Ultra-sensitive detection of tumor necrosis factor-alpha on gold nano-patterned protein chip formed via E-beam nanolithography by total internal reflection fluorescence microscopy.

We detected "tumor necrosis factor-alpha (TNF-alpha)" on a gold nanoarray protein chip at the attomolar (aM) concentration level (x10(-18)) by total internal reflection fluorescence microscopy (TIRFM). The 4 x 5 nanoarray patterning of gold with a spot diameter of 500 nm was successfully achieved on 10-mm square glass substrates using an electron beam evaporator. The dithiobis(succinimidyl propionate) as a protein linker and the Protein A/G to help oriented immobilization of antibody were used. The interactions of individual protein molecules were detected based on the sandwich fluorescence immunoassay by TIRFM. The linear regression equation for TNF-alpha in the range of 13 aM-130 fM was determined to be y = 6687.8x +126133 (R = 0.9938). The detection limit was 1.3 aM (S/N = 3). These results show that TNF-alpha as a tumor marker protein molecule was applied to gold nano-patterned protein chip.