Electrochemical detection of enzyme labeled DNA based on disposable pencil graphite electrode

Electrochemical biosensor for the detection of DNA hybridization using the reduction signal of α-naphthol is described. A pencil graphite electrode was used as a working electrode. Capture probes were covalently attached on to the pencil graphite electrode surface (PGE) at the 5′ end amino group by using N-(dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent on to PGE. After capture probe immobilization on to PGE surface; probe was hybridized with complementary biotinylated oligonucleotide. Alkaline phosphatase labeled with extravidin (Ex-AP) binds to biotinylated hybrid via biotin-avidin interaction. α-Naphthyl phosphate (α-NAP) was added and the reaction between alkaline phosphatase (AP) and α-NAP was occurred consequently as a substrate of AP, α-NAP reduction signal was obtained from this reaction, at −0.100 V by using differential pulse voltammetry (DPV). Other experimental parameters were studied such as; optimizations of hybridization time, and the concentrations of capture probe, biotinylated oligonucleotide and enzyme.