Single-nucleotide polymorphism genotyping using a novel multiplexed electrochemical biosensor with nonfouling surface.

A novel electrochemical DNA biosensor for single-nucleotide polymorphism (SNP) analysis was developed. In this work, an oligonucleotide-incorporated nonfouling surface (ONS) was constructed to resist nonspecific absorption. The biosensor was developed using a 16-electrode array for high-throughput SNP analysis. The proposed strategy was primarily based on specific oligonucleotide ligation. Fully matched target DNA templated the ligation between a capture probe assembled on gold electrodes and a tandem signal probe with a biotin moiety that could capture avidin-horseradish peroxidase and sequentially generate a catalysed amperometric signal. A pre-core mutation in the hepatitis B virus (HBV) genome at G1896A and two adjacent polymorphisms in the human CYP2C19 genome at C680T and G681A were analysed. Polymerase chain reaction (PCR) products were used as real-life samples and analysed. Our results showed that 10% of a single-mismatched mutant gene was clearly distinguished with a current signal 16 times higher than that of the blank sample, demonstrating the selectivity and practicability of the multiplexed electrochemical DNA biosensor.