A flash signal amplification approach for ultrasensitive and rapid detection of single nucleotide polymorphisms in tuberculosis

In recent years, the demand for rapid, sensitive, and simple methods for diagnosing deoxyribonucleic acid (DNA) has grown due to the increase in the variation of infectious diseases. This work aimed to develop a flash signal amplification method coupled with electrochemical detection for polymerase chain reaction (PCR)-free tuberculosis (TB) molecular diagnosis. We exploited the slightly miscible properties of butanol and water to instantly concentrate a capture probe DNA, a single-stranded mismatch DNA, and gold nanoparticles (AuNPs) to a small volume to reduce the diffusion and reaction time in the solution. In addition, the electrochemical signal was enhanced once two strands of DNA were hybridized and bound to the surface of the gold nanoparticle at an ultra-high density. To eliminate non-specific adsorption and identify mismatched DNA, the self-assembled monolayers (SAMs) and Muts proteins were sequentially modified on the working electrode. This sensitive and specific approach can detect as low as attomolar levels of DNA targets (18 aM) and is successfully applied to detecting tuberculosis-associated single nucleotide polymorphisms (SNPs) in synovial fluid. More importantly, as this biosensing strategy can amplify the signal in only a few seconds, it possesses a great potential for point-of-care and molecular diagnosis applications.