Isothermal detection of multiple point mutations by a surface plasmon resonance biosensor with Au nanoparticles enhanced surface-anchored rolling circle amplification

Abstract In this study, we developed a surface plasmon resonance (SPR) DNA biosensor method using surface-anchored rolling circle amplification (RCA) and Au nanoparticles modified probes (AuNPs) to isothermally detect multiple point mutations associated with drug-resistance in multidrug-resistant Mycobacterium Tuberculosis (MDRTB). A set of probes contains an allele-specific padlock probe (PLP), a capture probe and an AuNPs. The linear PLPs, circularized by ligation upon the recognition of the point mutation on DNA targets, hybridize to the capture probes via the specific tag/anti-tag recognition. Upon recognition each point mutation is identified by locating into the corresponding channel on the chip. Then the immobilized primer (capture probe)–template (circular PLP) complex are amplified isothermally as RCA and further amplified by AuNPs. The RCA products immobilized on the chip surface cause great SPR angle changes consequently. The 5 pM synthetic oligonucleotides and 8.2 pg uL −1 of genomic DNA from clinical samples can be detected by the method. The positive mutation detection is achieved with a wild-type to mutant ratio of 5000:1. The method was demonstrated by targeting five clinically meaningful mutations in MDRTB. Thirty clinical samples were identified and they were in good agreement with the results from sequencing.