On-chip enzymatic microreactor using trypsin-immobilized superparamagnetic nanoparticles for highly efficient...

Area of Science:

• Biochemistry and Analytical Chemistry
• Nanotechnology and Materials Science
• Proteomics and Mass Spectrometry

Background:

• Enzymatic microreactors are crucial for protein digestion in proteomics.
• Immobilization of enzymes on magnetic nanoparticles offers advantages in separation and reusability.
• Developing efficient and easily replaceable microreactor systems is essential for high-throughput proteomic analysis.

Purpose of the Study:

• To fabricate an easily replaceable microchip enzymatic microreactor.
• To immobilize trypsin onto superparamagnetic nanoparticles for on-chip protein digestion.
• To evaluate the performance of the microreactor for model proteins and complex biological samples.

Main Methods:

• Synthesis of amine-functionalized superparamagnetic nanoparticles (50 nm diameter).
• Functionalization of nanoparticles with aldehyde groups using glutaraldehyde.
• Immobilization of trypsin onto aldehyde-functionalized nanoparticles.
• Packing of nanoparticles onto a glass microchip using a magnetic field to form a microreactor.
• Protein digestion of cytochrome c, bovine serum albumin, and myoglobin, followed by mass spectrometry analysis.
• Application to a rat liver extract fraction for bottom-up proteomic analysis.

Main Results:

• Successful fabrication of a magnetic nanoparticle-based microchip enzymatic microreactor.
• High sequence coverage achieved for model proteins (cytochrome c: 83%, myoglobin: 79%, albumin: 43%).
• Complete protein digestion in 10 seconds at a flow rate of 5 µL/min.
• Demonstrated reusability of the packing bed (at least five times) and rapid replacement (<1 min).
• Identification of six proteins from a rat liver extract fraction, showcasing applicability in complex samples.

Conclusions:

• The developed microchip enzymatic microreactor offers an efficient, rapid, and easily replaceable platform for proteolysis.
• The low-cost and reproducible preparation of magnetic nanoparticles presents significant potential for proteomic research.
• This system opens new possibilities for on-chip proteolysis and bottom-up proteomic analysis.