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.
Lee
Kyuseo
K
Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, Jeonju 561-756, Korea.
Lee
Seungah
S
Yu
Hyunung
H
Kang
Seong Ho
SH
D002294:Carcinoma, Squamous Cell / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D009062:Mouth Neoplasms / Q000737:chemistry; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D013205:Staphylococcal Protein A; D014409:Tumor Necrosis Factor-alpha; D006046:Gold