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

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          Abstract

          The detection of single protein molecules 1, 2 in blood could help identify many new diagnostic protein markers. We report an approach for detecting hundreds to thousands of individual protein molecules simultaneously that enables the detection of very low concentrations of proteins. Proteins are captured on microscopic beads and labeled with an enzyme, such that each bead has either one or zero enzyme-labeled proteins. By isolating these beads in arrays of 50-femtoliter reaction chambers, single proteins can be detected by fluorescence imaging. By singulating molecules in these arrays, ~10–20 enzymes can be detected in 100 μL (~10 −19 M). Single molecule enzyme-linked immunosorbent assays (digital ELISA) based on singulation of enzyme labels enabled the detection of clinically-relevant proteins in serum at concentrations (<10 −15 M) much lower than conventional ELISA 3- 5. Digital ELISA detected prostate specific antigen in all tested sera from patients who had undergone radical prostatectomy, down to 14 fg/mL (0.4 fM).

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          Most cited references27

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          Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins.

          An ultrasensitive method for detecting protein analytes has been developed. The system relies on magnetic microparticle probes with antibodies that specifically bind a target of interest [prostate-specific antigen (PSA) in this case] and nanoparticle probes that are encoded with DNA that is unique to the protein target of interest and antibodies that can sandwich the target captured by the microparticle probes. Magnetic separation of the complexed probes and target followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows the determination of the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe. Because the nanoparticle probe carries with it a large number of oligonucleotides per protein binding event, there is substantial amplification and PSA can be detected at 30 attomolar concentration. Alternatively, a polymerase chain reaction on the oligonucleotide bar codes can boost the sensitivity to 3 attomolar. Comparable clinically accepted conventional assays for detecting the same target have sensitivity limits of approximately 3 picomdar, six orders of magnitude less sensitive than what is observed with this method.
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            Label-free, single-molecule detection with optical microcavities.

            Current single-molecule detection techniques require labeling the target molecule. We report a highly specific and sensitive optical sensor based on an ultrahigh quality (Q) factor (Q > 10(8)) whispering-gallery microcavity. The silica surface is functionalized to bind the target molecule; binding is detected by a resonant wavelength shift. Single-molecule detection is confirmed by observation of single-molecule binding events that shift the resonant frequency, as well as by the statistics for these shifts over many binding events. These shifts result from a thermo-optic mechanism. Additionally, label-free, single-molecule detection of interleukin-2 was demonstrated in serum. These experiments demonstrate a dynamic range of 10(12) in concentration, establishing the microcavity as a sensitive and versatile detector.
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              Drivers of biodiagnostic development.

              The promise of point-of-care medical diagnostics - tests that can be carried out at the site of patient care - is enormous, bringing the benefits of fast and reliable testing and allowing rapid decisions on the course of treatment to be made. To this end, much innovation is occurring in technologies for use in biodiagnostic tests. Assays based on nanomaterials, for example, are now beginning to make the transition from the laboratory to the clinic. But the potential for such assays to become part of routine medical testing depends on many scientific factors, including sensitivity, selectivity and versatility, as well as technological, financial and policy factors.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nature biotechnology
                1087-0156
                1546-1696
                24 May 2010
                23 May 2010
                June 2010
                1 December 2010
                : 28
                : 6
                : 595-599
                Affiliations
                [1 ]Quanterix Corporation, One Kendall Square, Suite B14201, Cambridge, MA 02139, USA
                [2 ]Department of Chemistry, Tufts University, Medford, MA 02155, USA
                Author notes
                Correspondence should be addressed to D.C.D. ( dduffy@ 123456quanterix.com )
                [*]

                These authors contributed equally to this work.

                Article
                nihpa200929
                10.1038/nbt.1641
                2919230
                20495550
                a9d73e71-dc53-45e4-8373-84cb2a2549ab
                History
                Funding
                Funded by: National Cancer Institute : NCI
                Award ID: R43 CA133987-02 ||CA
                Funded by: National Cancer Institute : NCI
                Award ID: R43 CA133987-01 ||CA
                Categories
                Article

                Biotechnology
                Biotechnology

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