Surface plasmon resonance biosensor for label-free and highly sensitive detection of point mutation using polymerization extension reaction.

A novel biosensing technique was developed for label-free and highly sensitive detection of point mutation using surface plasmon resonance (SPR) biosensor coupled with polymerization extension reaction. In this work, 3'-thiolated DNA probes with complementary sequences to target DNA were immobilized onto the sensor surface via molecular self-assembly. In the presence of wild target sequences, the primers can be selectively extended by DNA polymerase to form double-stranded DNA. In contrast, mutant target sequences, containing one mutation site mismatched with the 3'-end base of the primer, cannot be elongated. Thus, the extension reaction products can hybridize with the capture probes modified on the sensor surface to induce an SPR signal. The experimental results showed that the presented approach could detect the mutant sequences in BRCA1 gene related to inherited breast cancer, and the wild-type and mutant-type sequences were successfully discriminated. Using synthetic DNA sequences as targets, 100pM detection limits were achieved under the optimal reaction conditions. Hence, this highly sensitive and specific assay might have the potential to become an efficient alternative technique for point mutation detection in biomedical research and clinical diagnosis.