Gene mutation screening using single-molecule force spectroscopy: a novel way of PCR-independent genetic testing

Detection of systemic pathologies during their subclinical phases remains a global challenge, as current diagnostics often fail to correctly identify diseases in asymptomatic individuals. While conventional radiological imaging and histopathological tissue biopsies are considered clinical benchmarks, they are frequently hindered by ionizing radiation risks, invasiveness, and the inability to account for intratumoral heterogeneity. Consequently, the diagnostic landscape is shifting toward liquid biopsy, with salivary diagnostics emerging as a promising, non-invasive alternative that facilitates longitudinal surveillance and high patient compliance. Here, we propose that atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) could play a pivotal role in PCR-independent screening of genetic diseases like cancer at a subclinical stage using liquid biopsy samples. This is because SMFS is capable of discriminating between wild type and mutant nucleic acid sequences of pM-level concentrations that can be obtained from liquid biopsy samples and detected directly without PCR amplification. Sensing by SMFS is found to be precise since the signal transduction mode is based on mechanically induced dehybridization of nucleic acid duplexes, making it a "two-step" verification technique, as dehybridization cannot happen unless hybridization occurs in the first place. This reduces the chances of false-positive results that are often seen in techniques where just "one-step" hybridization transduces a signal. In this review, we discuss the advantages/disadvantages of different nucleic acid sensing techniques and compare to SMFS to assess the potential of SMFS to make its way into the translational healthcare system, especially when synthetic DNA analogues like locked nucleic acid (LNA) are employed as the capture probes.