High sensitive detection of C-reactive protein by total internal reflection fluorescence microscopy on rapidly making nanoarray protein chip

This study investigated a method for the high sensitivity detection and quantification of the C-reactive protein (CRP) in human serum using total internal reflection fluorescence microscopy (TIRFM) on a rapidly made nanoarray protein chip. The nanoarray biotin-probe was patterned onto 3-mercaptopropyl trimethoxysilane-coated cover glass with a spot diameter of ∼400 nm within 1 min using a NanoeNabler™-based surface patterning tool. The unlabeled CRP molecules were detected in human sera using TIRFM, based on a sandwich fluorescence immunoassay. The linear regression for standard CRP in the range of 50 zM–1 fM was determined using the equation y = 0.437x + 84.991 (R = 0.9993). This proposed method was ∼2000 times faster than conventional atomic force microscopy based dip-pen nanolithography in terms of the chip manufacturing process. Additionally this method was 6 × 106 times more sensitive than enzyme-linked immunosorbent assay and exhibited a wide dynamic linear range (50 zM–1 fM).