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

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.