Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

Abstract

The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.

Effect of Particle Size and Surface Chemistry of Photon‐Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

“…[25] Furthermore, the absence of optical background interferences enables the detection and counting of individual UCNP labels using wide-field optical microscopy. [19,26] This has led to the development of single-molecule (digital) immunoassays, [27] as opposed to analog immunoassays where the integrated signal generated by thousands of labels is measured. It is essential that the UCNP labels have the right size and are bright enough to be reliably detectable (and countable) at the single-nanoparticle level.…”

Single-Particle Counting Based on Digital Plasmonic Nanobubble Detection for Rapid and Ultrasensitive Diagnostics

“…Such paradigms prompt further innovations that develop various digital sensing platforms based on micro/nano-particles (5-7) . With its capability of examining individual particles’ changes upon recognizing target molecules, single-particle detection holds great potential to simplify digital assays (8-10) . Examples of single-particle digital assays include bright/dark-field imaging (11) , interferometric (12) or fluorescent imaging (13, 14) , surface-enhanced Raman scattering (15) , surface plasmon resonance microscopy imaging (16) , and particle mobility tracking (17-19) .…”

Nanoparticle‐Based Biosensing: En Route to Ultimate Sensitivity

“…In many critical situations, even a few toxin molecules can be harmful, a few microorganisms or viruses initiate infectious disease, and traces of cancer markers indicate the beginning of a malignant transformation. Thus, it is essential to develop new highly sensitive analytical methods that will reach as low detection limits as possible, ideally detecting individual analyte molecules [1] …”

“…Even though the currently used single‐molecule approaches are based on counting individual binding events, the detection limits are still typically in thousands of molecules. However, increasing the affinity of recognition molecules and decreasing the non‐specific binding might together lead to reaching the ultimate sensitivity – the ability to detect the individual molecule within the whole sample [1] …”