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A recent focus in clinical immunodiagnostics is to improve sensitivity of assays for rare circulating protein biomarkers. The ability to detect low concentrations of certain biomarkers translates to diagnosis of disease at an earlier stage, which can positively impact prognosis and disease management (1)(2)(3). In research (for example in the discovery and development of new biomarkers), targeted assays for individual proteins are often required before multiplexing strategies are developed. Cross-reactivity of antibodies often compromises assay sensitivity in multiplexed immunoassays(4). Ultrasensitive individual assays consume only small sample volumes, a characteristic that can be advantageous in the case of scarce archival clinical samples.The development of single-molecule detection approaches has provided new opportunities for improving immunoassay sensitivity and miniaturization (5). Fluorescence confocal microscopy with laser-induced excitation enables single-molecule detection in extremely small interrogation zones (on the order of femtoliters). By reducing interrogation zones to these dimensions and carefully tuning the timescales of the data acquisition and molecular flow rates, background interference is decreased and individual molecules are readily recorded as discrete fluorescence bursts above background(6). Counting these bursts, or molecular events, by applying fluorescence thresholds on the basis of the internally derived background noise generates experimental data whose precision is dictated by counting statistics. Hence, sample read times can be adjusted to achieve a desired counting precision. The combination of small interrogation volumes and short sample read times can also lead to rapid analysis.Despite the advantages of single-molecule detection and the development of single-molecule detection instruments, adaptation of this technology to quantification of serum or plasma analytes has only scarcely been reported in the literature [for example, (7)]. Building on our previous work(8), we constructed single-molecule immunoassays (SMIAs) that use immune capture of analytes on microparticles, tagging with biotinylated antibodies, and quantification of fluorophore-labeled …

Fluorescence Single-Molecule Counting Assays for High-Sensitivity Detection of Cytokines and Chemokines