Ultraspecific analyte detection by direct kinetic fingerprinting of single molecules.

The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes.