Advances in optical counting and imaging of micro/nano single-entity reactors for biomolecular analysis

Ultrasensitive detection of biomarkers is of paramount importance in various fields. Superior to the conventional ensemble measurement-based assays, single-entity assays, especially single-entity detection-based digital assays, not only can reach ultrahigh sensitivity, but also possess the potential to examine the heterogeneities among the individual target molecules within a population. In this review, we summarized the current biomolecular analysis methods that based on optical counting and imaging of the micro/nano-sized single entities that act as the individual reactors (e.g., micro-/nanoparticles, microemulsions, and microwells). We categorize the corresponding techniques as analog and digital single-entity assays and provide detailed information such as the design principles, the analytical performance, and their implementation in biomarker analysis in this work. We have also set critical comments on each technique from these aspects. At last, we reflect on the advantages and limitations of the optical single-entity counting and imaging methods for biomolecular assay and highlight future opportunities in this field.