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

Abstract

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.

Recent Developments in Single-Entity Electrochemistry

“…Optical microscopy has proven an ideal tool for nondestructively and nonintrusively studying SEs in biological and material applications. − The diversity of optical techniques, each with strengths and limitations, allows for flexibility in experimental design, making optical approaches to study SEs highly versatile . Further, the wide-field nature and high spatiotemporal resolution of optical microscopy allows for multiple SE processes to be monitored simultaneously, allowing for statistical analysis that reveals heterogeneous distributions and behaviors not captured in ensemble studies. − In tandem with SE electrochemistry methods, optical microscopy can be used to provide new, complementary information that can be hidden in electrochemical measurements alone, providing greater insight. − In the simplest form of SE opto-electrochemistry, optical microscopy is used to monitor and/or provide real-time time feedback about electrochemical-driven reactions and processes .…”

“…Optical microscopy has proven an ideal tool for nondestructively and nonintrusively studying SEs in biological and material applications. 194 − 197 The diversity of optical techniques, each with strengths and limitations, allows for flexibility in experimental design, making optical approaches to study SEs highly versatile. 198 Further, the wide-field nature and high spatiotemporal resolution of optical microscopy allows for multiple SE processes to be monitored simultaneously, allowing for statistical analysis that reveals heterogeneous distributions and behaviors not captured in ensemble studies.…”

Spherical Nucleic Acid‐Amplified Digital Flow Cytometric Bead Assay for the Ultrasensitive Detection of Protein and Exosome†

“…In this respect, most of the conventional bulk measurement‐based analytical methods, namely, the analog signal readout assays, are not quite satisfactory. In the past decade, with the rapid development of fluorescence imaging/counting technologies, [ 5‐8 ] a collection of elegant digital bioassays that can detect biomarkers at the single‐molecule level have opened a new way towards the quantification of trace amount of biomarkers. [ 9‐13 ] Totally different from the traditional analytical methods, in a typical digital bioassay, the low abundance of target molecules is isolated into numerous sealed microreactors, ensuring that at most one single target molecule is located in one microreactor.…”

Confinement-guided ultrasensitive optical assay with artificial intelligence for disease diagnostics

“…The screened biomarker can be used for toward separation of different groups in a cohort study to permit disease early diagnostics, stratification, therapy monitoring, etc. [53][54][55][56] So far, plasmonic-and nonplasmonicmaterials have been widely used as optical labels in cooperation with confinement strategy for ultrasensitive assay development, which will be discussed in the following sections.…”