Amperometric DNA sensor using gold electrode modified with polymerized mediator by layer-by-layer adsorption

An amperometric DNA sensing system is proposed based on the combination of sandwich hybridization of reporter probe, capture probe, and target DNA. InvA gene of Salmonella typhimurium was used for target DNA and glucose-6-phosphate dehydrogenase (G6PDH) was used for subsequent enzymatic electrochemical detection as reporter probe. DNA sensor was constructed as follows. At first, a gold electrode was modified with mercaptopropionic acid, then PEI-Fc (ferrocene immobilized polyethylenimine)/alginic acid, diaphorase/PEI, and PEI/streptavidin layers were formed on the surface of electrode by layer-by-layer adsorption. Finally, capture probe was immobilized on the electrode via streptavidin. Hybridization of target DNA and the both probe was carried at 56 °C. Hybridization product was immobilized on the DNA sensor surface by biotin-avidin bond. Electrochemical measurement was performed in the solution containing G6P as substrate and NAD+ as cofactor for enzyme reaction. The anodic current against glucose-6-phosphate was obtained. It indicates that reporter probe was immobilized on the electrode by hybridization with target DNA and G6PDH on the probe produced NADH. The detection limit of present DNA sensor was femto mol order of target DNA.