Microfluidic based immunosensor for detection and purification of carbonylated proteins

Abstract

A microchip has been developed on the basis of immno-precipitation approach for fast and sensitive enrichment of low abundant carbonylated proteins. This microfluidic method could enrich molecular biomarkers, which could be further analyzed in the proteomic study of age-related diseases and therapeutic development. In this study, an immunoaffinity-based PDMS micro-device was designed, fabricated, and chemically modified to specifically trap DNP-labeled PTM proteins of low abundance from a complex protein mixture. Carbonylated protein is selected as a representative PTM protein to illustrate the wide application of this immuno-based microchip for other PTMs which could be readily labeled by different antibody groups. Surface characterization methods such as atomic force microscopy and fluorescence microscopy were used to evaluate the construction of glutaraldehyde- and antibody- terminated PDMS substrates in the device fabrication. Quantitative study was also applied to study the target protein capture and elution efficiency of the device. In a testing mixture consisting of smaller amount of test model-In Vitro oxidized cytochrome c and large blocking protein BSA, a high sensitivity and specificity for only carbonylated protein biomarkers was demonstrated using this on-chip immnuoaffinity based extraction/enrichment. For this highly dense 193-post arrays μ-chip, a low abundance of 159 ng of standard in vitro test model- cytochrome c was enriched at flow speed of 5 μL/min within 110 min. We demonstrated that this nascent micro-immunoprecipitation (μ-IP) method is capable for enrichment of biomarkers in protein post-translation modification related diseases and promise great advance in early disease detection.