Quenching effect on gold nano-patterned cardiac troponin I chip by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy (TIRFM) was investigated for enhanced detection sensitivity of cardiac troponin I (cTnI) on a gold nano-patterned protein chip. A 4×5 nanoarray patterning of a gold chip based on a single-molecule sandwich immunoassay was prepared with a spot diameter of 500 nm on glass substrates formed via electron-beam nanolithography. Quantitative trace analysis was possible as a function of increasing total internal reflection fluorescence intensity in the range of 84 aM–350 pM at prism-type and 35 aM–350 pM at objective-type TIRFM. Although using same gold nano-patterned cTnI chips, however, the objective-type TIRFM showed enhanced detection sensitivities 2.4 times better than the prism-type due to weaker gold quenching. This result is also about 7000 times lower in the detection limit compared with those obtained by conventional commercial sandwich enzyme-linked immunosorbent assay techniques.