Capillary electrophoresis-based immobilized enzyme reactor using particle-packing technique

Area of Science:

• Analytical Chemistry
• Biochemistry
• Separation Science

Background:

• Capillary electrophoresis (CE) is a powerful separation technique.
• Immobilized enzyme reactors (IMERs) enhance CE applications for biochemical assays.
• Existing IMER fabrication methods can be complex or lack flexibility.

Purpose of the Study:

• To develop a novel, rapid, and simple method for fabricating CE-based IMERs.
• To evaluate the performance, reproducibility, and stability of the newly developed CE-IMERs.
• To demonstrate the application of these CE-IMERs for on-line enzyme assays and protein analysis.

Main Methods:

• Utilized a particle-packing technique with perfusive silica single particles as frits and large-pore beads as enzyme supports.
• Fabricated CE-IMERs with adjustable length and enzyme loading.
• Investigated performance using on-line trypsin digestion as a model reaction.
• Assessed reproducibility over 100 consecutive runs and enzyme stability over 10 days.
• Applied CE-IMERs for trypsin inhibition analysis and on-line digestion of standard proteins (myoglobin, BSA).

Main Results:

• Achieved high reproducibility for on-line enzyme assays (RSD < 4.1% for peak area, < 3.8% for migration time).
• Demonstrated remarkable enzyme stability, maintaining activity for at least 10 days.
• Successfully applied CE-IMERs for accurate trypsin inhibition analysis.
• Effectively performed on-line digestion of standard proteins like myoglobin and BSA.

Conclusions:

• The particle-packing technique offers a simple and rapid method for fabricating robust CE-IMERs.
• The developed CE-IMERs exhibit excellent reproducibility, stability, and performance for on-line enzymatic assays.
• This novel approach expands the utility of CE for on-line analysis, characterization of peptides and proteins, and enzyme inhibition studies.