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Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance.

Huiling Wu1, Jianjun Zhai, Yuping Tian

  • 1Department of Chemistry, Shanghai 200433, P. R. China.

Lab on a Chip
|December 1, 2004
PubMed
Summary
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Poly(dimethylsiloxane) (PDMS) microfluidic enzymatic-reactors were developed for efficient on-line protein analysis. These devices offer rapid digestion and high reusability, improving protein analysis workflows.

Area of Science:

  • Biochemical Engineering
  • Analytical Chemistry
  • Materials Science

Background:

  • Microfluidic devices offer advantages for biochemical analyses due to their small sample volumes and rapid reaction times.
  • Enzymatic reactors are crucial for protein digestion in proteomics workflows, but efficient immobilization and stability remain challenges.

Purpose of the Study:

  • To design and characterize two types of poly(dimethylsiloxane) (PDMS) microfluidic enzymatic-reactors.
  • To evaluate their analytical utility when coupled with MALDI TOF and ESI MS for on-line protein analysis.
  • To investigate methods for enzyme immobilization and reactor regeneration.

Main Methods:

  • Fabrication of PDMS microfluidic devices with integrated stainless steel tubing (SST) using a casting technique.

Related Experiment Videos

  • Surface modification of PDMS via ultraviolet graft polymerization of acrylic acid to introduce carboxyl groups.
  • Covalent immobilization of trypsin using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and physical immobilization using poly(diallyldimethylammonium chloride) (PDDA).
  • Characterization of immobilization success using contact angle, FTIR, AFM, and electropherograms.
  • On-line protein digestion and analysis coupled to mass spectrometry (MALDI TOF and ESI MS).

    • Main Results:

    • Successfully fabricated PDMS microfluidic enzymatic-reactors with distinct trypsin immobilization strategies.
    • Demonstrated excellent protein digestion efficiency at fast flow rates (3.5 µL/min for EDC/NHS, 0.8 µL/min for PDDA) with short residence times (5 s and 20 s).
    • Devices exhibited low memory effects, high reusability (≥50 runs), and retained activity over a week.
    • Regeneration of the PDDA-modified device was achieved with a simple HCl treatment.

    Conclusions:

    • The developed PDMS microfluidic enzymatic-reactors are highly effective for on-line protein analysis.
    • The reactors offer rapid digestion, excellent stability, and reusability, addressing key requirements for microfluidic protein analysis.
    • These devices represent a significant advancement for high-throughput proteomics and biochemical assays.