Method, system and devices for the assay and detection of biochemical molecules
First Claim
1. A colorimetric assay system which is designed to measure the presence or concentration of a biochemical analyte in a sample by a color change which is indicative of the presence or concentration of the analyte, wherein the system comprises,(a) an oxidase enzyme capable of oxidizing the analyte by removing electrons from the analyte without involving oxygen in the reaction, thereby yielding a reduced enzyme,(b) a non-chromogenic electron acceptor which is oxygen, having a half cell reduction potential greater than that of the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, and(c) a chromogen which is capable of accepting electrons from the reduced enzyme and changing color in a visible range upon reduction, wherein the change of color is less than one equivalent of color per equivalent of analyte oxidized and is indicative of the presence or the concentration of the analyte to be determined.
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Accused Products
Abstract
Oxygen-independent methods, systems and devices for the enzymatic colorimetric assay and detection of biochemical analytes. Two systems are described, both of which produce less than one equivalent of dye per equivalent of substrate, maintaining dye concentrations in the range where Beer'"'"'s law predicts a linear color-concentration relationship. One system produces an analog color signal from an analog analyte input, the other system produces a digital color signal from an analog analyte input.
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Citations
61 Claims
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1. A colorimetric assay system which is designed to measure the presence or concentration of a biochemical analyte in a sample by a color change which is indicative of the presence or concentration of the analyte, wherein the system comprises,
(a) an oxidase enzyme capable of oxidizing the analyte by removing electrons from the analyte without involving oxygen in the reaction, thereby yielding a reduced enzyme, (b) a non-chromogenic electron acceptor which is oxygen, having a half cell reduction potential greater than that of the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, and (c) a chromogen which is capable of accepting electrons from the reduced enzyme and changing color in a visible range upon reduction, wherein the change of color is less than one equivalent of color per equivalent of analyte oxidized and is indicative of the presence or the concentration of the analyte to be determined.
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5. A colorimetric assay system which is designed to measure the presence or concentration of a biochemical analyte by a color change which is determinative of the presence of concentration of the analyte, wherein the system comprises:
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(a) an oxidase enzyme capable of electron transferase activity with the analyte and capable of oxidizing the analyte by removing electrons from the analyte without involving oxygen in the reaction, thereby yielding a reduced enzyme, (b) a chromogen which is capable of accepting electrons derived from the reduced enzyme and is capable of changing color in a visible range upon reduction, the color change of the reduced chromogen being detectable and determinative of the presence or concentration of the analyte to be determined, and (c) a non-chromogenic electron acceptor which is not oxygen having a half cell potential greater than the reduced enzyme, which electron acceptor is capable of accepting electrons derived from the reduced enzyme in a functionally irreversible manner, and advantageously competes over any oxygen present to oxidize the reduced enzyme, and (d) an electron transfer agent which promotes the transfer of electrons from the reduced enzyme to the electron acceptor, the chromogen, or both. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 58, 59)
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51. A colorimetric assay system which is designed to measure the presence or concentration of an oxidase enzyme in a sample by a color change which is indicative of the presence or concentration of the oxidase, wherein the system comprises,
(a) a substrate which the oxidase is capable of oxidizing by removing electrons from the substrate without involving oxygen in the reaction, wherein the electrons are transferred to an electron acceptor, and wherein the electron acceptor is not oxygen, (b) a non-chromogenic electron acceptor which is not oxygen, having a half cell reduction potential greater than that of the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, (c) a chromogen which is capable of accepting electrons from the reduced enzyme and changing color in a visible range upon reduction, wherein the change of color is less than one equivalent of color per equivalent of substrate oxidized and is indicative of the presence or the concentration of the oxidase to be determined, and (d) an electron transfer agent which promotes the transfer of electrons from the reduced enzyme to the electron acceptor, the chromogen, or both the electron acceptor and the chromogen.
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52. A diagnostic colorimetric assay device which is designed to measure the concentration of a biochemical analyte in a sample by developing a color signal when the analyte is at or above a threshold concentration and by the absence of a color signal when the concentration of the analyte is below the threshold, wherein the device comprises:
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(a) physical support means, (b) an oxidase enzyme capable of oxidizing the analyte by removing electrons from the analyte without involving oxygen in the reaction, thereby yielding a reduced enzyme, (c) a non-chromogenic electron acceptor which is oxygen, having a half cell reduction potential greater than that of the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, (d) a chromogen which is capable of accepting electrons from the reduced enzyme and changing color in a visible range upon reduction, wherein the change of color is less than one equivalent of color per equivalent of analyte oxidized and is indicative of the concentration of the analyte to be determined, and (e) an electron transfer agent which promotes the transfer of electrons from the reduced enzyme to the electron acceptor, the chromogen, or both the electron acceptor and the chromogen. - View Dependent Claims (53, 54, 55)
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56. A method for measuring the presence or the concentration of a biochemical analyte in a sample by the development of a color change wherein the method comprises:
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(a) contacting the sample with an assay system comprising (i) an oxidase enzyme capable of oxidizing the analyte by removing electrons from the analyte without involving oxygen in the reaction, thereby yielding a reduced enzyme, (ii) a non-chromogenic electron acceptor which is oxygen, having a half cell reduction potential greater than that of the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, (iii) a chromogen which is capable of accepting electrons from the reduced enzyme and changing color in a visible range upon reduction, wherein the change of color is less than one equivalent of color per equivalent of analyte oxidized and is indicative of the concentration of the analyte to be determined, and (iv) an electron transfer agent which promotes the transfer of electrons from the reduced enzyme to the electron acceptor, the chromogen, or both the electron acceptor and the chromogen, (b) incubating to permit oxidation of the analyte, and (c) detecting a color change resulting from reduction of the chromogen, wherein a visible color change is indicative of the presence of the analyte or a concentration of the analyte above a predetermined threshold and wherein the absence of a visible color change is indicative of the absence of the analyte or a concentration of the analyte below a predetermined threshold.
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57. A method for measuring the presence or the concentration of an oxidase enzyme in a sample by the development of a color change wherein the color change is indicative of the presence or concentration of the oxidase, wherein the method comprises:
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(a) contacting the sample with an assay system comprising (i) a substrate which the oxidase is capable of oxidizing by removing electrons from the substrate without involving oxygen in the reaction, wherein the electrons are transferred to an electron acceptor, and wherein the electron acceptor is not oxygen, (ii) a non-chromogenic electron acceptor which is not oxygen, having a half cell reduction potential greater than that of the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, (iii) a chromogen which is capable of accepting electrons from the reduced enzyme and changing color in a visible range upon reduction, wherein the change of color is less than one equivalent of color per equivalent of substrate oxidized and is indicative of the concentration of the substrate to be determined, and (iv) an electron transfer agent which promotes the transfer of electrons from the reduced enzyme to the electron acceptor, the chromogen, or both the electron acceptor and the chromogen, (b) incubating to permit the oxidation of the substrate, and (c) detecting a color change resulting from reduction of the chromogen, wherein a visible color change is indicative of the presence of the oxidase or a concentration of the oxidase above a predetermined threshold and wherein the absence of a visible color change is indicative of the absence of the oxidase or a concentration of the oxidase below a predetermined threshold.
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60. A colorimetric assay system which is designed to measure the presence or concentration of a biochemical analyte in a sample by a color change which is determinative of the presence or concentration of the analyte, which system comprises:
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(a) a heme- or copper-containing protein capable of oxidizing the analyte by removing electrons from the analyte without involving oxygen in the reaction thereby yielding a reduced protein, (b) a chromogen which is capable of accepting electrons from the reduced protein and is capable of changing color in a visible range upon reduction, the change of the reduced chromogen being detectable and determinative of the presence or concentration of the analyte to be determined, and (c) a non-chromogenic electron acceptor which is not oxygen, having a half cell potential greater than the reduced enzyme, wherein the electron acceptor is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, and (d) an electron transfer agent which promotes the transfer of electrons from the reduced enzyme to the electron acceptor, the chromogen, or both. - View Dependent Claims (61)
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Specification