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David D. Thomas - One of the best experts on this subject based on the ideXlab platform.

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • Screening for SERCA Activators using a High-throughput Time-Resolved FRET Assay
    Biophysical Journal, 2013
    Co-Authors: Ji Li, Joseph M. Muretta, Kurt C. Peterson, Gregory D. Gillispie, Holly R. Langer, Razvan L Cornea, David D. Thomas
    Abstract:

    We used a prototype time-resolved fluorescence lifetime Microplate Reader to carry out a high-throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifetime plate Reader was made possible by our recent development of fast time-resolved fluorescence by direct waveform recording, which achieves 105 higher throughput than the conventional single-photon counting technology. using this plate Reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the time-resolved Microplate Reader has at least 10x higher precision than a conventional intensity-based Microplate Reader, raising the quality index (z') of our assay from marginal, in the intensity Reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

  • fluorescence lifetime Microplate Reader for structural biology high performance and high throughput in the same tool
    Biophysical Journal, 2013
    Co-Authors: Gregory D. Gillispie, Kurt C. Peterson, David D. Thomas
    Abstract:

    Microplate Readers are widely considered as designed and intended for high-throughput (HT) applications such as drug discovery, whereas “research-grade” (RG) fluorescence spectrometers are the tools of choice when quality and diversity of data are the prime considerations. However, a definite trend of the past decade is the commercial availability of Microplate Readers that come equipped with emission and excitation monochromators (even double monochromators) very similar to those found on research-grade spectrometers. The flashlamp sources used in most Microplate Readers are very similar or even identical in design to the flashlamp sources of the research-grade spectrometers. Microplate Readers that employ fluorescence lifetime as a primary readout represent another possible form of convergence between the high-throughput assay and research. An interesting question is to what degree the biggest difference between these two worlds is now simply the sample format, i.e., cuvette vs. Microplate. It seems reasonable to assume that the cuvette with its straightforward implementation of right angle excitation-emission geometry offers significant advantages over the epi-illumination geometry and uncontained sample imposed by a Microplate when it comes to data quality. We have implemented a prototype Microplate Reader equipped with a variety of pulsed laser sources for measurement of fluorescence spectra, fluorescence lifetimes, and anisotropy. The subject of this poster is benchmarking its performance relative to cuvette format. The plate Reader employs direct waveform recording as an alternative to TCSPC; studies to compare the speed, accuracy, and precision of the two lifetime approaches are presented along with several examples of titration curves for rapid determination of binding affinities via time-resolved FRET.

Gregory D. Gillispie - One of the best experts on this subject based on the ideXlab platform.

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • Screening for SERCA Activators using a High-throughput Time-Resolved FRET Assay
    Biophysical Journal, 2013
    Co-Authors: Ji Li, Joseph M. Muretta, Kurt C. Peterson, Gregory D. Gillispie, Holly R. Langer, Razvan L Cornea, David D. Thomas
    Abstract:

    We used a prototype time-resolved fluorescence lifetime Microplate Reader to carry out a high-throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifetime plate Reader was made possible by our recent development of fast time-resolved fluorescence by direct waveform recording, which achieves 105 higher throughput than the conventional single-photon counting technology. using this plate Reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the time-resolved Microplate Reader has at least 10x higher precision than a conventional intensity-based Microplate Reader, raising the quality index (z') of our assay from marginal, in the intensity Reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

  • fluorescence lifetime Microplate Reader for structural biology high performance and high throughput in the same tool
    Biophysical Journal, 2013
    Co-Authors: Gregory D. Gillispie, Kurt C. Peterson, David D. Thomas
    Abstract:

    Microplate Readers are widely considered as designed and intended for high-throughput (HT) applications such as drug discovery, whereas “research-grade” (RG) fluorescence spectrometers are the tools of choice when quality and diversity of data are the prime considerations. However, a definite trend of the past decade is the commercial availability of Microplate Readers that come equipped with emission and excitation monochromators (even double monochromators) very similar to those found on research-grade spectrometers. The flashlamp sources used in most Microplate Readers are very similar or even identical in design to the flashlamp sources of the research-grade spectrometers. Microplate Readers that employ fluorescence lifetime as a primary readout represent another possible form of convergence between the high-throughput assay and research. An interesting question is to what degree the biggest difference between these two worlds is now simply the sample format, i.e., cuvette vs. Microplate. It seems reasonable to assume that the cuvette with its straightforward implementation of right angle excitation-emission geometry offers significant advantages over the epi-illumination geometry and uncontained sample imposed by a Microplate when it comes to data quality. We have implemented a prototype Microplate Reader equipped with a variety of pulsed laser sources for measurement of fluorescence spectra, fluorescence lifetimes, and anisotropy. The subject of this poster is benchmarking its performance relative to cuvette format. The plate Reader employs direct waveform recording as an alternative to TCSPC; studies to compare the speed, accuracy, and precision of the two lifetime approaches are presented along with several examples of titration curves for rapid determination of binding affinities via time-resolved FRET.

Kurt C. Peterson - One of the best experts on this subject based on the ideXlab platform.

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • Screening for SERCA Activators using a High-throughput Time-Resolved FRET Assay
    Biophysical Journal, 2013
    Co-Authors: Ji Li, Joseph M. Muretta, Kurt C. Peterson, Gregory D. Gillispie, Holly R. Langer, Razvan L Cornea, David D. Thomas
    Abstract:

    We used a prototype time-resolved fluorescence lifetime Microplate Reader to carry out a high-throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifetime plate Reader was made possible by our recent development of fast time-resolved fluorescence by direct waveform recording, which achieves 105 higher throughput than the conventional single-photon counting technology. using this plate Reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the time-resolved Microplate Reader has at least 10x higher precision than a conventional intensity-based Microplate Reader, raising the quality index (z') of our assay from marginal, in the intensity Reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

  • fluorescence lifetime Microplate Reader for structural biology high performance and high throughput in the same tool
    Biophysical Journal, 2013
    Co-Authors: Gregory D. Gillispie, Kurt C. Peterson, David D. Thomas
    Abstract:

    Microplate Readers are widely considered as designed and intended for high-throughput (HT) applications such as drug discovery, whereas “research-grade” (RG) fluorescence spectrometers are the tools of choice when quality and diversity of data are the prime considerations. However, a definite trend of the past decade is the commercial availability of Microplate Readers that come equipped with emission and excitation monochromators (even double monochromators) very similar to those found on research-grade spectrometers. The flashlamp sources used in most Microplate Readers are very similar or even identical in design to the flashlamp sources of the research-grade spectrometers. Microplate Readers that employ fluorescence lifetime as a primary readout represent another possible form of convergence between the high-throughput assay and research. An interesting question is to what degree the biggest difference between these two worlds is now simply the sample format, i.e., cuvette vs. Microplate. It seems reasonable to assume that the cuvette with its straightforward implementation of right angle excitation-emission geometry offers significant advantages over the epi-illumination geometry and uncontained sample imposed by a Microplate when it comes to data quality. We have implemented a prototype Microplate Reader equipped with a variety of pulsed laser sources for measurement of fluorescence spectra, fluorescence lifetimes, and anisotropy. The subject of this poster is benchmarking its performance relative to cuvette format. The plate Reader employs direct waveform recording as an alternative to TCSPC; studies to compare the speed, accuracy, and precision of the two lifetime approaches are presented along with several examples of titration curves for rapid determination of binding affinities via time-resolved FRET.

Joseph M. Muretta - One of the best experts on this subject based on the ideXlab platform.

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • High throughput Time Resolved Fluorescence in a Microplate Reader
    Biophysical Journal, 2014
    Co-Authors: Karl J. Petersen, Joseph M. Muretta, Sutton E. Higgins, Kurt C. Peterson, Gregory D. Gillispie, David D. Thomas
    Abstract:

    We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)

  • Screening for SERCA Activators using a High-throughput Time-Resolved FRET Assay
    Biophysical Journal, 2013
    Co-Authors: Ji Li, Joseph M. Muretta, Kurt C. Peterson, Gregory D. Gillispie, Holly R. Langer, Razvan L Cornea, David D. Thomas
    Abstract:

    We used a prototype time-resolved fluorescence lifetime Microplate Reader to carry out a high-throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifetime plate Reader was made possible by our recent development of fast time-resolved fluorescence by direct waveform recording, which achieves 105 higher throughput than the conventional single-photon counting technology. using this plate Reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the time-resolved Microplate Reader has at least 10x higher precision than a conventional intensity-based Microplate Reader, raising the quality index (z') of our assay from marginal, in the intensity Reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

Gang Logan Liu - One of the best experts on this subject based on the ideXlab platform.

  • one step rapid quantification of sars cov 2 virus particles via low cost nanoplasmonic sensors in generic Microplate Reader and point of care device
    Biosensors and Bioelectronics, 2021
    Co-Authors: Liping Huang, Longfei Ding, Jun Zhou, Shuiliang Chen, Fang Chen, Chen Zhao, Gang Logan Liu
    Abstract:

    The spread of SARS-CoV-2 virus in the ongoing global pandemic has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemic. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. As low as 370 vp/mL were detected in one step within 15 min and the virus concentration can be quantified linearly in the range of 0 to 107 vp/mL. Measurements shown on both generic Microplate Reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.

  • Protein binding kinetics quantification via coupled plasmonic-photonic resonance nanosensors in generic Microplate Reader
    Biosensors & bioelectronics, 2019
    Co-Authors: Tang Dang, Wei Zhang, Zifang Song, Yi Wang, Mingqian Chen, Gang Logan Liu
    Abstract:

    Almost no analytical assays, either colorimetric or fluorescence assays, for generic Microplate Readers is capable of dynamic measurements of protein-protein binding or the quantification of kinetic association and dissociation constants of protein interactions. On the other hand, protein binding kinetics quantification can be uniquely done on special expensive surface plasmon resonance (SPR) sensing equipment. Here we report the integration of coupled plasmonic-photonic resonance nanosensors in standard 96-well plate format and by using which, for the very first time, the demonstration of label-free dynamic SPR-like protein binding measurement and kinetics quantification in a generic Microplate Reader. Our low-cost label-free nanosensor plate enables very sensitive detection of immobilized protein interactions based on the transmission optical density (OD) value changes at specific wavelengths measured in a generic Microplate Reader. The relative end-point OD value changes show a good linear response with protein concentrations (from 0.05 to 50 μg/ml). And the protein quantification in serum results are consistent with the concurrent hospital lab tests. Most importantly, the kinetic association and dissociation constants of protein interactions in our sensor plate wells are determined by time-lapse dynamic OD value measurement in the generic Microplate Reader. Enabled by our unique nanosensor plate, SPR-like measurement of protein binding kinetics is now available using generic Microplate Reader ubiquitous in many chemistry and biomedical research labs.