# US 9,435,794 B2 · Luminescent polymer cyclic amplification > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,435,794 B2 - Document ID: US9435794B2 - Status: Granted - Kind: B2 - Application number: 14/165,381 - Filed: 2014-01-27 - Published: 2015-10-01 - Granted: 2016-09-06 - Earliest claimed: 2011-04-20 - Assignees: Access Medical Systems, Ltd. - Applicants: Access Medical Systems, LTD. - Inventors: Robert F. Zuk; Hong Tan - Web page: https://patentgrep.com/patent/US9435794B2 ## CPC - G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding} - G01N 21/76: Chemiluminescence; Bioluminescence - G01N 27/3277: {being a redox reaction, e.g. detection by cyclic voltammetry (voltammetry per se G01N27/42, G01N27/48)} - G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding} - G01N 33/533: with fluorescent label - G01N 33/5438: {Electrodes} - G01N 33/553: Metal or metal coated - G01N 2021/757: {using immobilised reagents} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention is directed to a luminescent immunoassay method for detecting an analyte in a liquid sample with high sensitivity. The invention provides a unique combination of (i) using a probe having a small sensing surface area for binding analyte molecules, (ii) using a high molecular weight branched polymer conjugated with multiple binding molecules and multiple luminescent labels, and (iii) cycling the probe having immunocomplex formed back to the reagent vessel and amplification vessel 1-10 times and repeating the reaction with the reagent and the amplification polymer, to improve the sensitivity of detection level. For each cycling, the luminescent signal is increased significantly over the noise. ## Claims ### Claim 1 A method of detecting an analyte in a liquid sample, comprising the steps of: (a) obtaining a probe having a first antibody immobilized on the tip of the probe, wherein the diameter of the tip surface is ≦5 mm; (b) dipping the probe tip into a sample vessel containing a sample solution to bind an analyte, if present, in the sample solution to the first antibody on the probe tip; (c) dipping the probe tip into a reagent vessel containing a reagent solution comprising a reagent of a second antibody conjugated with a first member of a binding pair to bind the reagent to the analyte; (d) dipping the probe tip into a first wash vessel containing a first wash solution to wash the probe tip; (e) dipping the probe tip into an amplification vessel containing an amplification solution comprising a polymer conjugated with at least 5 molecules of second member of the binding pair and at least 25 luminescent labels, to form an immunocomplex comprising the analyte, the first antibody, the second antibody, the first and the second members of the binding pair, the polymer, and the luminescent labels on the probe tip, wherein the polymer has a molecular weight of at least 1 million daltons, and each of the luminescent labels has a molecular weight of less than 2,000 Daltons; (f) dipping the probe tip into a second wash vessel containing a second wash solution to wash the probe tip; (g) repeating steps (c) to (f) 1-10 times in the same reagent vessel and the same amplification vessel, and (h) detecting the immunocomplex formed by measuring the luminescent signal on the probe tip; wherein the first antibody and the second antibody are antibodies against the analyte; wherein the binding pair is a hapten and its antibody, a ligand and its receptor, complementary strands of nucleic acids, or lectin and carbohydrates. ### Claim 2 (depends on 1) The method according to claim 1, wherein the tip surface is ≦about 2 mm. ### Claim 3 (depends on 1) The method according to claim 1, wherein the binding pair is fluorescein and anti-fluorescein, digioxigenin/anti-digioxigenin, or DNP-anti-DNP. ### Claim 4 (depends on 1) The method according to claim 1, wherein the polymer is a polysaccharide, a polynucleotide, a dendrimer, a polyol, or polyethylene glycol. ### Claim 5 (depends on 1) The method according to claim 1, wherein the polymer is crosslinked copolymers of sucrose and epichlorohydrin. ### Claim 6 (depends on 1) The method according to claim 1, wherein the luminescent label is a fluorescent dye selected from the group consisting of: cyanine, coumarin, xanthene and a derivative thereof. ### Claim 7 (depends on 5) The method according to claim 5, wherein the luminescent label is a fluorescent dye selected from the group consisting of: cyanine, coumarin, xanthene and a derivative thereof. ### Claim 8 (depends on 1) The method according to claim 1, wherein the luminescent label is a chemiluminescent molecule selected from the group consisting of: ruthenium(II)tris-bipyridine, luminol, acridinium ester, and hemin. ### Claim 9 (depends on 1) The method according to claim 1, wherein the polymer is conjugated with 5-50 second member of the binding pair and 25-100 luminescent labels. ### Claim 10 (depends on 1) The method according to claim 1, wherein the polymer is crosslinked copolymers of sucrose and epichlorohydrin and is conjugated with 20-30 molecules of said second member of the binding pair and 40-90 Cy 5. ### Claim 11 (depends on 1) The method according to claim 1, wherein steps (c) to (f) are repeated 1-5 times. ### Claim 12 (depends on 1) The method according to claim 1, wherein steps (c) to (f) are repeated 2-3 times. ### Claim 13 (depends on 1) The method according to claim 1, wherein each binding step of steps (c) and (e) takes 30 seconds to 2 minutes. ### Claim 14 (depends on 1) The method according to claim 1, wherein the first member of the binding pair is biotin, and the second member of the binding pair is streptavidin. ### Claim 15 (depends on 1) The method according to claim 1, wherein each step (c) and (e) takes 20 seconds to 2 minutes. ### Claim 16 (depends on 1) The method according to claim 1, where in each step (b), (c) and (e), the solution in the vessel is agitated or mixed to accelerate the reaction. ### Claim 17 (depends on 16) The method according to claim 16, wherein a lateral flow of the solution across the probe tip is induced to accelerate the reaction.