Bioanalysis Using Surface-Enhanced Raman Spectroscopy: From the Perspective of Single Molecules.

Single-molecule surface-enhanced Raman spectroscopy (SM-SERS), with single-molecule sensitivity and fingerprint-like specificity, has become a rising tool for probing the physicochemical properties of biomolecules at a single-molecule level. Notably, SM-SERS is generally achieved by complex methodologies with the assistance of rationally designed substrates and single-molecule techniques, often accompanied by significant technical challenges. Given the persistent issues of reproducibility and reliability, this critical perspective does not aim to provide a comprehensive review but instead progresses from a selective overview of representative SM-SERS methods and features to a detailed discussion of its intrinsic and often overlooked limitations, including the long-existing signal intensity fluctuations (SIFs), sample degradation, and the emerging challenge of big data processing and analysis. It is aimed to provide a conceptual framework that advances both the fundamental understanding and practical application of SM-SERS in future research.