Ultrasensitive biosensing with single-molecule/particle digital counting

The goal of developing sensing methods for biomolecular assay is to offer new tools for the discrimination of trace molecules embedded in complex and crowding biological surrounding with high sensitivity and specificity. Single-molecule detection provides the capability to resolve the small and rare differences between ostensibly identical samples which is hard to realize with ensemble measurements. Great efforts have been paid on exploiting single-molecule detection methods for early disease diagnosis. In this review, we summarize recent achievements in single-molecule detection with optical microscopy, including statistical single-molecule counting with total internal reflection fluorescence (TIRF), spectrum-resolved single-molecule/particle imaging for multiplex assay, and label-free scattering detection in an interference mode for virus or protein counting. The mechanism and advantages of these methods for biomolecule detection are described. The challenges and prospects of adopting optical microscopy for single-molecule detection are also discussed.