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Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

Ee Xien Ng1, Miles A Miller2, Tengyang Jing3

  • 1Department of Biomedical Engineering, National University of Singapore, Block E4, #04-08, 4 Engineering Drive 3, Singapore 117583, Singapore.

Biosensors & Bioelectronics
|March 21, 2016
PubMed
Summary
This summary is machine-generated.

This study introduces a microfluidics platform with multi-color Förster resonance energy transfer (FRET) substrates to measure multiple protease activities in single cells. This enables high-throughput analysis of cellular enzyme function for applications in cancer research and systems biology.

Keywords:
Droplet microfluidicsFRET-sensorsProteolytic activity matrix analysisSingle cell assay

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Biotechnology

Background:

  • Cellular enzyme activity is crucial for cell behavior but difficult to measure simultaneously in single cells.
  • Existing technologies lack the ability to profile multiple enzyme activities within individual cells.
  • Proteases play key roles in cellular processes, including cancer metastasis.

Purpose of the Study:

  • To develop a novel platform for simultaneous measurement of multiple protease activities in single cells.
  • To characterize protease activity profiles at single-cell resolution in various cancer cell lines.
  • To enable high-throughput analysis of cellular enzyme heterogeneity.

Main Methods:

  • Development of multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates.
  • Integration of FRET substrates with a microfluidics platform for single-cell analysis.
  • Application of Proteolytic Activity Matrix Analysis (PrAMA) for computational analysis of protease signals.

Main Results:

  • Simultaneous measurement of six different protease activities from single cells was achieved.
  • Protease activity profiles were characterized for breast cancer (MDA-MB-231), lung cancer (PC-9), and leukemia (K-562) cell lines.
  • The platform demonstrated high-throughput analysis capabilities (~100 cells/run) in both live-cell and in-situ lysis formats.

Conclusions:

  • The developed microfluidics-FRET platform enables high-throughput, single-cell resolution measurement of multiple protease activities.
  • This technology facilitates the characterization of cell heterogeneity and protease roles in metastasis.
  • Potential applications include systems biology, pharmacology, cancer diagnostics, and stem cell research.