Chemical force titrations of antigen- and antibody-modified poly(methylmethacrylate)

Poly(methylmethacrylate) (PMMA) is a versatile polymer that displays desirable properties for development of cheap and disposable microfluidic devices for sensing biomolecular interactions. Atomic force microscopy (AFM) and chemical force titrations were used to determine the efficacy of surface modifications made to accommodate protein-substrate linkage. AFM images show the effects on surface morphology of carboxylated-, amine-, hCG antigen- and anti-hCG antibody-modified PMMA substrates. Confocal microscopy was used to determine the fluorescent intensity of labeled antibody species on the PMMA substrate, confirming the success of surface antigen/antibody immobilization. Surface pK(1/2) value for carboxylic acid and amine species grafted on PMMA were determined. When carboxylic acid or amine-terminated tips were titrated against PMMA samples terminated with the hCG antigen and anti-hCG antibody, peaks appeared in the force titration curve consistent with the pI range of the antigen or antibody species. Strong adhesive forces were present at pH values above 7.0 when the antigen was present on the PMMA substrate, and these were attributed to hydrophobic interactions between the antigen and the alkane "linker" chain attaching the amine or carboxylate group to the AFM tip. Such hydrophobic interactions were not observed with the carboxylic acid or amine/antibody combinations suggesting that the surface-linked antibody was more resistant to denaturation under higher pH. The results demonstrated the feasibility of using AFM approaches for interrogating protein grafting strategies in the fabrication of PMMA-based microsystems.