Real Time Detection of Lysozyme by Pulsed Streaming Potentials Using Polyclonal Antibodies Immobilized on a Renewable Nonfouling Surface Inside Plastic Microfluidic Channels

A composite surface was prepared on cyclic olefin copolymer (COC) microchannels by UV-photografting of polyethylene glycol acrylate (PEGA) and poly(acrylic acid) (PAA) films. A PEGA layer of globular particles with average thickness of 60 nm was formed after 15 min of polymerization. Real time monitoring by pulsed streaming potentials demonstrated the ability of the PEGA layer to inhibit the adhesion of five different nonspecific adsorbing proteins when compared with pristine COC. Roughness determined by atomic force microscopy (AFM) after PAA grafting on COC-PEGA at different UV illumination times suggests that PAA formation is initiated at the free space in between the PEGA particles. Carboxylic groups activated with N-hydroxysuccinimide and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide were used to bind anti-lysozyme polyclonal antibodies. The composite COC-PEGA-PAA-anti-lysozyme surface demonstrated its ability to detect lysozyme with a dynamic range between 140 and 860 nM. Linearity was maintained even when samples were spiked with 250 nM of cytochrome as interfering species. The equilibrium constant K-eq for the adsorption of Ly on COC-PEGA-PAA-anti-Ly was estimated to be 2.7 x 10(6) M-1, and its shows that this kinetic approach of monitoring the surface charge is also useful to estimate affinity interactions for proteins in label free fashion. The regeneration of the surface exhibited an average percentage of recovery of similar to 97% for each of six adsorption-regeneration cycles. This feature enables curve calibration on a single microfluidic chip because each point of the curve has a reproducible and renewable surface.