Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification

Area of Science:

• Biochemistry
• Analytical Chemistry
• Materials Science

Background:

• Protein digestion is crucial for peptide analysis.
• Existing methods can be time-consuming and lack sensitivity for low-abundance proteins.
• Microfluidic devices offer potential for enhanced analytical performance.

Purpose of the Study:

• To develop a microchip reactor for fast and sensitive protein digestion.
• To immobilize enzymes effectively within a microfluidic system.
• To improve peptide analysis through enhanced proteolytic efficiency.

Main Methods:

• Layer-by-layer assembly of chitosan (CS) and hyaluronic acid (HA) on a poly(ethylene terephthalate) (PET) microfluidic chip.
• Characterization of multilayer assembly using AFM, ATR-IR, and contact angle measurements.
• Enzyme immobilization and activity assay of adsorbed trypsin using quartz crystal microbalance.

Main Results:

• A biocompatible network for enzyme immobilization was successfully created on the microchip.
• Adsorbed trypsin exhibited significantly higher proteolytic velocity (approx. 600 mM/min µg) compared to solution-phase trypsin.
• Sensitive protein identification (low femtomole level) was achieved with digestion times under 5 seconds.

Conclusions:

• The developed microchip reactor provides a highly efficient platform for rapid protein digestion.
• This technique demonstrates potential for sensitive analysis of low-level proteins.
• The layer-by-layer approach offers a promising strategy for enzyme immobilization in microfluidic devices.