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Novel fluorescence sensing methods for high throughput screening.

J R Lakowicz1, I Gryczynski, Z Gryczynski

  • 1Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. lakowicz@cfs.umbi.umd.edu

Journal of Biomolecular Screening
|July 14, 2000
PubMed
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Two novel fluorescence sensing methods, modulation and polarization sensing, offer internally calibrated measurements for high-throughput screening (HTS). These techniques enable reliable quantification of analytes like pH and calcium without sample additions, improving assay standardization.

Area of Science:

  • Biophysical Chemistry
  • Analytical Chemistry
  • Biotechnology

Background:

  • High-throughput screening (HTS) requires robust and standardized analytical methods.
  • Existing fluorescence sensing techniques can be susceptible to variations in excitation and detection.
  • Internal calibration is crucial for improving the reliability of HTS assays.

Purpose of the Study:

  • To introduce two novel fluorescence sensing methods for HTS applications.
  • To demonstrate the utility of internal calibration using a reference film.
  • To enhance the standardization and reliability of HTS measurements.

Main Methods:

  • Development of modulation sensing to convert intensity changes into modulation signal variations.
  • Development of polarization sensing to convert intensity changes into polarization variations.

Related Experiment Videos

  • Integration of an adjacent reference film for internal intensity or polarization calibration.

    • Main Results:

    • Both modulation and polarization sensing successfully measured fluorophore concentrations, pH, and calcium concentrations in HTS plates.
    • Internal calibration via reference film eliminated the need for sample additions.
    • The methods demonstrated potential for standardizing HTS assays across different experimental runs.

    Conclusions:

    • Modulation and polarization sensing are effective fluorescence-based techniques for HTS.
    • Internal calibration with a reference film enhances assay robustness and reproducibility.
    • These methods offer a pathway to improved standardization in HTS workflows.