Demonstration of a direct capture immunoaffinity separation for C-reactive protein using a capillary-based microfluidic device.

C-reactive protein (CRP), a biomarker of inflammation and cardiovascular disease (CVD) risk assessment, was selected as a model antigen to demonstrate a direct labeling/direct capture immunoaffinity separation. The miniaturized device for immunoaffinity chromatography was constructed from two syringe pumps, a gradient mixing microchip, micro-injector with 250nL capillary injection loop, a capillary column, and a diode laser-induced fluorescence detector fitted with a fused-silica capillary flow cell. Monoclonal anti-CRP was biotinylated and attached to 5.0mum streptavidin-coated silica beads to make the solid support for separation columns. CRP in simulated serum matrix was fluorescently labeled in a one-step reaction and directly injected onto the immunoaffinity capillary. The purified antigen was then eluted in an acid gradient and measured. The antibody binding of CRP was evaluated in two physiological buffers, phosphate buffered saline (PBS) and Dulbecco's PBS (DPBS). A quadratic calibration model produced % relative errors of -15.9 to 12.6 for CRP concentration levels ranging from 0.47 to 95.0mug/mL. The accuracy (% difference from nominal) and precision (% relative standard deviation) of replicate injections were within 17.0%. The limit of detection was 57.2ng/mL and chromatographic run times were less than 10min. The instrument design is simple, and potentially portable, while the assay procedure may be modified for other clinically relevant markers by changing the capture antibody.