ArticleProceedings of the National Academy of Sciences of the United States of America2020
Direct kinetic fingerprinting and digital counting of single protein molecules.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 62 citations in OpenAlex.
- Dynamic Single-Binding Event Profiling With on-Chip Microlenses for Wash-Free Digital Biosensing.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection.Research square · 2026Article
- FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection.bioRxiv : the preprint server for biology · 2026Article
- Single-Molecule Localization Microscopy of Membrane Proteins using Single-Antibody Labeling.Journal of visualized experiments : JoVE · 2026Article
- Discriminating single-molecule binding events from diffraction-limited fluorescence.Nature communications · 2025Article
- Single Molecule Kinetic Fingerprinting of Glycans on IgA1 Antibodies.Analytical chemistry · 2025Article
- Chromato-Kinetic Fingerprinting Enables Multiomic Digital Counting of Single Disease Biomarker Molecules.ACS nano · 2025Article
- Chromato-kinetic fingerprinting enables multiomic digital counting of single disease biomarker molecules.bioRxiv : the preprint server for biology · 2025Article
- How Highly Heterogeneous Sensors with Single-Molecule Resolution can Result in Robust Continuous Monitoring Over Long Time Spans.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- Unveiling nanoparticle-immune interactions: how super-resolution imaging illuminates the invisible.Nanoscale · 2025Review
- Ultrasensitive Amplification-Free Quantification of a Methyl CpG-Rich Cancer Biomarker by Single-Molecule Kinetic Fingerprinting.Analytical chemistry · 2024Article
- Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.bioRxiv : the preprint server for biology · 2024Article
- Carbon-nanotube field-effect transistors for resolving single-molecule aptamer-ligand binding kinetics.Nature nanotechnology · 2024Article
- Misfolded protein oligomers: mechanisms of formation, cytotoxic effects, and pharmacological approaches against protein misfolding diseases.Molecular neurodegeneration · 2024Review
- Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices.ACS nano · 2024Article
- Application of microfluidic technology based on surface-enhanced Raman scattering in cancer biomarker detection: A review.Journal of pharmaceutical analysis · 2023Review
- Single-molecule fluorescence methods for protein biomarker analysis.Analytical and bioanalytical chemistry · 2023Review
- High-Throughput Single-Molecule Sensors: How Can the Signals Be Analyzed in Real Time for Achieving Real-Time Continuous Biosensing?ACS sensors · 2023Article
- Superresolution Imaging with Single-Antibody Labeling.Bioconjugate chemistry · 2023Article
- Digital and Absolute Assays for Low Abundance Molecular Biomarkers.Accounts of chemical research · 2023Article
Corrections and comments
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Authors and funding
11 authors at 3 institutions in 2 countries.
Funding
Abstract
The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.