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Related Experiment Videos

A fluorogenic assay using pressure-driven flow on a microchip.

M Kerby1, R L Chien

  • 1Caliper Technologies Corporation, Mountain View, CA, USA.

Electrophoresis
|November 10, 2001
PubMed
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This study developed a microchip assay for human T-cell phosphatase (TCPTP) using pressure-driven flow. The novel assay accurately measures enzyme kinetics, showing good agreement with traditional methods.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Microfluidics

Background:

  • Human T-cell phosphatase (TCPTP) is a key enzyme in cellular signaling.
  • Accurate measurement of TCPTP activity is crucial for understanding its role in various diseases.
  • Traditional enzyme assays can be time-consuming and require large sample volumes.

Purpose of the Study:

  • To develop and validate a novel microfluidic platform for quantifying human T-cell phosphatase (TCPTP) activity.
  • To demonstrate the capability of the microchip assay for real-time enzyme kinetics measurements.
  • To compare the performance of the microchip assay with conventional fluorometric methods.

Main Methods:

  • A custom-etched glass microchip was utilized with pressure-driven flow for precise fluid handling.

Related Experiment Videos

  • A fluorogenic substrate, 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP), was employed to detect TCPTP activity.
  • Real-time on-chip dilution and an 8-channel pressure controller were implemented to manage mixed viscosity fluids and reagent concentrations.
  • Kinetic parameters (Km, Vmax, Kcat) were determined by varying substrate concentration and flow rates.

    • Main Results:

    • The microchip assay successfully measured TCPTP activity in both low and high viscosity solutions.
    • Accurate control over reagent dilutions and enzyme concentration was achieved using the pressure controller.
    • The determined kinetic parameters (Km, Vmax, Kcat) closely matched those obtained from standard well-plate assays.
    • The system demonstrated robust performance across a two-order-of-magnitude range of substrate concentrations.

    Conclusions:

    • The developed microfluidic platform offers a sensitive and efficient method for TCPTP activity assays.
    • This technology enables precise kinetic analysis of enzymes in a microfluidic format.
    • The microchip assay provides a viable alternative to traditional methods, with potential applications in diagnostics and drug discovery.