Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers

Abstract

Conspectus Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next-generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. The analysis of these kinetic fingerprints enables nearly perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with a limit of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions of as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay desi...

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology

“…While the detection of antibodies outperforms the classical RBD-IgG ELISA assay by the measured parameters (dynamic range, sensitivity, and throughput), the sensitivity of singlemolecule hybridizations falls short of amplification-based PCR reactions, which can reach a detection limit of 100-1000 copies of viral RNA per milliliter of transport media [26]. Higher sensitivity may be achieved by implementing single-molecule kinetic fingerprinting [15,27], to be explored in future studies. Furthermore, additional studies on larger cohorts, as well as systematical comparisons to additional available serological tests are needed.…”

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology

“…While the detection of antibodies outperforms the classical ELISA assay by the measured parameters (dynamic range, sensitivity, and throughput), the sensitivity of singlemolecule hybridizations falls short of amplification-based PCR reactions. Higher sensitivity may be achieved by implementing single-molecule kinetic fingerprinting (14,24), to be explored in future studies.…”

“…While the detection of antibodies outperforms the classical RBD-IgG ELISA assay by the measured parameters (dynamic range, sensitivity, and throughput), the sensitivity of single-molecule hybridizations falls short of amplification-based PCR reactions, which can reach a detection limit of 100-1000 copies of viral RNA per milliliter of transport media [26]. Higher sensitivity may be achieved by implementing single-molecule kinetic fingerprinting [15, 27], to be explored in future studies. Furthermore, additional studies on larger cohorts, as well as systematical comparisons to additional available serological tests are needed.…”

Probing Biosensing Interfaces With Single Molecule Localization Microscopy (SMLM)

“…In SiMREPS, (A) using carefully designed molecular probes, specific interactions are associated with single molecule blinking signals that are homogeneous over time, while non-specific events are seen as heterogeneous blinking events. (B) Over time, better separation between specific and non-specific events are expected (Mandal et al, 2020 ). Copyright with permission from the American Chemical Society.…”