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Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations


The ability to detect single protein molecules1,2 in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as 10–20 enzyme-labeled complexes in 100 μl of sample (10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA3,4,5. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

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Figure 1: Digital ELISA based on arrays of femtoliter-sized wells.
Figure 2: Digitization of enzyme-linked complexes greatly increases sensitivity compared with bulk, ensemble measurements.
Figure 3: Subfemtomolar detection of proteins in serum using digital ELISA.
Figure 4: Digital detection of prostate-specific antigen (PSA) in serum samples of patients who had undergone radical prostatectomy.


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The project described was supported by Award Number R43CA133987 from the National Cancer Institute.

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Authors and Affiliations



D.M.R., C.W.K., D.R.F., D.R.W. and D.C.D. conceived the approach. D.R.F. built the imaging system. D.M.R., C.W.K., T.G.C., S.C.H., L.S., P.P.P., A.J.R., E.P.F., J.D.R. and G.K.P. conducted the experiments. T.P. wrote the image analysis software. L.C. prepared reagents. D.M.R. and D.C.D. wrote the manuscript. All authors were involved in designing experiments, reviewing and discussing data, and commenting on the manuscript.

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Correspondence to David C Duffy.

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Competing interests

All authors are employees or advisors of Quanterix Corporation who have a minority ownership or ownership option position in the company.

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Supplementary Tables 1–3, Supplementary Figs. 1–3 and Supplementary Methods (PDF 250 kb)

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Rissin, D., Kan, C., Campbell, T. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 28, 595–599 (2010).

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