Development of electrochemical genetic diagnosis sensor with mismatch-specific endonuclease

Abstract

Electrochemical method has been a good analytical technique for a miniaturized and portable biosensor, contributing to an achievement of point-of-care testing (POCT) or disease diagnosis. Particularly, due to the fact that it does not require any complicated equipment and it generates numerical signal without computational data analysis, rapid, simple and cost-effective detection could be achieved with electrochemistry. In this study, the electrochemical detection methods for the genetic diagnosis including Korean-specific breast cancer and sexually transmitted diseases were described. A new electrochemical detection method of DNA mutation on a gold electrode surface was developed utilizing mismatch-specific DNA cleavage and amplified electrocatalytic signaling. After target samples were hybridized with biotin-modified capture probes immobilized on the electrode, the hybridized double-stranded DNA (dsDNA) was treated with Surveyor™ nuclease. With subsequent addition of glucose oxidase (GOX)-avidin conjugate, electrocatalytic current was generated when the dsDNA was perfectly matched. Successive addition of biotin-modified polyamidoamime dendrimer (generation 4) caused densely assembled GOX on the electrode surface generating highly enhanced electrocatalytic current. On the other hand, there was no significant electrocatalytic current observed when the dsDNA contained base-mismatched site because it was cleaved by the nuclease and removed from the electrode including biotin tag. Using the method described above, we could successfully detect the insertion, deletion, and displacement of single base in real BRCA1 gene samples, and the results exhibited highly selective detection of DNA mutation on the electrode surface. We also detected gene of STD pathogen, Chlamydia trachomatis, using artificially designed capture probe having base-mismatched site when it was hybridized with target strand. As a result, we could successfully detect the target PCR produc...