Facile engineered polymeric microdevice via co-coupling of phenylboronic acid and Protein A for oriented antibody immobilization enables substantial signal enhancement for an enhanced fluorescence immunoassay

The powerful immunoassay has attracted increasing attention for detecting trace substances. Herein, based on the co-coupling of phenylboronic acid and Protein A for oriented antibodies immobilization, we proposed a polymeric device-based fluorescence immunoassay with enhanced assay performance for Alpha-fetoprotein (AFP) analysis. Unlike traditional physical absorption or chemical covalent conjugation to immobilize antibodies, in this immunoassay, the bioreactivity of AFP antibody was maximumly obtained owing to the oriented immobilization manner. The package density of AFP antibody on the polymethylmethacrylate (PMMA) surface is significantly improved because of the co-coupling case associated distance effect. In addition, the non-specific absorption is greatly inhibited with the combined using of zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) and bovine serum albumin (BSA). These integrated features facilitate the output of strong fluorescence in the presence of AFP but emit very weak background in the absence of AFP. After a typical sandwich immunoreaction, the recorded fluorescence intensity of the immunosensor is proportional to the AFP concentration, showing excellent sensitivity, specificity, and reliability for AFP determination. Comparative studies show the co-coupling is superior than its single coupling counterparts. We envision this unique immunoassay can provide a new avenue for building immunosensors in the fields of disease diagnosis, food safety detection, and environmental monitoring.

Automated summary

The study introduces a fluorescence immunoassay method based on co-coupling for detecting AFP. By immobilizing antibodies through co-coupling of phenylboronic acid and Protein A, the immunoassay achieves oriented antibody fixation. The recorded fluorescence intensity is proportional to AFP concentration after immunoreaction, demonstrating high sensitivity, specificity, and reliability.