Digital counting of single biomolecules: progress and perspectives with SERS

Abstract Single-molecule digital counting has become an essential analytical framework for ultrasensitive detection and absolute quantification of biomolecules in complex biological samples. This approach relies on isolating individual molecules into microscale reaction compartments, such as microdroplets or microchambers, thereby enabling direct enumeration of signal-positive compartments generated by target biomolecules. Fluorescence-based digital counting coupled to enzymatic reactions have achieved single-molecule sensitivity and have been widely applied to clinical diagnostics. However, their multiplexing capacity and molecular discrimination performance are fundamentally limited by spectral overlap. Recently, Raman scattering has emerged as a powerful alternative readout modality for single-molecule digital counting. In particular, integration of surface-enhanced Raman scattering (SERS) spectroscopy with metal nanoparticle–decorated microchamber arrays enables highly sensitive detection with narrow spectral signatures, substantially enhancing multiplexing capability and molecular discrimination. This review summarizes the principles of single-molecule digital counting, highlights recent advances enabled by SERS-based readouts, and discusses future perspectives for bioanalytical and diagnostic applications.