973-01-01

    Damage scar volume measurements taken from like metal fretting pairs, combined with scanning electron microscopy observations, showed that three sequentially operating mechanisms result in the fretting of titanium, Monel-400, and cobalt-25% molybdenum. Initially, adhesion and plastic deformation on the surface played an important role. This was followed after a few hundred cycles by a fatigue mechanism, producing spall-like pits in the damage scar. Finally, an oxidation-related mechanism became most significant. Damage scar measurements made on several elemental metals after 600,000 fretting cycles suggested that the ratio of oxide hardness to metal hardness was a measure of the susceptibility of a metal to progressive damage by fretting.

  14. Study of fretting wear in titanium, Monel-400, and cobalt-25 percent molybdenum using scanning electron microscopy.

    Science.gov (United States)

    Bill, R. C.

    1973-01-01

    Damage scar volume measurements taken from like metal fretting pairs, combined with scanning electron microscopy observations, showed that three sequentially operating mechanisms result in the fretting of titanium, Monel-400, and cobalt-25% molybdenum. Initially, adhesion and plastic deformation on the surface played an important role. This was followed after a few hundred cycles by a fatigue mechanism, producing spall-like pits in the damage scar. Finally, an oxidation-related mechanism became most significant. Damage scar measurements made on several elemental metals after 600,000 fretting cycles suggested that the ratio of oxide hardness to metal hardness was a measure of the susceptibility of a metal to progressive damage by fretting.

  15. Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications.

    Science.gov (United States)

    Tian, Feng; Lyu, Jing; Shi, Jingyu; Yang, Mo

    2017-03-15

    In the past decades, Förster resonance energy transfer (FRET) has been applied in many biological applications to reveal the biological information at the nanoscale. Recently, graphene and graphene-like two-dimensional (2D) nanomaterials started to be used in FRET assays as donors or acceptors including graphene oxide (GO), graphene quantum dot (GQD), graphitic-carbon nitride nanosheets (g-C3N4) and transition metal dichalcogenides (e.g. MoS2, MnO2, and WS2). Due to the remarkable properties such as large surface to volume ratio, tunable energy band, photoluminescence and excellent biocompatibility, these 2D nanomaterials based FRET assays have shown great potential in various biological applications. This review summarizes the recent development of graphene and graphene-like 2D nanomaterials based FRET assays in applications of biosensing, bioimaging, and drug delivery monitoring.

  16. Fretting corrosion behavior of nitinol spinal rods in conjunction with titanium pedicle screws.

    Science.gov (United States)

    Lukina, Elena; Kollerov, Mikhail; Meswania, Jay; Khon, Alla; Panin, Pavel; Blunn, Gordon W

    2017-03-01

    Untypical corrosion damage including erosions combined with the build-up of titanium oxide as a corrosion product on the surface of explanted Nitinol spinal rods in the areas where it was in contact with titanium pedicle screw head is reported. It was suggested that Nitinol rods might have inferior fretting corrosion resistance compared with that made of titanium or CoCr. Fretting corrosion of Nitinol spinal rods with titanium (Ti6Al4V) pedicle screws were tested in-vitro by conducting a series of potentiostatic measurements of the peak-to-peak values of fretting corrosion current under bending in a 10% solution of calf serum in PBS. The test included Nitinol rods locked in titanium pedicle screws of different designs. Performance of commercially available titanium (Ti6Al4V) and CoCr spinal rods was also investigated for a comparison. Corrosion damage observed after the in-vitro tests was studied using SEM and EDAX analysis and was compared with patterns on Nitinol rods retrieved 12months after initial surgery. Metal ions level was measured in the test media after in-vitro experiments and in the blood and tissues of the patients who had the rods explanted. The results of this study revealed that Nitinol spinal rods locked in Ti pedicle screws are susceptible to fretting corrosion demonstrating higher fretting corrosion current compared with commercially used Ti6Al4V and CoCr rods. On the surface of Nitinol rods after in-vitro tests and on those retrieved from the patients similar corrosion patterns were observed. Improved resistance to fretting corrosion was observed with Nitinol rods in the in-vitro tests where pedicle screws were used with a stiffer locking mechanism. Since the development of the localized corrosion damage might increase the risk of premature fatigue failure of the rods and result in leaching of Ni ions, it is concluded that Nitinol rods should not be used in conjunction with Ti pedicle screws without special protection especially where the

  17. Development of Hardware Dual Modality Tomography System

    Directory of Open Access Journals (Sweden)

    R. M. Zain

    2009-06-01

    Full Text Available The paper describes the hardware development and performance of the Dual Modality Tomography (DMT system. DMT consists of optical and capacitance sensors. The optical sensors consist of 16 LEDs and 16 photodiodes. The Electrical Capacitance Tomography (ECT electrode design use eight electrode plates as the detecting sensor. The digital timing and the control unit have been developing in order to control the light projection of optical emitters, switching the capacitance electrodes and to synchronize the operation of data acquisition. As a result, the developed system is able to provide a maximum 529 set data per second received from the signal conditioning circuit to the computer.

  18. Effect of frequency on fretting wear behavior of Ti/TiN multilayer film on depleted uranium.

    Science.gov (United States)

    Wu, Yan-Ping; Li, Zheng-Yang; Zhu, Sheng-Fa; Lu, Lei; Cai, Zhen-Bing

    2017-01-01

    The Ti/TiN multi-layer film was prepared on the depleted uranium (DU) substrate by cathodic arc ion plating equipment. The character of multi-layer film was studied by SEM, XRD and AES, revealed that the surface was composed of small compact particle and the cross-section had a multi-layer structure. The fretting wear performance under different frequencies was performed by a MFT-6000 machine with a ball-on-plate configuration. The wear morphology was analyzed by white light interferometer, OM and SEM with an EDX. The result shows the Ti/TiN multi-layer film could greatly improve the fretting wear performance compared to the DU substrate. The fretting wear running and damaged behavior are strongly dependent on the film and test frequency. The fretting region of DU substrate and Ti/TiN multi-layer under low test frequency is gross slip. With the increase of test frequency, the fretting region of Ti/TiN multi-layer change from gross slip to mixed fretting, then to partial slip.

  19. Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

    Directory of Open Access Journals (Sweden)

    Niko Hildebrandt

    2011-10-01

    Full Text Available Förster resonance energy transfer (FRET from luminescent terbium complexes (LTC as donors to semiconductor quantum dots (QDs as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.

  20. Evaluation of quantum dot-based concentric FRET configurations with a fluorescent dye and dark quencher for multiplexed bioanalyses

    Science.gov (United States)

    Conroy, Erin M.; Algar, W. Russ

    2014-03-01

    Semiconductor quantum dots (QDs) continue to emerge as a highly advantageous platform for bioanalysis. Their unique physical and optical properties are especially well suited for Förster resonance energy transfer (FRET)-based bioprobes. Concentric FRET configurations are a recent development in this area of research and are best described as QD bioconjugates where multiple energy transfer pathways have been assembled around the central QD. Concentric FRET configurations permit multiplexed bioanalysis using one type of QD vector, but require more sophisticated analyses than conventional FRET pairs. In this paper, we describe the design and characterization of a new concentric FRET configuration that assembles both a fluorescent dye, Alexa Fluor 555 or Alexa Fluor 647, and a dark quencher, QSY9, at different ratios around a central CdSeS/ZnS QD. It was found that the magnitudes of the total photoluminescence (PL) intensity and either the A555/QD or A647/QD PL ratio can be related to the number of QSY9 and A555 or A647 per QD. The trends in these parameters with changes in the number of each dye molecule per QD have both similarities and differences between configurations with A555 and A647. In each case, a system of equations can be defined to permit calculation of the number of each dye molecule per QD from PL measurements. Both of these dark quencher-based concentric FRET configurations are therefore good candidates for quantitative, multiplexed bioanalysis.

  1. Experimental and Numerical Investigations of Fretting Fatigue Behavior for Steel Q235 Single-Lap Bolted Joints

    Directory of Open Access Journals (Sweden)

    Yazhou Xu

    2016-01-01

    Full Text Available This work aims to investigate the fretting fatigue life and failure mode of steel Q235B plates in single-lap bolted joints. Ten specimens were prepared and tested to fit the S-N curve. SEM (scanning electron microscope was then employed to observe fatigue crack surfaces and identify crack initiation, crack propagation, and transient fracture zones. Moreover, a FEM model was established to simulate the stress and displacement fields. The normal contact stress, tangential contact stress, and relative slipping displacement at the critical fretting zone were used to calculate FFD values and assess fretting fatigue crack initiation sites, which were in good agreement with SEM observations. Experimental results confirmed the fretting fatigue failure mode for these specimens. It was found that the crack initiation resulted from wear regions at the contact surfaces between plates, and fretting fatigue cracks occurred at a certain distance away from hole edges. The proposed FFD-N relationship is an alternative approach to evaluate fretting fatigue life of steel plates in bolted joints.

  2. Fretting properties of biodegradable Mg-Nd-Zn-Zr alloy in air and in Hank’s solution

    Science.gov (United States)

    Li, Wenting; Li, Nan; Zheng, Yufeng; Yuan, Guangyin

    2016-11-01

    Fretting is a significant cause for the failure of orthopedic implants. Currently, since magnesium and its alloys have been developed as promising biodegradable implant materials, the fretting behavior of the Mg alloys is of great research significance. In this study, a Mg-Nd-Zn-Zr alloy (hereafter, denoted as JDBM alloy) was selected as experimental material, and its fretting behaviors were evaluated under 5 N, 10 N and 20 N normal loads with a displacement of 200 μm under the frequency of 10 Hz at 37 °C in air and in Hank’s solution, respectively. The results indicated that while the friction coefficient decreased with the increment of the normal load, the wear volume of the alloy increased with the increment of the normal load both in air and in Hank’s solution. Both the friction coefficients and the wear volume of the fretting in Hank’s solution were much lower than those in air environment. The evolution trend of friction coefficients with time had different performance in air environment and the Hank’s solution group. Although oxidation occurred during the fretting tests in Hank’s solution, the damage of JDBM alloy was still reduced due to the lubrication effects of Hank’s solution. Moreover, the addition of Fetal bovine serum (FBS) could act as lubrication and result in the reduction of the fretting damage.

  3. FRET-based nanoscale point-to-point and broadcast communications with multi-exciton transmission and channel routing.

    Science.gov (United States)

    Kuscu, Murat; Akan, Ozgur B

    2014-09-01

    Nanoscale communication based on Förster Resonance Energy Transfer (FRET) enables nanoscale single molecular devices to communicate with each other utilizing excitons generated on fluorescent molecules as information carriers. Based on the point-to-point single-exciton FRET-based nanocommunication model, we investigate the multiple-exciton case for point-to-point and broadcast communications following an information theoretical approach and conducting simulations through Monte Carlo approach. We demonstrate that the multi-exciton transmission significantly improves the channel reliability and the range of the communication up to tens of nanometers for immobile nanonodes providing high data transmission rates. Furthermore, our analyses indicate that multi-exciton transmission enables broadcasting of information from a transmitter nanonode to many receiver nanonodes pointing out the potential of FRET-based communication to extend over nanonetworks. In this study, we also propose electrically and chemically controllable routing mechanisms exploiting the strong dependence of FRET rate on spectral and spatial characteristics of fluorescent molecules. We show that the proposed routing mechanisms enable the remote control of information flow in FRET-based nanonetworks. The high transmission rates obtained by multi-exciton scheme for point-to-point and broadcast communications, as well as the routing opportunities make FRET-based communication promising for future molecular computers.

  4. Evaluation of a color fused dual-band NVG

    NARCIS (Netherlands)

    Hogervorst, M.A.; Toet, A.

    2009-01-01

    We designed and evaluated a dual-band Night Vision Goggles sensor system. The sensor system consists of two optically aligned NVGs fitted with filters splitting the sensitive range into a visual and a near-infrared band. The Color-the-night technique (Hogervorst & Toet, FUSION2008) was used to fuse

  5. Cold-Sprayed Cu-MoS2 and Its Fretting Wear Behavior

    Science.gov (United States)

    Zhang, Yinyin; Descartes, Sylvie; Vo, Phuong; Chromik, Richard R.

    2016-02-01

    Cu and Cu-MoS2 coatings were fabricated by cold spray, and the fretting wear performance of the two coatings was compared. A mixture (95 wt.% Cu + 5 wt.% MoS2) was used as feedstock for the composite coating. Coatings were sprayed with identical gas flow conditions on the substrates pre-heated to approximately 170 °C. The morphology of coating top surface and polished cross sections was analyzed by scanning electron microscopy (SEM) and light optical microscopy (LOM). The influence of MoS2 on Cu deposition was examined. The local MoS2 concentration within the coating was found to affect the hardness. Fretting tests were carried out at two different normal loads, and the influence of MoS2 on friction and wear was studied. The morphology and elemental compositions of the wear scars and wear debris were observed by SEM and energy dispersive x-ray spectroscopy (EDS), respectively.

  6. Ratiometric FRET-based detection of DNA and micro-RNA in solution

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, Evgenia G., E-mail: ematveev@hsc.unt.ed [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States); Gryczynski, Zygmunt [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States); Stewart, Donald R. [Omm Scientific, Inc., 2600 N. Stemmons Freeway, Suite 129, Dallas, TX 75207 (United States); Gryczynski, Ignacy [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States)

    2009-11-15

    A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

  7. Kinetic Studies of Lysine Riboswitch Folding Using Single-Molecule FRET

    Science.gov (United States)

    Fiegland, Larry; Garst, Andrew; Fiore, J.; Batey, Robert; Nesbitt, David

    2010-03-01

    Riboswitches regulate gene expression through conformational changes induced by metabolite binding. This regulation of gene expression depends on the kinetics of metabolite binding and structural changes. Therefore, an understanding of these dynamics is crucial to developing a compete knowledge of riboswitch functionality. To probe the binding of a metabolite and subsequent folding, a metabolite-binding domain of the Bacillus subtilis lysine riboswitch was transcribed and hybridized to a fluorescent-labeled RNA strand, which allows FRET monitoring of ligand-induced conformational changes. The RNA construct was studied using single-molecule FRET methods that allowed for characterization of the folding dynamics. In the presence of lysine, we observed two states, of which the relative populations are perturbed by lysine concentration. We measured the folding and unfolding rates of the inter-conversion between these states. We also observe that [Mg^2+] affects the lysine-free conformation and the lysine sensitivity of the riboswitch.

  8. High-efficiency FRET-enhanced photoacoustic probes for in vivo tumor imaging

    Science.gov (United States)

    Qin, Huan; Liu, Liming

    2017-01-01

    Photoacoustic imaging can provide high-resolution and high-contrast image under unprecedented depth compared with pure optical imaging techniques by making use of laser-induced ultrasound waves. Although a series of absorption-enhanced optical contrast agents for photoacoustic imaging were developed, the probe with fully conversion from absorbed light energy to acoustic energy has not been achieved so far. Here we develop a high-efficiency photoacoustic probes with fluorescence resonance energy transfer (FRET) effect for enhancement of nonradiative energy. Graphene oxide (GO) binding optical dyes (GO-dyes) were achieved to show highly fluorescence quenching and violently increased photoacoustic signal intensity. GO-dyes were constructed and testified for multi-spectral photoacoustic imaging. As a representative probe, GO-Cy7 nanoparticles were used to validate the feasibility of photoacoustic tumor molecular imaging in vivo. Our work demonstrated a new approach to construct high-efficiency FRET-enhanced multi-spectrum probes for photoacoustic molecular imaging.

  9. Photodynamic therapy via FRET following bioorthogonal click reaction in cancer cells.

    Science.gov (United States)

    Bio, Moses; Rajaputra, Pallavi; You, Youngjae

    2016-01-01

    Longer wavelength light (650-800nm) is desired to treat large tumors in photodynamic therapy (PDT). However, shorter wavelength light is needed in PDT for thin tumors, not to cause undesirable local side effects. We proposed a strategy for stepwise optical imaging and PDT using a bioorthogonal click chemistry and fluorescence resonance energy transfer (FRET). We prepared azidyl rhodamine (Rh-N3, clickable FD) and cyclooctynyl phthalocyanine [Pc-(DIBAC), clickable PS], with which, here, we demonstrate that the non-catalytic click chemistry is rapid and efficient in cancer cells and FRET from a fluorescence dye (FD) to a photosensitizer (PS) is sufficient to generate enough singlet oxygen killing cancer cells by using shorter wavelength light. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Coverage-dependent changes of cytochrome c transverse location in phospholipid membranes revealed by FRET.

    Science.gov (United States)

    Domanov, Yegor A; Molotkovsky, Julian G; Gorbenko, Galyna P

    2005-10-01

    The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.

  11. Topographical Analysis of Fretted Terrain: Implications for Ancient Martian Super-eruptions

    Science.gov (United States)

    Mason, K. A.; Bleacher, J. E.; Whelley, P.

    2016-12-01

    Recent work has identified Late Noachian to Early Hesperian plains-style caldera complexes on Mars in western Arabia Terra (AT), eruptions from which might have produced widespread volcanic deposits throughout AT and the entire planet. AT is composed of 2 km thick friable and fretted Late Noachian to Early Hesperian material. Although an ash deposit origin has been suggested for the friable material due to its low thermal inertia, layering, thickness, sulfate abundance, zonal indurations and draped morphology, prior to 2013 no volcanic sources with ability to produce such a deposit were known, and therefore the existence of extensive ash deposits on AT was thought unlikely. Due to recent identification of evidence for ancient super-eruptions in AT such as Eden Patera, the geomorphology of the fretted material warrants reexamination. This study involves a quantitative analysis of eroded valleys throughout the fretted terrain visible at Mars Orbiter Laser Altimeter (MOLA) Digital Elevation Model (DEM) resolution (463 m). These erosion features are mapped objectively via the D8 flow direction algorithm, a technique developed for delineating watersheds with DEMs. The features are then partitioned based on depth and location. Valley orientations are measured for each feature segment and plotted on rose diagrams. To study valley formation mechanisms we use the ratio between high and low stream order lengths (bifurcation ratio) as well as the variance between orientation and primary slope direction. On Earth, bifurcation ratios > 3 and 5 indicate other processes principally formed the valleys and are consistent with the deformation of welded and fractured volcanic deposits. Valleys east of Isidis basin (IB) have bifurcation ratios process throughout the study area. We propose that the AT fretted terrain resembles a network of valleys formed by exploiting angular cooling and compaction fractures in a Late Noachian to Early Hesperian volcanic deposit.

  12. FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

    OpenAIRE

    Raina, Shweta A.; Jeffrey Tsai; Montserrat Samsó; Fessenden, James D.

    2012-01-01

    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) inse...

  13. FRET enhancement close to gold nanoparticles positioned in DNA origami constructs.

    Science.gov (United States)

    Aissaoui, Nesrine; Moth-Poulsen, Kasper; Käll, Mikael; Johansson, Peter; Wilhelmsson, L Marcus; Albinsson, Bo

    2017-01-05

    Here we investigate the energy transfer rates of a Förster resonance energy transfer (FRET) pair positioned in close proximity to a 5 nm gold nanoparticle (AuNP) on a DNA origami construct. We study the distance dependence of the FRET rate by varying the location of the donor molecule, D, relative to the AuNP while maintaining a fixed location of the acceptor molecule, A. The presence of the AuNP induces an alteration in the spontaneous emission of the donor (including radiative and non-radiative rates) which is strongly dependent on the distance between the donor and AuNP surface. Simultaneously, the energy transfer rates are enhanced at shorter D-A (and D-AuNP) distances. Overall, in addition to the direct influence of the acceptor and AuNP on the donor decay there is also a significant increase in decay rate not explained by the sum of the two interactions. This leads to enhanced energy transfer between donor and acceptor in the presence of a 5 nm AuNP. We also demonstrate that the transfer rate in the three "particle" geometry (D + A + AuNP) depends approximately linearly on the transfer rate in the donor-AuNP system, suggesting the possibility to control FRET process with electric field induced by 5 nm AuNPs close to the donor fluorophore. It is concluded that DNA origami is a very versatile platform for studying interactions between molecules and plasmonic nanoparticles in general and FRET enhancement in particular.

  14. The stability of dual-taper modular hip implants: a biomechanical analysis examining the effect of impact location on component stability

    Directory of Open Access Journals (Sweden)

    Nicholas B. Frisch, MD

    2017-06-01

    Conclusions: Direction of impaction influences stability of the modular interface. The greatest stability was achieved with impaction directed in line with the longitudinal axis of the taper junction. Off-axis impaction of the 8° and 15° neck led to significantly reduced stability at the NS. Improving stability of dual-taper modular hip prostheses with appropriately directed impaction may help to minimize micromotion, component settling, fretting corrosion, and subsequent failure.

  15. Effects of Plasma ZrN Metallurgy and Shot Peening Duplex Treatment on Fretting Wear and Fretting Fatigue Behavior of Ti6Al4V Alloy

    Directory of Open Access Journals (Sweden)

    Jingang Tang

    2016-03-01

    Full Text Available A metallurgical zirconium nitride (ZrN layer was fabricated using glow metallurgy using nitriding with zirconiuming prior treatment of the Ti6Al4V alloy. The microstructure, composition and microhardness of the corresponding layer were studied. The influence of this treatment on fretting wear (FW and fretting fatigue (FF behavior of the Ti6Al4V alloy was studied. The composite layer consisted of an 8-μm-thick ZrN compound layer and a 50-μm-thick nitrogen-rich Zr–Ti solid solution layer. The surface microhardness of the composite layer is 1775 HK0.1. A gradient in cross-sectional microhardness distribution exists in the layer. The plasma ZrN metallurgical layer improves the FW resistance of the Ti6Al4V alloy, but reduces the base FF resistance. This occurs because the improvement in surface hardness results in lowering of the toughness and increasing in the notch sensitivity. Compared with shot peening treatment, plasma ZrN metallurgy and shot peening composite treatment improves the FW resistance and enhances the FF resistance of the Ti6Al4V alloy. This is attributed to the introduction of a compressive stress field. The combination of toughness, strength, FW resistance and fatigue resistance enhance the FF resistance for titanium alloy.

  16. Effects of Plasma ZrN Metallurgy and Shot Peening Duplex Treatment on Fretting Wear and Fretting Fatigue Behavior of Ti6Al4V Alloy.

    Science.gov (United States)

    Tang, Jingang; Liu, Daoxin; Zhang, Xiaohua; Du, Dongxing; Yu, Shouming

    2016-03-23

    A metallurgical zirconium nitride (ZrN) layer was fabricated using glow metallurgy using nitriding with zirconiuming prior treatment of the Ti6Al4V alloy. The microstructure, composition and microhardness of the corresponding layer were studied. The influence of this treatment on fretting wear (FW) and fretting fatigue (FF) behavior of the Ti6Al4V alloy was studied. The composite layer consisted of an 8-μm-thick ZrN compound layer and a 50-μm-thick nitrogen-rich Zr-Ti solid solution layer. The surface microhardness of the composite layer is 1775 HK0.1. A gradient in cross-sectional microhardness distribution exists in the layer. The plasma ZrN metallurgical layer improves the FW resistance of the Ti6Al4V alloy, but reduces the base FF resistance. This occurs because the improvement in surface hardness results in lowering of the toughness and increasing in the notch sensitivity. Compared with shot peening treatment, plasma ZrN metallurgy and shot peening composite treatment improves the FW resistance and enhances the FF resistance of the Ti6Al4V alloy. This is attributed to the introduction of a compressive stress field. The combination of toughness, strength, FW resistance and fatigue resistance enhance the FF resistance for titanium alloy.

  17. Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.

    Science.gov (United States)

    Bienert, Roland; Zimmermann, Boris; Rombach-Riegraf, Verena; Gräber, Peter

    2011-02-25

    H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

  18. 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.

  19. Single-molecule FRET reveals hidden complexity in a protein energy landscape.

    Science.gov (United States)

    Tsytlonok, Maksym; Ibrahim, Shehu M; Rowling, Pamela J E; Xu, Wenshu; Ruedas-Rama, Maria J; Orte, Angel; Klenerman, David; Itzhaki, Laura S

    2015-01-06

    Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

  20. Influence of surface coating on Ti811 alloy resistance to fretting fatigue at elevated temperature

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiaohua; LIU Daoxin

    2009-01-01

    An extensive study of the composition distribution, bonding strength, hardness, and wear resistance of a 0Cr18Ni9 film deposited on a Ti811 titanium alloy surface by ion beam enhanced deposition (IBED) is presented. Shot peening was introduced to post-treat the modified surface to synergistically improve the fretting fatigue resistance of the Ti811 alloy at 350~C. The results indicate that the 0Cr18Ni9 film with high density, small grain size, low void radio, and high bonding strength can be prepared using IBED. As a result, the hardness, wear resistance, and fretting fatigue resistance of the Ti811 alloy are increased to a remarkable extent. Compared with shot peening treatment or IBED 0Cr18Ni9 film alone, the Ti811 titanium alloy with an IBED 0Cr18Ni9 film combined with shot peening shows a higher fretting fatigue resistance at 350℃. This is due to the synergistic effect of the high wear resistance of the film surface and the residual compressive stress induced by shot peening.

  1. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.

    Science.gov (United States)

    Day, Richard N; Tao, Wen; Dunn, Kenneth W

    2016-11-01

    Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.

  2. FRET Fluctuation Spectroscopy of Diffusing Biopolymers: Contributions of Conformational Dynamics and Translational Diffusion

    Science.gov (United States)

    Gurunathan, Kaushik; Levitus, Marcia

    2009-01-01

    The use of Fluorescence Correlation Spectroscopy (FCS) to study conformational dynamics in diffusing biopolymers requires that the contributions to the signal due to translational diffusion are separated from those due to conformational dynamics. A simple approach that has been proposed to achieve this goal involves the analysis of fluctuations in Fluorescence Resonance Energy Transfer (FRET) efficiency. In this work, we investigate the applicability of this methodology by combining Monte Carlo simulations and experiments. Results show that diffusion does not contribute to the measured fluctuations in FRET efficiency in conditions where the relaxation time of the kinetic process is much shorter than the mean transit time of the molecules in the optical observation volume. However, in contrast to what has been suggested in previous work, the contributions of diffusion are otherwise significant. Neglecting the contributions of diffusion can potentially lead to an erroneous interpretation of the kinetic mechanisms. As an example, we demonstrate that the analysis of FRET fluctuations in terms of a purely kinetic model would generally lead to the conclusion that the system presents complex kinetic behavior even for an idealized two-state system PMID:20030305

  3. FRET based biosensor for detection of active NF-kB

    Science.gov (United States)

    Baldini, Francesco; Citti, Lorenzo; Domenici, Claudio; Giannetti, Ambra; Tedeschi, Lorena; Vo-Dinh, Tuan; Wabuyele, Musundi B.

    2005-05-01

    The Nuclear Factor kB is a transcription factor, ubiquitously expressed, involved in the regulation of a large number of genes and in a variety of human disease including inflammation, asthma, atherosclerosis, AIDS, septic shock, arthritis and cancer. The critical need for a simple and direct method to evaluate the quantity of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. A single-stranded DNA (ssDNA) with auto-complementary sequence has been properly designed and synthesized. In order to evaluate FRET due to the DNA/protein binding interaction taking place between double-stranded DNA (dsDNA) immobilized in a capillary wall and NF-kB proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used. Preliminary results show that our system was capable of detecting the active form of NF-kB protein with a detection efficiency of about 90% and that the system has a good regenerability.

  4. Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

    Science.gov (United States)

    Briggman, Kevin L; Kristan, William B; González, Jesús E; Kleinfeld, David; Tsien, Roger Y

    2015-01-01

    Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

  5. DNA quantification based on FRET realized by combination with surfactant CPB.

    Science.gov (United States)

    Liu, Chunxia; Wang, Lei; Jiang, Wei

    2010-04-15

    In this work, we developed a novel DNA quantitative analysis based on fluorescence resonance energy transfer (FRET) realized by combination with a surfactant CPB. The approach was capable of detecting long-stranded DNA in a separation-free format. A sandwich-type FAM-c-DNA-t-DNA-r-DNA-TAMRA conjugate was first formed by the capture probe tagged with FAM, the reporter probe tagged with TAMRA and the target DNA through hybridization. The donor (FAM) and the acceptor (TAMRA) were bridged to afford a FRET system. Subsequently, an addition of the cationic surfactant CPB to the system resulted in a substantial change of the microenvironment and an effective condensation of DNA strands. Consequently, without altering the component of the double strands, an enhanced acceptor fluorescence signal from FRET was achieved and a quantification of the target DNA containing 30 bases was enabled. Under the optimal experimental conditions, an excellent linear relationship between the increase of acceptor fluorescent peak area and the target DNA concentration was obtained over the range from 1.0 x 10(-7) to 3.0 x 10(-9) mol L(-1). The proposed approach offered adequate sensitivity for the detection of the target DNA at 1.0 x 10(-9) mol L(-1).

  6. Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning.

    Science.gov (United States)

    Qiu, Xue; Guo, Jiajia; Jin, Zongwen; Petreto, Alexandra; Medintz, Igor L; Hildebrandt, Niko

    2017-07-01

    Multiplexed photoluminescence (PL) detection plays an important role in chemical and biological sensing. Here, it is shown that time-gated (TG) detection of a single terbium-donor-based Förster resonance energy transfer (FRET) pair can be used to selectively quantify low nanomolar concentrations of multiple DNAs or microRNAs in a single sample. This study demonstrates the applicability of single-TG-FRET-pair multiplexing for molecular (Tb-to-dye) and nanoparticle (Tb-to-quantum-dot) biosensing. Both systems use acceptor-sensitization and donor-quenching for quantifying biomolecular recognition and modification of the donor-acceptor distance for tuning the PL decays. TG intensity detection provides extremely low background noise and a quick and simple one-step assay format. Single-TG-FRET-pair multiplexing can be combined with spectral and spatial resolution, paving the way for biosensing with unprecedented high-order multiplexing capabilities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Classic maximum entropy recovery of the average joint distribution of apparent FRET efficiency and fluorescence photons for single-molecule burst measurements.

    Science.gov (United States)

    DeVore, Matthew S; Gull, Stephen F; Johnson, Carey K

    2012-04-05

    We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions.

  8. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    Science.gov (United States)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  9. Design improvement for fretting-wear reduction of HANARO fuel assembly

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Yeong Garp; Chae, H. T.; Ryu, J. S.; Kim, H. R

    2000-06-01

    In the course of the visual inspection of the fuel assemblies un-loaded from the reactor core in December 1996, it was observed that many of fuel assemblies had mechanical damages on some components. The major damage was the freting-wear on spacer plates and endplates due to the flow induced vibration of the fuel assembly in the flow tube. Since the reactor is activated and the system modification for complete removal of the driving factors of the vibration of fuel assemblies is practically very difficult, the focus has been on the design change of the fuel assemblies. Consequently, various design changes were proposed to strengthen the wear resistance of the components based on the evaluation of the visual inspection results. The validity of the proposals was verified through the performance tests for the modified components, and the vibration test and endurance test for the fuel assemblies using the single-channel test rig(SCTR) in AECL.The subsequent design changes were additionally proposed based on the visual inspections for the fuel assemblies that had been fabricated according to the first design change and loaded in the core. As the effects of the first design change, the fretting-wear of spacer plates was remarkably reduced and the period until fretting-wear damage was extended by 60% for the first modified 36-rod fuel assembly. It is too early to say the endurance life time for the first modified 18-rod fuel assembly because of insufficient statistical data of only two bundles damaged, but the fretting-wear at the bottom endplate slot was reduced to about 50%. The second modified fuel assemblies, that were not loaded into the core yet, are expected to meet the design requirements for the core residence time due to strengthening the weak parts from the fretting-wear point of view. This report describes design changes and tests for fuel assemblies of HANARO to reduce the fretting-wear, and estimates the effects of design improvement quantitatively compared

  10. FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

    Science.gov (United States)

    Markovic, Danijela; Holdich, Jonathan; Al-Sabah, Suleiman; Mistry, Rajendra; Krasel, Cornelius; Mahaut-Smith, Martyn P.; Challiss, R. A. John

    2012-01-01

    Background and Objective Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET). Methods Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M1 mAChR, respectively. The optimized FRET receptor construct (M1-cam5) was expressed stably in HEK293 cells. Results The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gαq/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M1 FRET (FEYFP/FECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine. Conclusion The M1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for

  11. FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists.

    Directory of Open Access Journals (Sweden)

    Danijela Markovic

    Full Text Available BACKGROUND AND OBJECTIVE: Muscarinic acetylcholine receptors (mAChRs are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET. METHODS: Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1 mAChR, respectively. The optimized FRET receptor construct (M(1-cam5 was expressed stably in HEK293 cells. RESULTS: The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα(q/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1 FRET (F(EYFP/F(ECFP that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine. CONCLUSION: The M(1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a

  12. Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements.

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    Gil Rahamim

    Full Text Available Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM, and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro7 and the second polypeptide was a flexible molecule consisting of (Gly-Ser7 repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.

  13. ETP-0474: Evaluation of Electroless Nickel Coatings to Achieve Interference Fit in the RSRM Without Fretting

    Science.gov (United States)

    Schaffnit, William O.

    1997-01-01

    Part of the redesign of the SRMs for the Space Shuttle involved the substitution of three new capture cylinders for three of the previously used cylinders. These new cylinders mate with the old standard case segments in each of the three field joints. The new capture cylinders contain an integral capture latch on the tang end which mates with a case clevis during stackup at KSC. The capture cylinders also contain a groove in the capture latch to provide for a third 0-ring in the joint and are designed to achieve a metal-to- metal interference fit between the capture latch and the mating clevis. An unexpected fretting problem has occurred on the tang capture feature and the inner clevis leg interference fit surfaces on flight hardware since STS-26. Varying degrees of fretting damage have been found on the case segments from different flight motors. Fretting is a wear phenomena that occurs when two tightly fitting metal surfaces are subject to cyclic relative motion of extremely small amplitudes (generally less than 0.010-inch) in the absence of adequate lubrication. It is adhesive ("cold" - welding) in nature and vibration is its essential causative factor. This problem has manifested itself on the flight motors as a series of pits and axial gouges on the inside diameter (ID) surfaces of the inner clevis legs and the outside diameter (OD) surfaces of the tang capture features. The problem occurs in varying degrees of severity in all of the field joints. It is not believed that fretting is a flight safety issue. However, it could become a reusability issue if left unattended. Fretting has been encountered in other industries for many years and measures that will prevent or reduce it have been devised. These include: elimination or reduction of vibration (amplitudes and/or frequencies), elimination of slip, improved lubrication between parts, increased surface separation, increased interference, inducing residual compressive stresses in the surfaces of the mating

  14. Proposal of a New Method for Measuring Förster Resonance Energy Transfer (FRET Rapidly, Quantitatively and Non-Destructively

    Directory of Open Access Journals (Sweden)

    Paul Johannes Helm

    2012-09-01

    Full Text Available The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor” to another chromophore (an “acceptor”, in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET. The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies ƒ1 and ƒ2 by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at ƒ1, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at ƒ2. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at ƒ1 but also at the beat frequency Δƒ: = |ƒ1 − ƒ2|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.

  15. A graphene quantum dot-based FRET system for nuclear-targeted and real-time monitoring of drug delivery.

    Science.gov (United States)

    Chen, Hui; Wang, Zhuyuan; Zong, Shenfei; Chen, Peng; Zhu, Dan; Wu, Lei; Cui, Yiping

    2015-10-07

    A graphene quantum dot-based FRET system is demonstrated for nuclear-targeted drug delivery, which allows for real-time monitoring of the drug release process through FRET signals. In such a system, graphene quantum dots (GQDs) simultaneously serve as the carriers of drugs and donors of FRET pairs. Additionally, a peptide TAT as the nuclear localization signal is conjugated to GQDs, which facilitates the transportation of the delivery system to the nucleus. We have demonstrated that: (a) both the conjugated TAT and small size of GQDs contribute to targeting the nucleus, which results in a significantly enhanced intranuclear accumulation of drugs; (b) FRET signals being extremely sensitive to the distance between donors and acceptors are capable of real-time monitoring of the separation process of drugs and GQDs, which is more versatile in tracking the drug release dynamics. Our strategy for the assembly of a FRET-based drug delivery system may be unique and universal for monitoring the dynamic release process. This study may give more exciting new opportunities for improving the therapeutic efficacy and tracking precision.

  16. Development of a Fluorescence Resonance Energy Transfer (FRET-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

    Directory of Open Access Journals (Sweden)

    Noremylia Mohd Bakhori

    2013-12-01

    Full Text Available An optical DNA biosensor based on fluorescence resonance energy transfer (FRET utilizing synthesized quantum dot (QD has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

  17. Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair.

    Science.gov (United States)

    Dumat, Blaise; Larsen, Anders Foller; Wilhelmsson, L Marcus

    2016-06-20

    Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins.

  18. Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

    Science.gov (United States)

    Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

    2012-03-01

    Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

  19. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET).

    Science.gov (United States)

    Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon

    2016-07-01

    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

  20. Assessment of Corrosion, Fretting, and Material Loss of Retrieved Modular Total Knee Arthroplasties.

    Science.gov (United States)

    Martin, Audrey J; Seagers, Kirsten A; Van Citters, Douglas W

    2017-07-01

    Modular junctions in total hip arthroplasties have been associated with fretting, corrosion, and debris release. The purpose of this study is to analyze damage severity in total knee arthroplasties of a single design by qualitative visual assessment and quantitative material loss measurements to evaluate implant performance and patient impact via material loss. Twenty-two modular knee retrievals of the same manufacturer were identified from an institutional review board-approved database. Junction designs included tapers with an axial screw and tapers with a radial screw. Constructs consisted of 2 metal alloys: CoCr and Ti6Al4V. Components were qualitatively scored and quantitatively measured for corrosion and fretting. Negative values represent adhered material. Statistical differences were analyzed using sign tests. Correlations were tested with a Spearman rank order test (P material loss and the maximum linear depth for the total population were -0.23 mm(3) and 5.84 μm, respectively. CoCr components in mixed metal junctions had higher maximum linear depth (P = .007) than corresponding Ti components. Fretting scores of Ti6Al4V alloy components in mixed metal junctions were statistically higher than the remaining groups. Taper angle did not correlate with material loss. Results suggest that CoCr components in mixed metal junctions are more vulnerable to corrosion than other components, suggesting preferential corrosion when interfacing with Ti6Al4V. Overall, although corrosion was noted in this series, material loss was low, and none were revised for clinical metal-related reaction. This suggests the clinical impact from corrosion in total knee arthroplasty is low. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Advanced non-linear flow-induced vibration and fretting-wear analysis capabilities

    Energy Technology Data Exchange (ETDEWEB)

    Toorani, M.; Pan, L.; Li, R.; Idvorian, N. [Babcock and Wilcox Canada Ltd., Cambridge, Ontario (Canada); Vincent, B.

    2009-07-01

    Fretting wear is a potentially significant degradation mechanism in nuclear steam generators and other shell and tube heat transfer equipment as well. This paper presents an overview of the recently developed code FIVDYNA which is used for the non-linear flow-induced vibration and fretting wear analysis for operating steam generators (OTSG and RSG) and shell-and-tube heat exchangers. FIVDYNA is a non-linear time-history Flow-Induced Vibration (FIV) analysis computer program that has been developed by Babcock and Wilcox Canada to advance the understanding of tube vibration and tube to tube-support interaction. In addition to the dynamic fluid induced forces the program takes into account other tube static forces due to axial and lateral tube preload and thermal interaction loads. The program is capable of predicting the location where the fretting wear is most likely to occur and its magnitude taking into account the support geometry including gaps. FIVDYNA uses the general purpose finite element computer code ABAQUS as its solver. Using ABAQUS gives the user the flexibility to add additional forces to the tube ranging from tube preloads and the support offsets to thermal loads. The forces currently being modeled in FIVDYNA are the random turbulence, steady drag force, fluid-elastic forces, support offset and pre-strain force (axial loads). This program models the vibration of tubes and calculates the structural dynamic characteristics, and interaction forces between the tube and the tube supports. These interaction forces are then used to calculate the work rate at the support and eventually the predicted depth of wear scar on the tube. A very good agreement is found with experiments and also other computer codes. (author)

  2. The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

    Directory of Open Access Journals (Sweden)

    Shaoying Lu

    Full Text Available Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP experiments, we have developed a finite element (FE method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2/sec than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2/sec and outside: 0.18+/-0.02 microm(2/sec. The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

  3. PEG-Phospholipids Coated Quantum Rods as Amplifiers of the Photosensitization Process by FRET.

    Science.gov (United States)

    Timor, Reut; Weitman, Hana; Waiskopf, Nir; Banin, Uri; Ehrenberg, Benjamin

    2015-09-30

    Singlet oxygen ((1)O2) generated upon photostimulation of photosensitizer molecules is a highly reactive specie which is utilized in photodynamic therapy. Recent studies have shown that semiconductor nanoparticles can be used as donors in fluorescence resonance energy transfer (FRET) process to excite attached photosensitizer molecules. In these studies, their unique properties, such as low nanoscale size, long-term photostability, wide broad absorbance band, large absorption cross section, and narrow and tunable emission bands were used to provide advantages over the traditional methods to produce singlet oxygen. Previous studies that achieved this goal, however, showed some limitations, such as low FRET efficiency, poor colloidal stability, nonspecific interactions, and/or complex preparation procedure. In this work, we developed and characterized a novel system of semiconductor quantum rods (QRs) and the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC), as a model system that produces singlet oxygen without these limitations. A simple two-step preparation method is shown; Hydrophobic CdSe/CdS QRs are solubilized in aqueous solutions by encapsulation with lecithin and PEGylated phospholipid (PEG-PL) of two lipid lengths: PEG350 or PEG2000. Then, the hydrophobic photosensitizer mTHPC, was intercalated into the new amphiphilic PEG-PL coating of the QR, providing a strong attachment to the nanoparticle without covalent linkage. These PEGylated QR (eQR)-mTHPC nanocomposites show efficient FRET processes upon light stimulation of the QR component which results in efficient production of singlet oxygen. The results demonstrate the potential for future use of this concept in photodynamic therapy schemes.

  4. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Directory of Open Access Journals (Sweden)

    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  5. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Science.gov (United States)

    Raina, Shweta A; Tsai, Jeffrey; Samsó, Montserrat; Fessenden, James D

    2012-01-01

    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) insertions within the ryanodine receptor type 1 (RyR1), a large intracellular Ca(2+) release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK)-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  6. Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Viviane Devauges

    Full Text Available We present a novel imaging system combining total internal reflection fluorescence (TIRF microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

  7. Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane.

    Science.gov (United States)

    Gordon, Sharona E; Senning, Eric N; Aman, Teresa K; Zagotta, William N

    2016-02-01

    Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane.

  8. Evaluation of fretting failures on PWR fuel by post-irradiation examinations and modeling in the DEGRAD-1 code

    Energy Technology Data Exchange (ETDEWEB)

    Castanheira, Myrthes; Silva, Jose Eduardo Rosa da; Lucki, Georgi; Terremoto, Luis A.A.; Silva, Antonio Teixeira e; Teodoro, Celso A.; Damy, Margaret de A. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: myrthes@ipen.br

    2007-07-01

    One of the major recognized causes of fuel rod failures is fretting of the clad due to the entrapment of debris in a fuel rod spacer. Such debris, inadvertently dropped into the primary system during maintenance operations, includes various sizes of particles. Intermediate size particles, such as metal cuttings, electrical connectors, metal fittings, pieces of wire, and small nuts and bolts can become trapped between fuel rods in a spacer where hydraulically induced vibrations can cause fretting failure of the fuel rod. An evaluation of debris fretting failure on PWR fuel is presented. The inquiries on fuel rods failures are based on results of analysis using post-irradiation non-destructive examination. The complementary analysis includes a modeling approach by code DEGRAD-1 to characterize the degradation phenomenon after primary failure integrated in the reactor operational history. (author)

  9. Modeling of Complex Wear Behavior Associated with Grid-to-Rod Fretting in Light Water Nuclear Reactors

    Science.gov (United States)

    Blau, P. J.; Qu, J.; Lu, R.

    2016-09-01

    Fretting wear damage to fuel cladding from flow-induced vibrations can be a significant concern in the operation of light water nuclear reactors. For years, research on the grid-to-rod fretting (GTRF) phenomena has been underway in countries where nuclear power production is a significant industry. Under the auspices of the U.S. Department of Energy Consortium for Advanced Simulation of Light Water Reactors, an effort has been underway to develop and test an engineering wear model for zirconium alloy fuel rod cladding against a supporting grid. The multi-stage model accounts for oxide layers and wear rate transitions. This paper describes the basis for a GTRF engineering wear model, the physical significance of the wear factor it contains, and recent progress toward model validation based on a fretting wear testing apparatus that accounts for coolant temperature, pressure, and the presence of periodic impacts (gaps) in grid/rod contact.

  10. Modeling of Complex Wear Behavior Associated with Grid-to-Rod Fretting in Light Water Nuclear Reactors

    Science.gov (United States)

    Blau, P. J.; Qu, J.; Lu, R.

    2016-11-01

    Fretting wear damage to fuel cladding from flow-induced vibrations can be a significant concern in the operation of light water nuclear reactors. For years, research on the grid-to-rod fretting (GTRF) phenomena has been underway in countries where nuclear power production is a significant industry. Under the auspices of the U.S. Department of Energy Consortium for Advanced Simulation of Light Water Reactors, an effort has been underway to develop and test an engineering wear model for zirconium alloy fuel rod cladding against a supporting grid. The multi-stage model accounts for oxide layers and wear rate transitions. This paper describes the basis for a GTRF engineering wear model, the physical significance of the wear factor it contains, and recent progress toward model validation based on a fretting wear testing apparatus that accounts for coolant temperature, pressure, and the presence of periodic impacts (gaps) in grid/rod contact.

  11. TECHNIQUE OF TESTING ON FRETTING AT THE SPHERE-TO-PLANE CONTACT

    Directory of Open Access Journals (Sweden)

    А. Khimko

    2012-12-01

    Full Text Available  The methodology of conducting tests on fretting at the sphere-to-plane contact was developed for the wing mechanization unit, namely for screw-nut pair with intermediate balls. Wearability tests were conducted on a modified installation МФК-1, the feature of which is the designed holder that allows testing with real balls. It was found that at the dry contact of ШХ-15 and 30Х2НВФA materials, surface microcracks are formed due to welding of microasperities areas and their rupture under the influence of vibration.

  12. Protein-protein interactions in the plant Golgi apparatus, studied with FRET acceptor photobleaching technique

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter

    to the plant Golgi apparatus and involved mainly in arabinogalactan protein (AGP) biosynthesis. Co-expression analysis identified 4 GTs and 4 NSTs possibly involved in AGP biosynthesis. As part of the method development, the cytoskeleton-acting agent Cytochalasin D was tested as an inhibitor...... of the actinomyosin based movement of Golgi vesicles, and was proved to be superior to commonly used fixatives such as the cross-linking agent paraformaldehyde which causes quenching of the fluorophores. According to FRET analysis, the results showed association between two galactosyltransferases, AtGALT29A and At...

  13. Multiphoton STED and FRET in human skin: Resolving the skin barrier

    DEFF Research Database (Denmark)

    Antonescu, Irina; Dreier, Jes; Brewer, Jonathan R.

    Understanding the penetration properties of substances across biological bar- riers and membranes is vital for many areas of research. In the case of human skin, the barrier is primarily found in the stratum corneum and consists of protein-enriched cells surrounded by a lipid membrane -enriched...... excited STED and Forster Resonance Energy Transfer (FRET) microscopy to probe the structure of human skin. Super resolution optical microscopy enables resolving structures in the skin below to 60 nm allowing visualization of the stratum corneum intercellular lipid matrix and individual proteins...

  14. Experimental evaluation of the fretting fatigue behavior of high-strength steel monostrands

    DEFF Research Database (Denmark)

    Winkler, Jan; Fischer, Gregor; Georgakis, Christos T.

    2013-01-01

    . Moreover, the paper provides relevant information about the monostrand bending stiffness and the extent of relative displacement between core and outer wires of the monostrand undergoing flexural deformations. The results presented herein are of special interest for the fatigue analysis of modern stay......In this paper, the fretting fatigue behavior of pretensioned high-strength steel monostrands is investigated. A method based on the digital image correlation (DIC) technique was used to quantify the relative movement between individual wires along the length of the monostrand. The experimental data...

  15. Analysis of temporal and spatial contact voltage fluctuation during fretting in automotive connectors

    OpenAIRE

    El Mossouess, Sofiane; Benjemâa, N; Carvou, E; El Abdi, R; Obame, H; Doublet, L; Rodari, T

    2014-01-01

    International audience; Our study is focused on contact voltage fluctuations during fretting with small amplitudes of a few tens microns which generate damage of the contact of connectors. A contact composed by a pin and a curve female part are submitted to vibration cyclic of 25µm at 100Hz and supplied with current ramp from 0.1mA to 3A in two directions. With the help of fast devices, the voltage and position data acquisition are conjointly made with the common DC contact voltage dur-ing fr...

  16. Development of an AP-FRET based analysis for characterizing RNA-protein interactions in myotonic dystrophy (DM1.

    Directory of Open Access Journals (Sweden)

    Shagufta Rehman

    Full Text Available Förster Resonance Energy Transfer (FRET microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1 is caused by a (CTGn repeat expansion in the 3' UTR of the DMPK gene which results in nuclear retention of mutant DMPK transcripts in RNA foci. This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins (e.g. MBNL1, hnRNPH, CUGBP1. In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.

  17. Imaging of caspase-3 activation by a novel FRET probe composed of CFP and DsRed

    Science.gov (United States)

    Lin, Juquiang; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming

    2006-01-01

    Caspases-3 is a kind of cysteine proteases and plays an important role in cell apoptosis. It has been reported that caspase-3 activation can be real-time detected in living cells by fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein and enhanced yellow fluorescent protein. However, the large spectral overlap between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) emission and the highly sensitivity to pH of YFP restricted their detecting sensitivity and reliability. CFP and red fluorescent protein (DsRed) possess superb wavelength separation of donor and acceptor emission spectra and DsRed was insensitive to pH, so the FRET probe composed of CFP and DsRed would be more suitable for imaging caspase-3 activation than the FRET probe composed of CFP and YFP. We constructed a vector that encoded CRS (caspase-3 recognition site) fused with CFP and DsRed (CFP-CRS-DsRed). In CFP-CRS-DsRed expressing tumor cells, FRET from CFP to DsRed could be detected. In the Clinical applications of cancer chemotherapy, cisplatin is one of the most broadly used drugs. It was already confirmed that caspase-3 was activated in HeLa cell treated by cisplatin. When the cells were stimulated with cisplatin, we found that the FRET efficient was remarkably decreased and then disappeared. It indicated that actived caspase-3 cleaved the CFP-CRS-DsRed fusion protein at CRS site. Thus, the FRET probe of CFP-CRS-DsRed could sensitively and reliably monitor caspase-3 activation in living cell. This probe will be highly useful for rapid-screening potential drugs that may target the apoptotic process and for imaging tumors in vivo.

  18. Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain.

    Science.gov (United States)

    Lamboy, Jorge A; Kim, Hajin; Dembinski, Holly; Ha, Taekjip; Komives, Elizabeth A

    2013-07-24

    Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1-4), at ARs 2 and 5 (AR 2-5), and at ARs 3 and 6 (AR 3-6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 "loses grip". AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.

  19. The effect of plasma nitriding and post oxidation on fretting wear behaviour of a high strength alloy steel

    Science.gov (United States)

    Prakash, N. Arun; Bennett, C. J.

    2017-05-01

    The fretting wear performance of the non-nitrided, nitrided and nitrided-post oxidized high strength alloy steel, W460 were investigated in the gross slip regime at ambient condition. Fretting wear tests were performed with an applied normal load of 250 and 650 N at a displacement amplitude of 100 μm using a cylinder-on-flat configuration. X-ray analysis (XRD) revealed the formation of the iron-nitrided Fe3N and Fe4N during plasma nitriding and iron oxide phases of hematite (Fe2O3) and magnetite (Fe3O4) during post-oxidation of the cylindrical steel samples. The steady state tangential force coefficient decreases when the nitrided and post-oxidized samples were fretted against the non-nitrided steel material when compared to the non-nitrided steel contact pair. The steady state tangential force coefficient decreased with an increase in applied normal load across all of the fretting conditions. The total dissipated energy and the total wear volume increased with an increase in applied normal load with total wear volume of the non-nitrided vs nitrided and non-nitrided vs nitrided post-oxidized sample pairs, showing a reduction in the wear volume of approximately 50% compared to the non-nitrided vs non-nitrided combination under the fretting conditions examined. The worn surface morphology of the fretted samples examined using a scanning electron microscope showed the presence of loose wear debris in the wear track, fragmented wear debris, delamination cracks, delamination with large discontinuities, plate-like wear debris, oxide patches and formation of large cavities.

  20. Ambient Sensors

    NARCIS (Netherlands)

    Börner, Dirk; Specht, Marcus

    2014-01-01

    This software sketches comprise two custom-built ambient sensors, i.e. a noise and a movement sensor. Both sensors measure an ambient value and process the values to a color gradient (green > yellow > red). The sensors were built using the Processing 1.5.1 development environment. Available under th

  1. Ambient Sensors

    NARCIS (Netherlands)

    Börner, Dirk; Specht, Marcus

    2014-01-01

    This software sketches comprise two custom-built ambient sensors, i.e. a noise and a movement sensor. Both sensors measure an ambient value and process the values to a color gradient (green > yellow > red). The sensors were built using the Processing 1.5.1 development environment. Available under

  2. Fretting Wear Behavior of Medium Carbon Steel Modified by Low Temperature Gas Multi-component Thermo-chemical Treatment

    Institute of Scientific and Technical Information of China (English)

    LUO Jun; ZHENG Jianfeng; PENG Jinfang; HE Liping; ZHU Minhao

    2010-01-01

    The introduction of surface engineering is expected to be an effective strategy against fretting damage. A large number of studies show that the low gas multi-component (such as carbon, nitrogen, sulphur and oxygen, etc) thermo-chemical treatment(LTGMTT) can overcome the brittleness of nitriding process, and upgrade the surface hardness and improve the wear resistance and fatigue properties of the work-pieces significantly. However, there are few reports on the anti-fretting properties of the LTGMTT modified layer up to now, which limits the applications of fretting. So this paper discusses the fretting wear behavior of modified layer on the surface of LZ50 (0.48%C) steel prepared by low temperature gas multi-component thermo-chemical treatment (LTGMTT) technology. The fretting wear tests of the modified layer flat specimens and its substrate (LZ50 steel) against 52100 steel balls with diameter of 40 mm are carried out under normal load of 150 N and displacement amplitudes varied from 2 μm to 40 μm. Characterization of the modified layer and dynamic analyses in combination with microscopic examinations were performed through the means of scanning electron microscope(SEM), optical microscope(OM), X-ray diffraction(XRD) and surface profilometer. The experimental results showed that the modified layer with a total thickness of 60 μm was consisted of three parts, i.e., loose layer, compound layer and diffusion layer. Compared with the substrate, the range of the mixed fretting regime(MFR) of the LTGMTT modified layer diminished, and the slip regime(SR) of the modified layer shifted to the direction of smaller displacement amplitude. The coefficient of friction(COF) of the modified layer was lower than that of the substrate in the initial stage. For the modified layer, the damage in partial slip regime(PSR) was very slight. The fretting wear mechanism of the modified layer both in MFR and SR was abrasive wear and delamination. The modified layer presented better wear

  3. Fluorescent sensor for Cu2+ with a tunable emission wavelength.

    Science.gov (United States)

    Mokhir, Andriy; Kiel, Alexander; Herten, Dirk-Peter; Kraemer, Roland

    2005-08-08

    A concept of fluorescent metal ion sensing with an easily tunable emission wavelength is presented and its principle demonstrated by detection of Cu(2+). A fluorescein dye was chemically modified with a metal chelating group and then attached to the terminus of ss-DNA. This was combined with a complementary ss-DNA modified with another fluorescent dye (ATTO 590), emitting at a longer wavelength. In the assembled duplex, fluorescence resonance energy transfer (FRET) between the fluorescein donor (excited at 470 nm) and the ATTO 590 acceptor (emitting at 624 nm) is observed. Proper positioning within the rigid DNA double helix prevents intramolecular contact quenching of the two dyes. Coordination of paramagnetic Cu(2+) ions by the chelating unit of the sensor results in direct fluorescence quenching of the fluorescein dye and indirect (by loss of FRET) quenching of the ATTO 590 emission at 624 nm. As a result, emission of the acceptor dye can be used for monitoring of the concentration of Cu(2+), with a 20 nM detection limit. The emission wavelength is readily tuned by replacement of ATTO-DNA by other commercially available DNA-acceptor dye conjugates. Fluorescent metal ion sensors emitting at >600 nm are very rare. The possibility of tuning the emission wavelength is important with respect to the optimization of this sensor type for application to biological samples, which usually show broad autofluorescence at <550 nm.

  4. Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Wendy E Kaman

    Full Text Available Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM. In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility.

  5. Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter

    Science.gov (United States)

    Billesbølle, Christian B.; Mortensen, Jonas S.; Sohail, Azmat; Schmidt, Solveig G.; Shi, Lei; Sitte, Harald H.; Gether, Ulrik; Loland, Claus J.

    2016-01-01

    Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle. PMID:27678200

  6. Transition metal ion FRET uncovers K(+) regulation of a neurotransmitter/sodium symporter.

    Science.gov (United States)

    Billesbølle, Christian B; Mortensen, Jonas S; Sohail, Azmat; Schmidt, Solveig G; Shi, Lei; Sitte, Harald H; Gether, Ulrik; Loland, Claus J

    2016-09-28

    Neurotransmitter/sodium symporters (NSSs) are responsible for Na(+)-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K(+) inhibits Na(+)-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K(+)-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni(2+) bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K(+)-induced closure of the transporter to the outside, which was counteracted by Na(+) and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K(+)-effect. The K(+)-effect depended on an intact Na1 site and mutating the Na2 site potentiated K(+) binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K(+) to regulate the LeuT transport cycle.

  7. Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection.

    Science.gov (United States)

    Benz, Christian; Retzbach, Heiko; Nagl, Stefan; Belder, Detlev

    2013-07-21

    Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.

  8. FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Hanna

    2011-08-26

    The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.

  9. Intravital imaging of fluorescent markers and FRET probes by DNA tattooing

    Directory of Open Access Journals (Sweden)

    Spencer David M

    2007-01-01

    Full Text Available Abstract Background Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment. Results By DNA tattooing we transfect keratinocytes of living mice with DNA encoding fluorescent biosensors. Subsequently, the behavior of individual cells expressing these biosensors can be visualized within hours and using conventional microscopy equipment. Using this "instant transgenic" model in combination with a corrected coordinate system, we followed the in vivo behavior of individual cells expressing either FRET- or location-based biosensors for several days. The utility of this approach was demonstrated by assessment of in vivo caspase-3 activation upon induction of apoptosis. Conclusion This "instant skin transgenic" model can be used to follow the in vivo behavior of individual cells expressing either FRET- or location-based probes for several days after tattooing and provides a rapid and inexpensive method for intravital imaging in murine skin.

  10. Maximum likelihood-based analysis of photon arrival trajectories in single-molecule FRET

    Energy Technology Data Exchange (ETDEWEB)

    Waligorska, Marta [Adam Mickiewicz University, Faculty of Chemistry, Grunwaldzka 6, 60-780 Poznan (Poland); Molski, Andrzej, E-mail: amolski@amu.edu.pl [Adam Mickiewicz University, Faculty of Chemistry, Grunwaldzka 6, 60-780 Poznan (Poland)

    2012-07-25

    Highlights: Black-Right-Pointing-Pointer We study model selection and parameter recovery from single-molecule FRET experiments. Black-Right-Pointing-Pointer We examine the maximum likelihood-based analysis of two-color photon trajectories. Black-Right-Pointing-Pointer The number of observed photons determines the performance of the method. Black-Right-Pointing-Pointer For long trajectories, one can extract mean dwell times that are comparable to inter-photon times. -- Abstract: When two fluorophores (donor and acceptor) are attached to an immobilized biomolecule, anti-correlated fluctuations of the donor and acceptor fluorescence caused by Foerster resonance energy transfer (FRET) report on the conformational kinetics of the molecule. Here we assess the maximum likelihood-based analysis of donor and acceptor photon arrival trajectories as a method for extracting the conformational kinetics. Using computer generated data we quantify the accuracy and precision of parameter estimates and the efficiency of the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) in selecting the true kinetic model. We find that the number of observed photons is the key parameter determining parameter estimation and model selection. For long trajectories, one can extract mean dwell times that are comparable to inter-photon times.

  11. The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Watkins, Jennifer L.; Kim, Hanseong [Arizona State University, Tempe, AZ 85287-1604 (United States); Markwardt, Michele L. [University of Maryland School of Medicine, Baltimore, MD 21201-1559 (United States); Chen, Liqing; Fromme, Raimund [Arizona State University, Tempe, AZ 85287-1604 (United States); Rizzo, Mark A. [University of Maryland School of Medicine, Baltimore, MD 21201-1559 (United States); Wachter, Rebekka M., E-mail: rwachter@asu.edu [Arizona State University, Tempe, AZ 85287-1604 (United States)

    2013-05-01

    The high resolution X-ray structure of the cyan fluorescent protein mCerulean3 demonstrates that different combinations of correlated residue substitutions can provide near optimum quantum yield values for fluorescence. Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

  12. Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits.

    Science.gov (United States)

    Quan, Ke; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Ying, Le; Wang, He; Xie, Nuli; Ou, Min; Wang, Kemin

    2016-06-07

    Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers' attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.

  13. An all-in-one numerical methodology for fretting wear and fatigue life assessment

    Directory of Open Access Journals (Sweden)

    I. Llavori

    2016-07-01

    Full Text Available Many mechanical components such as, bearing housings, flexible couplings and spines or orthopedic devices are simultaneously subjected to a fretting wear and fatigue damage. For this reason, the combined study on a single model of wear, crack initiation and propagation is of great interest. This paper presents an all-in-one 2D cylinder on flat numerical model for life assessment on coupled fretting wear and fatigue phenomena. In the literature, two stages are usually distinguished: crack nucleation and its subsequent growth. The method combines the Archard wear model, a critical-plane implementation of the Smith-Watson- Topper (SWT multiaxial fatigue criterion coupled with the Miner-Palmgren accumulation damage rule for crack initiation prediction. Then, the Linear Elastic Fracture Mechanics (LEFM via eXtended Finite Element Method (X-FEM embedded into the commercial finite element code Abaqus FEA has been employed to determine the crack propagation stage. Therefore, the sum of the two stages gives a total life prediction. Finally, the numerical model was validated with experimental data reported in the literature and a good agreement was obtained.

  14. Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.

    Directory of Open Access Journals (Sweden)

    Katrin Gruenwald

    Full Text Available Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.

  15. Using spread spectrum for AMR magnetic sensor

    Science.gov (United States)

    Vala, David

    2016-09-01

    This contribution describe invention of Magnetometer with protection against detection by electronic counter- measure (ECM) registered by Czech patent office as patent no. 305322.1 Magnetic sensors are often part of dual use or security instruments and equipment. For this purpose is very interesting to build sensor with is hidden against electronic countermeasure. In this case is very important level and behavior of electromagnetic noise produced by sensor. And also electromagnetic compatibility of electronic devices is the area which significant grows nowadays too. As the consequence of this growth there is a continuous process of making more strict standards focused on electromagnetic radiation of electronic devices. Sensors technology begins to be a part of these issues due sensors bandwidth increasing and approaching to frequency of radio communication band. Nowadays microcontrollers and similar digital circuits are integrated into sensors devices and it brings new sources of electromagnetic radiation in modern smart sensors.

  16. A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection.

    Science.gov (United States)

    Chakraborty, Smarajit; Mizusaki, Hideaki; Kenney, Linda J

    2015-04-01

    In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor ("I-switch") to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad

  17. A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range.

    Directory of Open Access Journals (Sweden)

    Jeffrey B Klarenbeek

    Full Text Available FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs. Through a conformational change in Epac1, alterations in cellular cAMP levels lead to a change in FRET that is most commonly detected by either Fluorescence Lifetime Imaging (FLIM or by Sensitized Emission (SE, e.g., by simple ratio-imaging. We recently reported a range of different Epac-based cAMP sensors with high dynamic range and signal-to-noise ratio. We showed that constructs with cyan FP as donor are optimal for readout by SE, whereas other constructs with green FP donors appeared much more suited for FLIM detection. In this study, we present a new cAMP sensor, termed (TEpac(VV, which employs mTurquoise as donor. Spectrally very similar to CFP, mTurquoise has about doubled quantum efficiency and unlike CFP, its fluorescence decay is strictly single-exponential. We show that (TEpac(VV appears optimal for detection both by FLIM and SE, that it has outstanding FRET span and signal-to-noise ratio, and improved photostability. Hence, (TEpac(VV should become the cAMP sensor of choice for new experiments, both for FLIM and ratiometric detection.

  18. Semiconductor Laser Lidar Wind Velocity Sensor for Turbine Control

    DEFF Research Database (Denmark)

    Rodrigo, Peter John; Hu, Qi; Pedersen, Christian

    2014-01-01

    A dual line-of-sight CW lidar that measures both wind speed and direction is presented . The wind lidar employs a semiconductor laser, which allows for inexpensive remote sensors geared towards enhanced control of wind turbines .......A dual line-of-sight CW lidar that measures both wind speed and direction is presented . The wind lidar employs a semiconductor laser, which allows for inexpensive remote sensors geared towards enhanced control of wind turbines ....

  19. Preparation of small amounts of sterile siRNA-liposomes with high entrapping efficiency by dual asymmetric centrifugation (DAC).

    Science.gov (United States)

    Hirsch, Markus; Ziroli, Vittorio; Helm, Mark; Massing, Ulrich

    2009-04-02

    Liposomal formulation of siRNA is an attractive approach for improving its delivery in vivo, shielding the RNA from nucleases and promoting tumor targeting. Here, the production of very small batch sizes of siRNA-liposomes by using the "dual asymmetric centrifugation (DAC)" technique was investigated. This new technique combines rapid and sterile liposome preparation with very high entrapping efficiencies. DAC is here presented in conjunction with a non-destructive microscale analysis based on double fluorescence labeling, which enables monitoring of siRNA integrity during the liposomal preparation. Integrity is reflected in spatial proximity of the dyes, which results in measurable fluorescence resonance energy transfer (FRET). The combination of DAC and the sensitive FRET analysis allows the handling of batch sizes down to 20 mg of conventional liposomes (CL) and sterically stabilized liposomes (SL). These were prepared in common 2 ml reaction tubes and loaded with calcein or labeled siRNA. Liposome sizes were 79+/-16 nm for CL and 109+/-9 nm for SL loaded with siRNA. Trapping efficiencies ranged from 43 to 81%, depending on batch size, enclosed compound, and liposome composition. FRET monitoring showed that the siRNA remained intact throughout DAC and that liposomal formulations protected the siRNA from nucleases. siRNA-liposomes remained stable for at least 3 months.

  20. A Wireless Sensor Enabled by Wireless Power

    Science.gov (United States)

    Lee, Da-Sheng; Liu, Yu-Hong; Lin, Chii-Ruey

    2012-01-01

    Through harvesting energy by wireless charging and delivering data by wireless communication, this study proposes the concept of a wireless sensor enabled by wireless power (WPWS) and reports the fabrication of a prototype for functional tests. One WPWS node consists of wireless power module and sensor module with different chip-type sensors. Its main feature is the dual antenna structure. Following RFID system architecture, a power harvesting antenna was designed to gather power from a standard reader working in the 915 MHz band. Referring to the Modbus protocol, the other wireless communication antenna was integrated on a node to send sensor data in parallel. The dual antenna structure integrates both the advantages of an RFID system and a wireless sensor. Using a standard UHF RFID reader, WPWS can be enabled in a distributed area with a diameter up to 4 m. Working status is similar to that of a passive tag, except that a tag can only be queried statically, while the WPWS can send dynamic data from the sensors. The function is the same as a wireless sensor node. Different WPWSs equipped with temperature and humidity, optical and airflow velocity sensors are tested in this study. All sensors can send back detection data within 8 s. The accuracy is within 8% deviation compared with laboratory equipment. A wireless sensor network enabled by wireless power should be a totally wireless sensor network using WPWS. However, distributed WPWSs only can form a star topology, the simplest topology for constructing a sensor network. Because of shielding effects, it is difficult to apply other complex topologies. Despite this limitation, WPWS still can be used to extend sensor network applications in hazardous environments. Further research is needed to improve WPWS to realize a totally wireless sensor network. PMID:23443370

  1. A Wireless Sensor Enabled by Wireless Power

    Directory of Open Access Journals (Sweden)

    Da-Sheng Lee

    2012-11-01

    Full Text Available Through harvesting energy by wireless charging and delivering data by wireless communication, this study proposes the concept of a wireless sensor enabled by wireless power (WPWS and reports the fabrication of a prototype for functional tests. One WPWS node consists of wireless power module and sensor module with different chip-type sensors. Its main feature is the dual antenna structure. Following RFID system architecture, a power harvesting antenna was designed to gather power from a standard reader working in the 915 MHz band. Referring to the Modbus protocol, the other wireless communication antenna was integrated on a node to send sensor data in parallel. The dual antenna structure integrates both the advantages of an RFID system and a wireless sensor. Using a standard UHF RFID reader, WPWS can be enabled in a distributed area with a diameter up to 4 m. Working status is similar to that of a passive tag, except that a tag can only be queried statically, while the WPWS can send dynamic data from the sensors. The function is the same as a wireless sensor node. Different WPWSs equipped with temperature and humidity, optical and airflow velocity sensors are tested in this study. All sensors can send back detection data within 8 s. The accuracy is within 8% deviation compared with laboratory equipment. A wireless sensor network enabled by wireless power should be a totally wireless sensor network using WPWS. However, distributed WPWSs only can form a star topology, the simplest topology for constructing a sensor network. Because of shielding effects, it is difficult to apply other complex topologies. Despite this limitation, WPWS still can be used to extend sensor network applications in hazardous environments. Further research is needed to improve WPWS to realize a totally wireless sensor network.

  2. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Science.gov (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

  3. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

    Science.gov (United States)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

    2006-09-01

    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  4. Single-Molecule Confocal FRET Microscopy to Dissect Conformational Changes in the Catalytic Cycle of DNA Topoisomerases.

    Science.gov (United States)

    Hartmann, S; Weidlich, D; Klostermeier, D

    2016-01-01

    Molecular machines undergo large-scale conformational changes during their catalytic cycles that are linked to their biological functions. DNA topoisomerases are molecular machines that interconvert different DNA topoisomers and resolve torsional stress that is introduced during cellular processes that involve local DNA unwinding. DNA gyrase catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. During its catalytic cycle, gyrase undergoes large-scale conformational changes that drive the supercoiling reaction. These conformational changes can be followed by single-molecule Förster resonance energy transfer (FRET). Here, we use DNA gyrase from Bacillus subtilis as an illustrative example to present strategies for the investigation of conformational dynamics of multisubunit complexes. We provide a brief introduction into single-molecule FRET and confocal microscopy, with a focus on practical considerations in sample preparation and data analysis. Different strategies in the preparation of donor-acceptor-labeled molecules suitable for single-molecule FRET experiments are outlined. The insight into the mechanism of DNA supercoiling by gyrase gained from single-molecule FRET experiment is summarized. The general strategies described here can also be applied to investigate conformational changes and their link to biological function of other multisubunit molecular machines. © 2016 Elsevier Inc. All rights reserved.

  5. Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

    Science.gov (United States)

    Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

    2017-02-01

    To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

  6. The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues

    Directory of Open Access Journals (Sweden)

    Anna-Karin E. Johnsson

    2014-03-01

    Full Text Available The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.

  7. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Science.gov (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  8. Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET.

    Science.gov (United States)

    Zagotta, William N; Gordon, Moshe T; Senning, Eric N; Munari, Mika A; Gordon, Sharona E

    2016-02-01

    Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co(2+) bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

  9. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET)

    NARCIS (Netherlands)

    Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon

    2016-01-01

    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of

  10. A Quantitative Theoretical Framework for Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET)

    NARCIS (Netherlands)

    Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon

    2016-01-01

    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements

  11. Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data.

    Science.gov (United States)

    Bronson, Jonathan E; Fei, Jingyi; Hofman, Jake M; Gonzalez, Ruben L; Wiggins, Chris H

    2009-12-16

    Time series data provided by single-molecule Förster resonance energy transfer (smFRET) experiments offer the opportunity to infer not only model parameters describing molecular complexes, e.g., rate constants, but also information about the model itself, e.g., the number of conformational states. Resolving whether such states exist or how many of them exist requires a careful approach to the problem of model selection, here meaning discrimination among models with differing numbers of states. The most straightforward approach to model selection generalizes the common idea of maximum likelihood--selecting the most likely parameter values--to maximum evidence: selecting the most likely model. In either case, such an inference presents a tremendous computational challenge, which we here address by exploiting an approximation technique termed variational Bayesian expectation maximization. We demonstrate how this technique can be applied to temporal data such as smFRET time series; show superior statistical consistency relative to the maximum likelihood approach; compare its performance on smFRET data generated from experiments on the ribosome; and illustrate how model selection in such probabilistic or generative modeling can facilitate analysis of closely related temporal data currently prevalent in biophysics. Source code used in this analysis, including a graphical user interface, is available open source via http://vbFRET.sourceforge.net.

  12. 一种采用双视场星敏感器的飞行器姿态角快速、高精度测量方法%A Fast and Accurate Method for Spacecraft Attitude Determination Based on a Dual-Field Star Sensor

    Institute of Scientific and Technical Information of China (English)

    范宏深; 王同权; 佛显超

    2011-01-01

    To determine attitudes of spacecrafts with laser communications under the condition of their frequently attitude maneuvering, a new method to measure the spacecraft attitude accurately and swiftly based on a dual-field star sensor is put forward and applied to a zero orbit spacecraft. The star sensor has an optical system with a wide and a narrow field of view and two sets of CCDs. The wide field sensor is used to detect the seven stars near the North Pole and to recognize the North Star. The narrow field sensor is used to track the North Star and to measure North Star' s direction vector, and then to compare with North Star' s theoretically direction vector in any orbit position. The spacecraft attitude is calculated by using the difference of the two vectors. It is educed from the model that measurement accuracy of attitude angles can be better than Ⅰ". No star searching and no database is need and the time consumed is just the time to process a frame of image of the star sensor.%针对姿态经常机动的飞行器之间建立激光通信问题,研究了一种针对这种飞行器的姿态角高精度、快速测量新方法.这种姿态角测量方法采用一种由宽、窄两种视场组成的双视场星敏感器,并以0轨道飞行器应用为例进行了姿态角测量仿真:其中的宽视场用于识别北极星,窄视场用于跟踪北极星;通过对北极星方向矢量测量值与其理论值进行比较精确求出了偏航、俯仰和滚动三个姿态角.分析表明,姿态角的角分辨率小于1″;测量过程不需要耗时的星图数据库搜索,一帧图像就可以完成一次快速测量.

  13. Influence of thermal oxidation duration on the microstructure and fretting wear behavior of Ti6Al4V alloy

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Song [State Key Laboratory of Tribology, Tsinghua University, Beijing 100084 (China); Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Liao, Zhenhua [Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Biomechanics and Biotechnology Lab, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Liu, Yuhong [State Key Laboratory of Tribology, Tsinghua University, Beijing 100084 (China); Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Liu, Weiqiang, E-mail: weiqliu@hotmail.com [State Key Laboratory of Tribology, Tsinghua University, Beijing 100084 (China); Biomechanics and Biotechnology Lab, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China)

    2015-06-01

    Thermal oxidation under water oxidizing atmosphere was performed on Ti6Al4V alloy under different durations from 2 h to 8 h. Surface characterizations were performed using X-ray diffractometery (XRD), scanning electron microscopy (SEM), Raman spectroscopy, nanoindentation and nano scratch testing. Fretting wear behaviors of untreated and oxidized samples were also examined. The formed oxide coating mainly included rutile TiO{sub 2} as well as a little alumina. The weight gain with respect to the oxidation duration obeyed the linear oxidation kinetics law. The growth of oxide grains was in inadequate growth state of incomplete scale coverage from 2nd to 4th hour duration, in normal growth state from 4th to 6th hour duration while in excessive growth state of oxide particle agglomeration and surface roughening from 6th to 8th (or more than 8th) hour duration. The coating thickness increased from 5 μm to 12 μm as oxidation duration increased from 2 h to 8 h. The increase in duration also increased surface roughness and nano hardness as well as adhesion strength of the film/substrate for oxidized samples. The nano hardness value was 10.06 ± 2.15 GPa and the critical load of failure during nano scratch testing was 554.3 ± 6.44 mN for 4 h treated sample. The untreated and oxidized samples showed a same fretting running status and fretting regime with a displacement amplitude of 200 μm while revealing different fretting failure mechanisms. It was mainly abrasive and adhesive wear under ploughing force for untreated sample, while a mix of 3-body abrasion by rolling oxide particles and severe plastic deformation under high contact stress between two ceramic materials for the oxidized samples. The oxide coating was not worn out and improved the fretting wear resistance of titanium alloy. - Highlights: • A thickness of 5–12 μm rutile TiO{sub 2} coating formed under different oxidation durations. • Weight gain with respect to oxidation duration obeyed linear

  14. A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

    Directory of Open Access Journals (Sweden)

    Pedersen Erik B

    2010-01-01

    Full Text Available Abstract Background Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH. Hoogsteen based parallel triplex helix formation requires protonation of cytosines in the triplex forming strand. Therefore, nucleic acid triplexes show strong pH dependence and are stable only at acidic pH. This led us to establish a new pH independent fluorophore based measuring system on the LightCycler for thermal stability studies of parallel triplexes. Results A novel LightCycler FRET pair labelled with ATTO495 and ATTO647N was established for parallel triplex detection with antiparallel duplex as a control for the general applicability of these fluorophores for Tm determination. The ATTO fluorophores were pH stable from pH 4.5 to 7.5. Melting of triplex and duplex structures were accompanied by a large decrease in fluorescence intensity leading to well defined Tm and high reproducibility. Validation of Tm showed low intra- and inter-assay coefficient of variation; 0.11% and 0.14% for parallel triplex and 0.19% and 0.12% for antiparallel duplex. Measurements of Tm and fluorescence intensity over time and multiple runs showed great time and light stability of the ATTO fluorophores. The variance on Tm determinations was significant lower on the LightCycler platform compared to UV absorbance measurements, which enable discrimination of DNA structures with very similar Tm. Labelling of DNA probes with ATTO fluorophore increased Tm of antiparallel duplexes significantly, but not Tm of parallel triplexes. Conclusions We have established a novel pH independent FRET pair with high fluorescence signals on the Light

  15. Sliding Wear and Fretting Wear of DLC-Based, Functionally Graded Nanocomposite Coatings

    Science.gov (United States)

    Miyoshi, K.; Pohlchuck, B.; Street, Kenneth W.; Zabinski, J. S.; Sanders, J. H.; Voevodin, A. a.; Wu, R. L. C.

    1999-01-01

    Improving the tribological functionality of diamondlike carbon (DLC) films--developing, good wear resistance, low friction, and high load-carrying capacity-was the aim of this investigation. Nanocomposite coatings consisting of an amorphous DLC (a-DLC) top layer and a functionally graded titanium-titanium carbon-diamondlike carbon (Ti-Ti(sub x) C(sub y)-DLC) underlayer were produced on AISI 440C stainless steel substrates by the hybrid technique of magnetron sputtering and pulsed-laser deposition. The resultant DLC films were characterized by Raman spectroscopy, scanning electron microscopy, and surface profilometry. Two types of wear experiment were conducted in this investioation: sliding friction experiments and fretting wear experiments. Unidirectional ball-on-disk sliding friction experiments were conducted to examine the wear behavior of an a-DLC/Ti-Ti(sub x) C(sub y)-DLC-coated AISI 440C stainless steel disk in sliding contact with a 6-mm-diameter AISI 440C stainless steel ball in ultrahigh vacuum, dry nitrogen, and humid air. Although the wear rates for both the coating and ball were low in all three environments, the humid air and dry nitrogen caused mild wear with burnishing, in the a-DLC top layer, and the ultrahigh vacuum caused relatively severe wear with brittle fracture in both the a-DLC top layer and the Ti-Ti(sub x) C(sub y)-DLC underlayer. For reference, amorphous hydrogenated carbon (H-DLC) films produced on a-DLC/Ti-Ti(sub x) C(sub y)-DLC nanocomposite coatings by using an ion beam were also examined in the same manner. The H-DLC films markedly reduced friction even in ultrahigh vacuum without sacrificing wear resistance. The H-DLC films behaved much like the a-DLC/Ti-Ti(sub x) C(sub y)-DLC nanocomposite coating in dry nitrogen and humid air, presenting low friction and low wear. Fretting wear experiments were conducted in humid air (approximately 50% relative humidity) at a frequency of 80 Hz and an amplitude of 75 micron on an a

  16. Friction and wear properties of high-velocity oxygen fuel sprayed WC-17Co coating under rotational fretting conditions

    Science.gov (United States)

    Luo, Jun; Cai, Zhenbing; Mo, Jiliang; Peng, Jinfang; Zhu, Minhao

    2016-05-01

    Rotational fretting which exist in many engineering applications has incurred enormous economic loss. Thus, accessible methods are urgently needed to alleviate or eliminate damage by rotational fretting. Surface engineering is an effective approach that is successfully adopted to enhance the ability of components to resist the fretting damage. In this paper, using a high-velocity oxygen fuel sprayed (HVOF) technique WC-17Co coating is deposited on an LZ50 steel surface to study its properties through Vickers hardness testing, scanning electric microscope (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffractrometry (XRD). Rotational fretting wear tests are conducted under normal load varied from 10 N to 50 N, and angular displacement amplitudes vary from 0.125° to 1°. Wear scars are examined using SEM, EDX, optical microscopy (OM), and surface topography. The experimental results reveal that the WC-17Co coating adjusted the boundary between the partial slip regime (PSR) and the slip regime (SR) to the direction of smaller amplitude displacement. As a result, the coefficients of friction are consistently lower than the substrate's coefficients of friction both in the PSR and SR. The damage to the coating in the PSR is very slight. In the SR, the coating exhibits higher debris removal efficiency and load-carrying capacity. The bulge is not found for the coating due to the coating's higher hardness to restrain plastic flow. This research could provide experimental bases for promoting industrial application of WC-17Co coating in prevention of rotational fretting wear.

  17. Metamaterial Sensors

    Directory of Open Access Journals (Sweden)

    Jing Jing Yang

    2013-01-01

    Full Text Available Metamaterials have attracted a great deal of attention due to their intriguing properties, as well as the large potential applications for designing functional devices. In this paper, we review the current status of metamaterial sensors, with an emphasis on the evanescent wave amplification and the accompanying local field enhancement characteristics. Examples of the sensors are given to illustrate the principle and the performance of the metamaterial sensor. The paper concludes with an optimistic outlook regarding the future of metamaterial sensor.

  18. Dual Income Taxes

    DEFF Research Database (Denmark)

    Sørensen, Peter Birch

    This paper discusses the principles and practices of dual income taxation in the Nordic countries. The first part of the paper explains the rationale and the historical background for the introduction of the dual income tax and describes the current Nordic tax practices. The second part...... of the paper focuses on the problems of taxing income from small businesses and the issue of corporate-personal tax integration under the dual income tax, considering alternative ways of dealing with these challenges. In the third and final part of the paper, I briefly discuss whether introducing a dual income...... tax could be relevant for New Zealand....

  19. Dual Income Taxes

    DEFF Research Database (Denmark)

    Sørensen, Peter Birch

    This paper discusses the principles and practices of dual income taxation in the Nordic countries. The first part of the paper explains the rationale and the historical background for the introduction of the dual income tax and describes the current Nordic tax practices. The second part...... of the paper focuses on the problems of taxing income from small businesses and the issue of corporate-personal tax integration under the dual income tax, considering alternative ways of dealing with these challenges. In the third and final part of the paper, I briefly discuss whether introducing a dual income...... tax could be relevant for New Zealand....

  20. Attention Sensor

    NARCIS (Netherlands)

    Börner, Dirk; Kalz, Marco; Specht, Marcus

    2014-01-01

    This software sketch was used in the context of an experiment for the PhD project “Ambient Learning Displays”. The sketch comprises a custom-built attention sensor. The sensor measured (during the experiment) whether a participant looked at and thus attended a public display. The sensor was built us

  1. Attention Sensor

    NARCIS (Netherlands)

    Börner, Dirk; Kalz, Marco; Specht, Marcus

    2014-01-01

    This software sketch was used in the context of an experiment for the PhD project “Ambient Learning Displays”. The sketch comprises a custom-built attention sensor. The sensor measured (during the experiment) whether a participant looked at and thus attended a public display. The sensor was built us

  2. Dual-fuel, dual-throat engine preliminary analysis

    Science.gov (United States)

    Obrien, C. J.

    1979-01-01

    A propulsion system analysis of the dual fuel, dual throat engine for launch vehicle applications was conducted. Basic dual throat engine characterization data were obtained to allow vehicle optimization studies to be conducted. A preliminary baseline engine system was defined.

  3. Structural dynamics of potassium-channel gating revealed by single-molecule FRET.

    Science.gov (United States)

    Wang, Shizhen; Vafabakhsh, Reza; Borschel, William F; Ha, Taekjip; Nichols, Colin G

    2016-01-01

    Crystallography has provided invaluable insights regarding ion-channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites and then examined structural dynamics within lipid membranes by single-molecule fluorescence resonance energy transfer (FRET). We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states, both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and in cation channels in general.

  4. Structural dynamics of potassium channel gating revealed by single molecule FRET

    Science.gov (United States)

    Borschel, William F.; Ha, Taekjip; Nichols, Colin G.

    2016-01-01

    Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general. PMID:26641713

  5. Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy

    Directory of Open Access Journals (Sweden)

    Daniel Sultanov

    2017-01-01

    Full Text Available DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose polymerase 1 increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA.

  6. A Study on Fretting Wear Property of CVD SiC and Sintered SiC

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Hyun-Jin; Jang, Ki-Nam; An, Ji-Hyeong; Kim, Kyu-Tae [Dongguk University, Gyeongju (Korea, Republic of)

    2015-10-15

    Silicon Carbide is broadly used as high temperature structure material because of its high temperature tolerance and superior mechanical properties. After the Fukushima nuclear power plant accident, SiC proposed as one of the alternative materials for LWR fuel cladding to provide enhanced safety margin. Grid-to-rod fretting wear-induced fuel failure is known to occur due to flow-induced vibration of the reactor core and grid to- rod gap. In this paper, wear tests for CVD SiC plate and sintered SiC tube were performed with two types of spacer grids. Wear test of corroded and non-corroded CVD SiC plates indicate that wear resistance of corroded specimen is lower than one of non-corroded specimen in contrast with zirconium alloy cladding tube. It may be affected by rough surface of corroded specimen caused by grain boundary attack.

  7. Multirate Simulations of String Vibrations Including Nonlinear Fret-String Interactions Using the Functional Transformation Method

    Science.gov (United States)

    Trautmann, L.; Rabenstein, R.

    2004-12-01

    The functional transformation method (FTM) is a well-established mathematical method for accurate simulations of multidimensional physical systems from various fields of science, including optics, heat and mass transfer, electrical engineering, and acoustics. This paper applies the FTM to real-time simulations of transversal vibrating strings. First, a physical model of a transversal vibrating lossy and dispersive string is derived. Afterwards, this model is solved with the FTM for two cases: the ideally linearly vibrating string and the string interacting nonlinearly with the frets. It is shown that accurate and stable simulations can be achieved with the discretization of the continuous solution at audio rate. Both simulations can also be performed with a multirate approach with only minor degradations of the simulation accuracy but with preservation of stability. This saves almost 80% of the computational cost for the simulation of a six-string guitar and therefore it is in the range of the computational cost for digital waveguide simulations.

  8. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays.

    Science.gov (United States)

    von der Haar, Marcel; Heuer, Christopher; Pähler, Martin; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2016-11-30

    The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs.

  9. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Marcel von der Haar

    2016-11-01

    Full Text Available The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS. Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs.

  10. FRET study in oligopeptide-linked donor–acceptor system in PVA matrix

    Science.gov (United States)

    Shah, Sunil; Mandecki, Wlodek; Li, Ji; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

    2016-12-01

    An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.

  11. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes.

    Science.gov (United States)

    Buning, Ruth; Kropff, Wietske; Martens, Kirsten; van Noort, John

    2015-02-18

    Chromatin, the structure in which DNA is compacted in eukaryotic cells, plays a key role in regulating DNA accessibility. FRET experiments on single nucleosomes, the basic units in chromatin, have revealed a dynamic nucleosome where spontaneous DNA unwrapping from the ends provides access to the nucleosomal DNA. Here we investigated how this DNA breathing is affected by extension of the linker DNA and by the presence of a neighboring nucleosome. We found that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Interactions between neighboring nucleosomes are more likely in dinucleosomes spaced by 55 bp of linker DNA than in dinucleosomes spaced by 50 bp of linker DNA. Such increased unwrapping may not only increase the accessibility of nucleosomal DNA in chromatin fibers, it may also be key to folding of nucleosomes into higher order structures.

  12. CASL Structural Mechanics Modeling of Grid-to-Rod Fretting (GTRF)

    Science.gov (United States)

    Lu, Wei; Thouless, M. D.; Hu, Zupan; Wang, Hai; Ghelichi, Ramin; Wu, Chen-Hung; Kamrin, Ken; Parks, David

    2016-09-01

    Fluid-induced grid-to-rod fretting (GTRF) wear is responsible for over 70% of fuel leaks in pressurized water reactors (PWRs) in the US. The Consortium for Advanced Simulation of Light Water Reactors (CASL) has identified GTRF as a challenge problem that is very important to nuclear plants. GTRF is a complex problem that involves multiple physical phenomena. This paper summarizes several GTRF-related problems being addressed by the University of Michigan and the Massachusetts Institute of Technology, CASL partners. These include analyses of cladding creep, wear, structural mechanics, and the effects of the rod-to-grid gap. Also outlined are additional aspects of material science and computational modeling that will be needed to realize the ultimate goal of high-fidelity predictive modeling and design tools to address GTRF.

  13. Maximum likelihood-based analysis of photon arrival trajectories in single-molecule FRET

    Science.gov (United States)

    Waligórska, Marta; Molski, Andrzej

    2012-07-01

    When two fluorophores (donor and acceptor) are attached to an immobilized biomolecule, anti-correlated fluctuations of the donor and acceptor fluorescence caused by Förster resonance energy transfer (FRET) report on the conformational kinetics of the molecule. Here we assess the maximum likelihood-based analysis of donor and acceptor photon arrival trajectories as a method for extracting the conformational kinetics. Using computer generated data we quantify the accuracy and precision of parameter estimates and the efficiency of the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) in selecting the true kinetic model. We find that the number of observed photons is the key parameter determining parameter estimation and model selection. For long trajectories, one can extract mean dwell times that are comparable to inter-photon times.

  14. TR-FRET binding assay targeting unactivated form of Bruton's tyrosine kinase.

    Science.gov (United States)

    Asami, Tokiko; Kawahata, Wataru; Sawa, Masaaki

    2015-01-01

    Bruton's Tyrosine Kinase (BTK) is one of the crucial kinases for the B cell maturation and mast cell activation, and specific inhibitors of BTK are considered to be attractive targets in drug discovery research. In this Letter, we have designed and synthesized a new fluorescent probe for TR-FRET-based high-throughput screening, to identify compounds that preferentially bind to an inactive conformation of BTK which has a unique structural feature. A set of kinase-focused compound library was screened using this assay method, and compound 31 was successfully identified as a potent inhibitor which preferentially bind to the inactive conformation of BTK. These results suggest that this screening method has a great potential for the discovery of novel selective BTK inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints.

    Directory of Open Access Journals (Sweden)

    Jayant James Jayasundar

    Full Text Available Cardiac troponin (cTn is the Ca(2+-sensitive molecular switch that controls cardiac muscle activation and relaxation. However, the molecular detail of the switching mechanism and how the Ca(2+ signal received at cardiac troponin C (cTnC is communicated to cardiac troponin I (cTnI are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET measurements and molecular dynamic (MD simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca(2+-free and saturating Ca(2+ conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca(2+-saturated structure, the absence of regulatory Ca(2+ perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca(2+, induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca(2+ the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

  16. Quantitative co-expression of proteins at the single cell level - application to a multimeric FRET sensor

    NARCIS (Netherlands)

    Goedhart, J.; van Weeren, L.; Adjobo-Hermans, M.J.W.; Elzenaar, I.; Hink, M.A.; Gadella (jr.), T.W.J.

    2011-01-01

    Background Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideall

  17. Quantitative co-expression of proteins at the single cell level--application to a multimeric FRET sensor.

    NARCIS (Netherlands)

    Goedhart, J.; Weeren, L. van; Adjobo-Hermans, M.J.W.; Elzenaar, I.; Hink, M.A.; Gadella, T.W.

    2011-01-01

    BACKGROUND: Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideal

  18. The Dual Career Family.

    Science.gov (United States)

    Gurtin, Lee

    1980-01-01

    The dual career couple is forced to make a series of choices and compromises that impact the realms of marriage and career. The dilemmas that confront dual career marriages can be overcome only by compromise, accommodation, and mutual understanding on the part of the individuals involved. A revamping of human resources and recruitment programs is…

  19. Dual Credit Report

    Science.gov (United States)

    Light, Noreen

    2016-01-01

    In 2015, legislation to improve access to dual-credit programs and to reduce disparities in access and completion--particularly for low income and underrepresented students--was enacted. The new law focused on expanding access to College in the High School but acknowledged issues in other dual-credit programs and reinforced the notion that cost…

  20. Dual Youla parameterization

    DEFF Research Database (Denmark)

    Niemann, Hans Henrik

    2003-01-01

    A different aspect of using the parameterisation of all systems stabilised by a given controller, i.e. the dual Youla parameterisation, is considered. The relation between system change and the dual Youla parameter is derived in explicit form. A number of standard uncertain model descriptions are...