Single-microchannel, multistep assay reports protein size and immunoaffinity.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Microfluidics

Background:

• Protein identification and sizing are crucial for understanding biological processes.
• Existing methods may lack integration or require complex sample preparation.
• Follistatin is a key glycoprotein regulating cell proliferation and differentiation.

Purpose of the Study:

• To introduce a fully integrated, single-channel assay for simultaneous protein identification and size determination.
• To demonstrate the utility of this assay (PLE-IA) for analyzing follistatin.
• To enhance protein detection sensitivity and efficiency.

Main Methods:

• Integration of pore limit electrophoresis (PLE) with heterogeneous immunoassay (IA) in a single microfluidic channel (PLE-IA).
• Immobilization of antibodies within a polyacrylamide PLE gradient gel via pore-limit exclusion.
• Electrophoretic introduction of target proteins and size ladders for simultaneous detection and separation.
• Utilized an electrophoretic continuous injection technique for improved detection limits.

Main Results:

• The PLE-IA assay successfully identified and sized follistatin, with molecular weight measurements aligning with reported values.
• Achieved high separation resolution (SR=1) with a peak capacity of 18 in 10 minutes within a 10 mm separation distance.
• Demonstrated a >50-fold decrease in detection limit for follistatin (0.078 nM) using continuous injection.
• Antibody immobilization strategy proved straightforward and customizable without chemical functionalization.

Conclusions:

• The integrated PLE-IA assay is a powerful tool for simultaneous protein identity and size analysis.
• This method offers high efficiency, sensitivity, and straightforward customization.
• PLE-IA holds significant potential for applications in cell signaling studies, including aging and regeneration research.