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Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule

Xinyu Zhou1,2, Rui Wang1, Zijian Wan1,3

  • 1Biodesign Center for Bioelectronics and Biosensors, Arizona State University, Tempe, Arizona85287, United States.

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Evanescent scattering microscopy (ESM) enables label-free, digital single-molecule counting for multiplexed protein detection and interaction analysis. This method accurately tracks protein binding kinetics, improving drug and biomarker screening efficiency.

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Area of Science:

  • Biophysics
  • Biochemistry
  • Analytical Chemistry

Background:

  • Multiplexed protein detection is crucial for efficient drug discovery and biomarker screening.
  • Current methods often require labels or lack the precision for real-time kinetic analysis.

Purpose of the Study:

  • To demonstrate multiplexed protein detection and parallel protein interaction analysis using evanescent scattering microscopy (ESM).
  • To develop and implement an automatic single-molecule counting strategy for precise binding kinetics measurement.

Main Methods:

  • Utilized evanescent scattering microscopy (ESM) for label-free, digital single-molecule counting.
  • Implemented an automatic single-molecule counting strategy with high temporal resolution.
  • Real-time tracking of free and bound proteins on a sensor surface.

Main Results:

  • Achieved accurate binding kinetics measurement through label-free digital single-molecule counting.
  • Demonstrated recognition of proteins with different molecular weights.
  • Successfully monitored protein binding processes in solution and simultaneously analyzed kinetics of two different protein interactions.

Conclusions:

  • ESM with digital single-molecule counting offers a powerful platform for multiplexed protein detection and interaction analysis.
  • This approach enhances efficiency and accuracy in drug and biomarker screening.
  • Potential applications include investigating complex biological interactions like competitive binding in biofluids.