Single-molecule detection for femtomolar quantification of proteins in heterogeneous immunoassays.

There is growing interest in the development of ultrasensitive immunoassays to facilitate validation of novel biomarkers, especially serum biomarkers, and the application of those assays in clinical settings (1). Current assays focus predominantly on abundantly produced protein biomarkers, such as cardiovascular structural proteins that leak into the bloodstream, and less so on lower abundance cytokines and chemokines (2). Even rarer markers, such as those released from cancerous cells into the bloodstream, remain more elusive (3). Approaches that exploit recent technological advances in molecular imaging, especially at the single-molecule level (4), are likely to improve the detection limits of the immunoassay, and therefore enable improved detection of these markers. Microfluidic sample delivery and confocal microscopic detection of labeled biomolecules are useful for detecting targets of interest (5)(6). Two-color correlation counting derived from focused laser-induced excitation allows for high signal-to-noise detection even in the presence of large excesses of free probes, enabling measurement of single molecular events (6). Flow velocity and data acquisition rates can be matched to provide highly accurate quantification, even for rapid analyses on the order of seconds per sample, and analysis time can easily be extended to provide increased detection efficiency of rare targets. Development and application of new assays are straightforward processes based on well-understood fluorescence methods. We describe application of the Trilogy® 2020 Single Molecule Analyzer for analysis of heterogeneous immunoassays. The human gonadotropin follicle-stimulating hormone (FSH), which plays a central role in fertility (7), was chosen as a representative analyte because of its clinical importance and current clinical requirements for high-sensitivity detection (8). The Trilogy 2020 instrument uses laser beams shaped into stripes for fluorescence illumination and focuses the beams through a microscope objective into the center of a sample-delivering microcapillary (see the Data Supplement that accompanies the online version of this …