Electrochemical DNA sensor using genetically engineered thermostable pyrroloquinoline quinone glucose dehydrogenase

Genetically engineered thermostable pyrroquinoline quinone glucose dehydrogenase (S415CGDH) was used for labeling probe DNA and amperometric DNA sensor was constructed and utilized for the detection of PCR amplified Salmonella virulence invA gene. The invA gene from Salmonella which accounts for many cases of food poisoning was targeted and the DNA bearing a specific sequence complementary to the invA gene was immobilized onto an Au electrode as a capture DNA. S415CGDH labeled probe DNA was hybridized with the immobilized DNA at 60degreesC for 10 min and then the resulting electric current generated from S415CGDH by glucose addition was measured. The electric current was obtained when S415CGDH was used for labeling probe DNA but not when the native enzyme was used. The sensor response increased with the addition of glucose and 4.0 x 10(-9) M of the S415CGDH labeled target DNA was detected in the presence of 29 mM glucose. The detection of PCR product was also investigated and it was successfully detected using asymmetric PCR product with sandwich method.