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US5036000A Expired / Lapsed US ⏱️ 0.0 years remaining

Threshold color control system

Assignee: Orasure Technologies Inc
•
Filing: 1987-07-20
•
Granted: 1991-07-30
•
Expires: 2007-07-20
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Abstract

Abstract A system for quantitative colorimetric analysis of biological fluids or organic compounds, including NAD(P)H, or a substrate of an enzyme which reacts with the formation or consumption of NAD(P)H. Concentrations of organic substrates for example alcohol, cholesterol, uric acid, in a biological fluid such as saliva, blood or urine may be determined. The system gives a digital reading of the organic material the concentration which is sought to be determined; the concentration of NAD(P)H is determined by a color change or color "signal" when the NAD(P)H is above a threshold concentration and by the absence of a color signal when the concentration of NAD(P)H is below the threshold concentration. The system includes a chromogen, an electron-accepting reactant which, until exhausted, prevents a visible color change due to accumulation of reduced chromogen, and a catalyst. The system is capable of measuring colorimetrically without dilution concentrations of organic compounds in biological fluids which previously could not be measured in such concentration.

Inventors

  • 👤 John L. Palmer
  • 👤 Marsha W. Timmerman

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-11-02 20:23
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/52; G01N33/521; C; C12; C12Q; C12Q1/00; C12Q1/008; Y; Y10; Y10S; Y10S436/00; Y10S436/904

Claims Analysis

Total Claims: 90
Claim 1
Independent Claim 166 words
A colorimetric assay system for measurement of the concentration of NAD(P)H, NAD(P), or an analyte in a sample, which analyte reacts to form or consume NAD(P)H, wherein the assay system comprises: a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant capable of accepting electrons from NAD(P)H, in an amount sufficient to increase the range of concentrations that can be measured by the color change of the chromogen when the non-chromogenic competing reactant is reduced, wherein the non-chromogenic competing reactant prevents a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, and an electron carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, wherein the change in color caused by the reduction of the chromogen occurs in a ratio of less than one molecule of dye for each molecule of NAD(P)H produced and is indicative of the concentration to be determined.
Claim 2
Dependent Claim 166 words
A colorimetric assay system for measurement of the concentration of NAD(P)H, NAD(P), or an analyte in a sample, which analyte reacts to form or consume NAD(P)H, wherein the assay system comprises: a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant capable of accepting electrons from NAD(P)H, in an amount sufficient to increase the range of concentrations that can be measured by the color change of the chromogen when the non-chromogenic competing reactant is reduced, wherein the non-chromogenic competing reactant prevents a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, and an electron carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, wherein the change in color caused by the reduction of the chromogen occurs in a ratio of less than one molecule of dye for each molecule of NAD(P)H produced and is indicative of the concentration to be determined.
Claim 3
Dependent Claim 29 words
The assay system of claim 1 wherein the non-chromogenic competing reactint is capable of accepting electrons directly from NAD(P)H, from reduced chromogen, or from both NAD(P)H and reduced chromogen.
Claim 4
Dependent Claim 18 words
The assay system of claim 1 wherein the non-chromogenic competing reactant is capable of oxidizing the reduced chromogen.
Claim 5
Dependent Claim 20 words
The assay system of claim 2 wherein the reactant accepts electrons directly from NAD(P)H and reacts preferentially with the NAD(P)H.
Claim 6
Dependent Claim 21 words
The assay system of claim 2 wherein the non-chromogenic competing reactant accepts electrons from both the reduced chromogen and the NAD(P)H.
Claim 7
Dependent Claim 23 words
The assay system of claim 1 wherein the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).
Claim 8
Dependent Claim 43 words
The assay system of claim 6 wherein the non-chromogenic competing reactant is selected from the group consisting of triethylenediamine chloride salt of Fe(III), Fe(III) citrate complex, Fe(III) EDTA complex, Fe(III) sorbitol complex, hexamino cobalt (III) chloride, potassium hexcacyanocobaltate (III) and sodium hexanitrocobaltate (III).
Claim 9
Dependent Claim 27 words
The assay system of claim 6 wherein the non-chromogenic competing reactant is selected from the group consisting of sodium potassium periodate, paranitrobenzyl disulfide, 2,2'-dithiobis-(pyride-N-oxide), and 2,2'-dithiobis(4-tert-butyl-1-isopropyl imidazole).
Claim 10
Dependent Claim 20 words
The assay system of claim 6 wherein the non-chromogenic competing reactant is a water soluble alkali metal salt of ferricyanide.
Claim 11
Dependent Claim 29 words
The assay system of claim 6 wherein the non-chromogenic competing reactant is a weak oxidizing agent selected from the group consisting of trichloro-melanine, hydrogen peroxide, and an organic peroxide.
Claim 12
Dependent Claim 15 words
The assay system of claim 9 wherein the salt is a sodium or potassium salt.
Claim 13
Dependent Claim 24 words
The assay system of claim 10 wherein the organic peroxide is selected from the group consisting of a lower alkyl peroxide and benzoyl perioxide.
Claim 14
Dependent Claim 22 words
The assay system of claim 1 wherein the catalyst is diaphorase and non-chromogenic competing reactant is not a substrate for the diaphorase.
Claim 15
Dependent Claim 14 words
The assay system of claim 1 wherein the catalyst is an organic polycyclic compound.
Claim 16
Dependent Claim 50 words
The assay system of claim 14 wherein the catalyst is an organic polycyclic compound selected from the group consisting of medola blue, phenazonium methosulfate, N-methyl-phenazonium methosulfate, methylene blue and anthraquinones substituted with a member selected from the group consisting of methyl-, chloro-, bromo-, amino-, hydroxy-, nitro-, methoxy and sulfonic acid.
Claim 17
Dependent Claim 20 words
The assay system of claim 1 which further comprises a metal salt which enhances the catalytic activity of the catalyst.
Claim 18
Dependent Claim 18 words
The assay system of claim 16 wherein the catalyst is an organic ligand complex of the metal salt.
Claim 19
Dependent Claim 11 words
The assay system of claim 7 wherein the catalyst is ferrocene.
Claim 20
Dependent Claim 13 words
The assay system of claim 1 wherein the chromogen is a tetrazolium salt.
Claim 21
Dependent Claim 21 words
The assay system of claim 1 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.
Claim 22
Dependent Claim 24 words
The assay system of claim 20 wherein the analyte is selected form the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.
Claim 23
Dependent Claim 28 words
The assay system of claim 21 wherein the analyte is generated in situ, and wherein the assay system further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.
Claim 24
Dependent Claim 94 words
The assay system of claim 1 wherein said assay system comprises a series of defined areas, and wherein each area contains the non-chromogenic competing reactant in different preselected concentrations, wherein each different preselected concentration is capable of preventing a visible color change due to accumulation of reduced chromogen until a different predetermined threshold amount of NAD(P)H is exceeded, and wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold and the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.
Claim 25
Dependent Claim 21 words
The assay system of claim 23 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.
Claim 26
Dependent Claim 20 words
The assay system of claim 23, wherein the assay system further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.
Claim 27
Dependent Claim 24 words
The assay system of claim 23 wherein the analyte is selected from the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.
Claim 28
Dependent Claim 13 words
The assay system of claim 26 wherein the analyte is generated in situ.
Claim 29
Dependent Claim 37 words
The assay system of claim 23 wherein the catalyst is an organic polycyclic compound, the chromogen is a tetrazolium salt and the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).
Claim 30
Dependent Claim 39 words
The assay system of claim 28 wherein the non-chromogenic competing reagent is selected from the group consisting of triethyldiamine salt of Fe(III), Fe(III) EDTA complex, Fe(III) sorbitol complex, hexamino cobalt (III) chloride, potassium hexacyanocobaltate (III) and sodium hexanitrocobaltate (III).
Claim 31
Dependent Claim 27 words
The assay system of claim 28 wherein the non-chromogenic competing reactant is selected from the group consisting of sodium potassium periodate, paranitrobenzyl disulfide and 2,2'-diathiobis (4-tert-butyl-1-isopropyl imidazole).
Claim 32
Dependent Claim 19 words
The assay system of claim 28 wherein the non-chromogenic competing reactant is a water-soluble alkali metal salt of ferricyanide.
Claim 33
Dependent Claim 15 words
The assay system of claim 31 wherein the salt is a sodium or potassium salt.
Claim 34
Dependent Claim 28 words
The assay system of claim 28 wherein the non-chromogenic competing reactant is a weak oxidizing agent selected from the group consisting of trichloro-melanine, hydrogen perioxide and organic peroxides.
Claim 35
Dependent Claim 24 words
The assay system of claim 33 wherein the organic peroxide is selected from the group consisting of a lower alkyl peroxide and benzoyl peroxide.
Claim 36
Dependent Claim 23 words
The assay system of claim 1 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.
Claim 37
Dependent Claim 24 words
The assay system of claim 1 wherein the sample is urine, the analyte is uric acid, and the concentration of uric acid is determined.
Claim 38
Dependent Claim 22 words
The assay system of claim 1 wherein the sample is saliva, the analyte is alcohol, and the concentration of alcohol is determined.
Claim 39
Dependent Claim 22 words
The assay system of claim 1 wherein the sample is blood, the analyte is cholesterol, and the concentration of cholesterol is determined.
Claim 40
Dependent Claim 33 words
The assay system of claim 1, wherein the concentration of an analyte which reacts to form NAD(P)H is determined and the assay system further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.
Claim 41
Dependent Claim 145 words
A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises: a physical support means, a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, a coenzyme selected from the group consisting of NAD(P) and NAD(P)H, wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold and the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.
Claim 42
Dependent Claim 145 words
A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises: a physical support means, a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, a coenzyme selected from the group consisting of NAD(P) and NAD(P)H, wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold and the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.
Claim 43
Dependent Claim 10 words
The device of claim 40 wherein the coenzyme is NAD(P).
Claim 44
Dependent Claim 14 words
The device of claim 40 wherein the physical support means is a water-absorbing material.
Claim 45
Dependent Claim 23 words
The device of claim 42 wherein the water-absorbing material is selected from the group consisting of chromogenic pater, gelatin and a synthetic resin.
Claim 46
Dependent Claim 25 words
The device of claim 40 wherein the physical support means is a synthetic resin selected from the group consisting of polyethylene, polypropylene, polyalkyleneacetate and polycarbonate.
Claim 47
Dependent Claim 19 words
The device of claim 42 wherein the water-absorbing material is a multilayer dry gel inside a controlled volume capillary.
Claim 48
Dependent Claim 30 words
The device of claim 45 wherein the gel is selected from the group consisting of gelatin, agarose, agar, polyvinyl alcohol, polyvinyl pyrolidone, alginate, carrageenan, dextran, xanthan gum and mixtures thereof.
Claim 49
Dependent Claim 26 words
The device of claim 42 wherein the physical support means are in a form selected from the group consisting of sheets, rods, webs, filters and strips.
Claim 50
Dependent Claim 14 words
The device of claim 42 wherein the water absorbing material is a metal oxide.
Claim 51
Dependent Claim 18 words
The device of claim 48 wherein the water absorbing metal oxide is a controlled pore alumina catalytic ring.
Claim 52
Dependent Claim 13 words
The device of claim 42 wherein the water-absorbing material is a hybrid ceramic/polymer.
Claim 53
Dependent Claim 20 words
The device of claim 50 wherein the hybrid ceramic/polymer material is a polyvinyl chloride sheet that contains embedded silica particles.
Claim 54
Dependent Claim 23 words
The device of claim 42 wherein the physical support means is selected from the group consisting of glass, cellulose, wood, metal and textiles.
Claim 55
Dependent Claim 15 words
The device of claim 42 wherein the physical support means is a controlled-pore polycarbonate membrane.
Claim 56
Dependent Claim 26 words
The device of claim 52 wherein the concentration of non-chromogenic competing reactant to NAD(P)H at the threshold is either 1 to 1 or 2 to 1.
Claim 57
Dependent Claim 22 words
The device of claim 40 wherein the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).
Claim 58
Dependent Claim 20 words
The device of claim 55 wherein the catalyst is an organic polycyclic compound, and the chromogen is a tetrazolium salt.
Claim 59
Dependent Claim 20 words
The device of claim 40 which further comprises a metal salt capable of enhancing the catalytic activity of the catalyst.
Claim 60
Dependent Claim 20 words
The device of claim 40 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.
Claim 61
Dependent Claim 16 words
The device of claim 58 which further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.
Claim 62
Dependent Claim 23 words
The device of claim 59 wherein the analyte is selected from the group consisting of alcohol, lactic acid, glucose, glycerol, ketones and beta-hydroxybutyrate.
Claim 63
Dependent Claim 12 words
The device of claim 60 wherein the analyte is generated in situ.
Claim 64
Dependent Claim 14 words
The device of claim 58 wherein the analyte is generated in situ from triglyceride.
Claim 65
Dependent Claim 12 words
The device of claim 59 wherein the analyte is generated in situ.
Claim 66
Dependent Claim 22 words
The device of claim 40 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.
Claim 67
Dependent Claim 154 words
A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises: a physical support means, a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, wherein the physical support means has a series of defined areas, each area containing a different pre-selected concentration of the non-chromogenic competing reactant and wherein each pre-selected concentration of non-chromogenic competing reactant is capable of preventing a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded.
Claim 68
Dependent Claim 154 words
A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises: a physical support means, a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, wherein the physical support means has a series of defined areas, each area containing a different pre-selected concentration of the non-chromogenic competing reactant and wherein each pre-selected concentration of non-chromogenic competing reactant is capable of preventing a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded.
Claim 69
Dependent Claim 22 words
The device of claim 65 wherein the non-chromogenic competing reactant is a weak oxidizing agent which accepts electrons more readily than NAD(P).
Claim 70
Dependent Claim 20 words
The device of claim 65 wherein the concentration of an analyte which reacts to form or consume NAD(P)H is measured.
Claim 71
Dependent Claim 16 words
The device of claim 67 which further comprises an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte.
Claim 72
Dependent Claim 23 words
The device of claim 68 wherein the analyte is selected from the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.
Claim 73
Dependent Claim 12 words
The device of claim 68 wherein the analyte is generated in situ.
Claim 74
Dependent Claim 22 words
The device of claim 65 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.
Claim 75
Dependent Claim 174 words
A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises: a physical support means, a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, NAD(P), and an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte to produce NAD(P)H in an amount proportional to the analyte, wherein the physical support means has a series of defined areas, each area containing a different pre-selected concentration of the non-chromogenic competing reactant and wherein each pre-selected concentration of non-chromogenic competing reactant is capable of preventing a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded.
Claim 76
Dependent Claim 174 words
A diagnostic colorimetric device for determination of the concentration of NAD(P)H, NAD(P), or an analyte which reacts to form or consume NAD(P)H, wherein the device comprises: a physical support means, a chromogen which is capable of accepting electrons from NAD(P)H and which produces a color change upon reduction by NAD(P)H, a non-chromogenic competing reactant which is capable of accepting electrons from NAD(P)H, in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded, an electron-carrier catalyst capable of transferring electrons from NAD(P)H to the chromogen, NAD(P), and an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte to produce NAD(P)H in an amount proportional to the analyte, wherein the physical support means has a series of defined areas, each area containing a different pre-selected concentration of the non-chromogenic competing reactant and wherein each pre-selected concentration of non-chromogenic competing reactant is capable of preventing a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P)H is exceeded.
Claim 77
Dependent Claim 22 words
The device of claim 72, wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.
Claim 78
Dependent Claim 161 words
A digital colorimetric method for determining the amount of an organic analyte in a sample, wherein the method comprises: (a) contacting the sample with an assay system comprising an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte to produce NAD(P)H in an amount proportional to the analyte, NAD(P), a chromogen capable of producing a color change upon reduction by NAD(P)H in the presence of an electron-carrier catalyst, an electron-carrier catalyst, and a non-chromogenic competing reactant capable of accepting electrons from NAD(P)H, wherein the non-chromogenic competing reactant is present in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P) is exceeded, and (b) determining the amount of NAD(P)H as an indirect determination of the analyte, wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold an the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.
Claim 79
Dependent Claim 161 words
A digital colorimetric method for determining the amount of an organic analyte in a sample, wherein the method comprises: (a) contacting the sample with an assay system comprising an NAD(P)-dependent dehydrogenase capable of oxidizing the analyte to produce NAD(P)H in an amount proportional to the analyte, NAD(P), a chromogen capable of producing a color change upon reduction by NAD(P)H in the presence of an electron-carrier catalyst, an electron-carrier catalyst, and a non-chromogenic competing reactant capable of accepting electrons from NAD(P)H, wherein the non-chromogenic competing reactant is present in an amount sufficient to prevent a visible color change due to accumulation of reduced chromogen until a predetermined threshold amount of NAD(P) is exceeded, and (b) determining the amount of NAD(P)H as an indirect determination of the analyte, wherein a visible color change is indicative of an amount of NAD(P)H above the predetermined threshold an the absence of a visible color change is indicative of an amount of NAD(P)H below the predetermined threshold.
Claim 80
Dependent Claim 23 words
The method of claim 74 wherein the analyte is selected from the group consisting of glucose, alcohol, lactic acid, glycerol, ketones and beta-hydroxybutyrate.
Claim 81
Dependent Claim 22 words
The method of claim 74 wherein the non-chromogenic competing reactant is capable of reoxidizing dye formed upon the reduction of the chromogen.
Claim 82
Dependent Claim 12 words
The method of claim 74 wherein the analyte is generated in situ.
Claim 83
Dependent Claim 20 words
The method of claim 74 wherein the sample is not diluted prior to adding the sample to the assay system.
Claim 84
Dependent Claim 16 words
The method of claim 74 wherein the sample is urine and the analyte is uric acid.
Claim 85
Dependent Claim 15 words
The method of claim 74 wherein the sample is saliva and the analyte is alcohol.
Claim 86
Dependent Claim 15 words
The method of claim 74 wherein the sample is blood and the analyte is cholesterol.
Claim 87
Dependent Claim 22 words
The device of claim 40 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.
Claim 88
Dependent Claim 22 words
The device of claim 65 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.
Claim 89
Dependent Claim 22 words
The device of claim 72 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.
Claim 90
Dependent Claim 22 words
The method of claim 75 wherein the catalyst is diaphorase and the non-chromogenic competing reactant is not a substrate for the diaphorase.

References Cited (Backward Citations)

US3964871A Cited
US4629697A Cited

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