PLASMONIC BEADS FOR MULTIPLEXED ANALYSIS BY FLOW DETECTION SYSTEMS
First Claim
1. A method for detection of analytes in a sample, comprising:
- (a) contacting the sample with a population of microparticles in suspension, said microparticles comprising a plasmonically active surface providing near-infrared (“
NIR”
) enhanced fluorescence, said fluorescence optionally provided by gold nano-island covering a portion of said microparticles, said population of microparticles further comprising subpopulations comprising different analyte capture molecules bound to said plasmonically active surface;
(b) allowing said different analyte capture molecules to form complexes with different analytes that may be present in the sample;
(c) labeling complexes formed in step (b) with fluorescent labels;
(d) detecting labeled complexes by irradiating said fluorescent labels and sensing metal enhanced fluorescence from labeled complexes, whereby NIR enhanced fluorescence from said microparticles indicates detection of analytes; and
(e) distinguishing multiple analytes in the sample, if present, by different NIR enhanced fluorescence signals.
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Abstract
Disclosed are methods and assays for detection of low concentration analytes such as proteins in a sample, using beads. Specially coated beads allow for femtomolar sensitivity through strong near-infrared fluorescence enhancement on plasmonic beads having gold nanostructures in a coating. By selecting different bead sizes and labeling with different fluorophores of plasmonic beads for immobilization of different capture antibodies, multiplexed plasmonic beads can be used for simultaneous quantification of various markers down to 0.01 pg/mL sensitivity. Exemplified are human cytokine IL-6, IFN-gamma, IL-1 beta, VEGF and ovarian cancer biomarker CA-125. Using flow cytometry, a detection limit below that of glass bead based immunoassays by 2-3 orders of magnitude was achieved. The multiplexed plasmonic bead assay was used to simultaneously quantify cytokines and CA125 of ovarian cancer cell culture medium, demonstrating the potential of plasmonic bead based immunoassay for sensitive biological detection relevant to human diseases.
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Citations
36 Claims
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1. A method for detection of analytes in a sample, comprising:
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(a) contacting the sample with a population of microparticles in suspension, said microparticles comprising a plasmonically active surface providing near-infrared (“
NIR”
) enhanced fluorescence, said fluorescence optionally provided by gold nano-island covering a portion of said microparticles, said population of microparticles further comprising subpopulations comprising different analyte capture molecules bound to said plasmonically active surface;(b) allowing said different analyte capture molecules to form complexes with different analytes that may be present in the sample; (c) labeling complexes formed in step (b) with fluorescent labels; (d) detecting labeled complexes by irradiating said fluorescent labels and sensing metal enhanced fluorescence from labeled complexes, whereby NIR enhanced fluorescence from said microparticles indicates detection of analytes; and (e) distinguishing multiple analytes in the sample, if present, by different NIR enhanced fluorescence signals. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 29, 30, 31, 32, 33, 34, 35)
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- 13. A product comprising a population of beads wherein each bead comprises, on an outer surface thereof, gold islands separated by gaps of between 5 and 100 nm, and said gold islands have an area between either 1,000 and 2,500 nm2, or 25 and 250,000 nm2, said population of beads further having coupled thereto analyte capture molecules of at least two different specificities.
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28. A method of making a bead-based immunoassay product comprising a population of beads, comprising the steps of:
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(a) modifying beads having a material that is one of a silica-based, polymer-based, or magnetic material with an amine functionality (b) coupling the beads to a plasmonically active layer comprising gold islands separated by gaps of 5 and 100 nm and wherein the islands are between 1,000 and 2,500 nm2, or between 25 and 250,000 nm2, in area; (c) applying to the beads a functionality for coupling thereto a population of detection molecules; and (d) coupling detection molecules of different specificities to the beads as prepared in steps (a)-(c).
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Specification