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Ultrasensitive Single-Molecule Enzyme Detection and Analysis Using a Polymer Microarray.

Barrett K Duan1, Peter E Cavanagh1, Xiang Li1

  • 1Department of Pathology , Harvard Medical School , Brigham and Women's Hospital, Wyss Institute for Biologically Inspired Engineering, Building for Transformative Medicine, 60 Fenwood Road , Boston , Massachusetts 02115 , United States.

Analytical Chemistry
|February 10, 2018
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method using polymer microwell arrays to isolate and detect single enzyme molecules. This technique simplifies experiments and achieves sensitive enzyme detection in the low-femtomolar range.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Materials Science

Background:

  • Detecting individual enzyme molecules is crucial for understanding enzyme kinetics and activity.
  • Existing methods often require complex device preparation and surface functionalization.
  • There is a need for simpler, more accessible techniques for single-molecule enzyme analysis.

Purpose of the Study:

  • To develop a novel method for isolating and detecting individual enzyme molecules.
  • To utilize polymer arrays of picoliter microwells for simplified enzyme analysis.
  • To achieve sensitive detection of enzyme concentrations in the low-femtomolar range.

Main Methods:

  • Fabrication of a fluidic flow-cell device with picoliter microwell arrays in cyclic olefin polymer (COP).
  • A simple and robust loading method to introduce reaction solutions into the microwells.
  • Isolation and stable sealing of individual enzyme molecules using fluorinated oil within the COP microwells.

Main Results:

  • Demonstrated a simplified experimental workflow by eliminating extensive device preparation and surface functionalization.
  • Achieved stable sealing of picoliter microwells for several hours.
  • Enabled measurement of enzyme concentrations in the low-femtomolar range through digital counting of active wells.

Conclusions:

  • The novel COP microwell array method offers a simplified and robust approach for single-molecule enzyme detection.
  • This technique allows for sensitive quantification of enzyme concentrations.
  • The microwell arrays are reusable, offering potential for cost-effective applications in biochemical analysis.