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Related Experiment Videos

A microchip-based enzyme assay for protein kinase A.

C B Cohen1, E Chin-Dixon, S Jeong

  • 1Caliper Technologies Corporation, 605 Fairchild Drive, Mountain View, California 94043, USA.

Analytical Biochemistry
|August 24, 1999
PubMed
Summary

This study introduces a novel microchip-based enzyme assay for protein kinase A (PKA). The assay utilizes microfluidics and electroosmosis for precise reagent handling and kinetic analysis, offering an alternative for assays lacking fluorogenic substrates.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Microfluidics

Background:

  • Protein kinase A (PKA) plays a crucial role in cellular signaling pathways.
  • Developing efficient and precise assays for enzyme activity is essential for biochemical research.
  • Existing enzyme assays may face limitations, particularly when fluorogenic substrates are not readily available.

Purpose of the Study:

  • To develop and validate a microchip-based enzyme assay for quantifying protein kinase A activity.
  • To demonstrate the utility of microfluidic devices for enzymatic reactions and kinetic analysis.
  • To establish an alternative assay method for enzymes where fluorogenic substrates are challenging to design.

Main Methods:

  • Microchips were fabricated using standard photolithographic techniques.

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  • Electroosmosis was employed for precise liquid handling and reagent transport within microchannels.
  • On-chip capillary electrophoresis was used to separate and detect fluorescently labeled peptide substrates and products.
  • Kinetic parameters (Km, Ki) were determined through on-chip dilutions and enzymatic reactions.

    • Main Results:

    • The microchip assay successfully measured protein kinase A activity.
    • Electroosmotic flow enabled precise control over all liquid-handling steps, including dilutions.
    • Kinetic constants for peptide substrate, ATP, and the inhibitor H-89 were accurately determined on-chip.
    • The assay proved effective even without the need for specialized fluorogenic substrates.

    Conclusions:

    • Microchip-based assays offer a powerful platform for enzymatic studies, particularly for enzymes lacking suitable fluorogenic substrates.
    • Electroosmosis provides a robust mechanism for automated liquid handling in microfluidic biochemical assays.
    • This technology enables efficient determination of enzyme kinetics and inhibitor efficacy in a miniaturized format.