Detection of single nucleotide polymorphism using tension-dependent stochastic behavior of a single-molecule template

Single nucleotide polymorphism (SNP) is the most common genetic variation among individuals. The association of SNP with individual's response to pathogens, phenotypic variations, and gene functions emphasizes the importance of sensitive and reliable SNP detection for biomedical diagnosis and therapy. To increase sensitivity, most approaches employ amplification steps, such as PCR, to generate detectable signals that are usually ensemble-averaged. Introduction of amplification steps increases the complexity of a system, whereas ensemble averaging of signals often suffers from background interference. Here, we have exploited the stochastic behavior of a single-molecule probe to recognize SNP sequence in a microfluidic platform using a laser-tweezers instrument. The detection relies on on-off mechanical signals that provide little background interference and high specificity between wild type and SNP sequences. The microfluidic setting allows multiplex sensing and in situ recycling of the SNP probe. As a proof-of-concept, we have detected as low as 100 pM of an SNP target associated with coronary heart diseases within half an hour without any amplification steps. The mechanical signal permits the detection of single mutations involving either G/C or A/T pairs. We anticipate this system has the capacity to function as a highly sensitive generic biosensor after incorporation of a specific recognition element, such as an aptamer for example.