Quantitative antibody immobilization using hetero-and homo-bifunctional crosslinkers for analytical biosensing

Advances in various scientific fields are providing biosensing developers the capabilities to detect analytes at picomolar concentrations and lower. In optical biosensing, particularly in immunoassays like ELISA, there is a benefit to increasing and amplifying a series of reactions that includes antibodies, antigens, and various other crosslinkers. Here, we quantitatively analyze the effects of various polymer crosslinking combinations on their ability to immobilize antibody on poly(methyl methacrylate) (PMMA) in a sandwich-type immunoassay. PMMA activation with sodium hydroxide and oxygen plasma treatment are quantitatively compared with for surface carboxyl group density and hydrophilicity. Several hetero- and homo-bifunctional crosslinkers of different molecular weight, including polyethyleneimines (PEI) and poly(ethylene glycols) (PEG), as well as glutaraldehyde (GA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide with sulpho-N-hydroxysulfosuccinimide (EDC/sNHS), are quantitatively assessed for their capabilities to capture antibody using fluorescence microscopy. Our findings indicate sodium hydroxide is preferred over oxygen plasma treatement and a sodium hydroxide treatment of 30 minutes maximizes carboxyl groups while minimizing oxygen radical formation. Further, a PEG (irrespective of molecular weight) and GA crosslinking combination is optimal for immobilizing antibody at a mximum density of 8ng/cm².