Digital colorimetric quantitative assay system

Claim 2

A colorimetric assay system which is designed to measure the concentration of hydrogen peroxide by developing a color signal when the hydrogen peroxide is above a threshold concentration and by the absence of color signal when the concentration of hydrogen peroxide is below the threshold, which system comprises a chromogen capable of reducing hydrogen peroxide and which is capable of changing color in a visible range to yield an oxidized chromogen of a different color, and an air-stable first reductant of hydrogen peroxide that has a larger reduction potential than that of the chromogen, which first reductant prevents accumulation of oxidized chromogen and thereby a visible color change until the amount of first reductant is essentially depleted at which threshold a color is developed when hydrogen peroxide is present, and in the absence of hydrogen peroxide, no color is developed.

Claim 3

The assay system of claim 1 wherein the first reductant is capable of reducing hydrogen peroxide, or oxidized chromogen, or both.

Claim 4

The assay system of claim 2 wherein the first reductant is capable of reducing oxidized chromogen. and thereby regenerates the chromogen.

Claim 5

The assay system of claim 1 wherein the reduction of hydrogen peroxide by either the first reductant or the oxidized chromogen is promoted by a catalyst and optionally a metal salt which enhances the cataiytic activity of the catalyst.

Claim 6

The assay system of claim 4 wherein the catalyst promoting the reduction of hydrogen peroxide or the oxidized chromogen does not react or interact with any other component of the system to produce appreciable visible color in the absence of hydrogen peroxide.

Claim 7

The assay system of claim 5 wherein the catalyst promoting the reduction of hydrogen peroxide or the oxidized chromogen is selected from the group oonsisting of peroxidase or horseradish peroxidase.

Claim 8

The assay system of claim 5 wherein the catalyst promoting the reduction of hydrogen peroxide or the oxidized chromogen is a water soluble derivative of ferrous ion.

Claim 9

The assay system of claim 1 wherein the first reductant is bisulfate or a water soluble alkali metal salt of ferrocyanide.

Claim 10

The assay system of olaim 5 wherein the first reductant is NAD(P)H and the catalyst is a compound capable of accepting electrons from NAD(P)H, such as NAD(P)H peroxidase, diaphorase or an organic polycyclic compound.

Claim 11

The assay system of olaim 5 wherein the first reductant is NAD(P)H and the catalyst is a compound capable of accepting electrons from NAD(P)H, such as NAD(P)H peroxidase, diaphorase or an organic polycyclic compound.

Claim 12

The assay system of claim 13 wherein the organic polycyclic compound catalyst capable of accepting electrons from NAD(P)H is selected from the group consisting of meldola blue. phenazine methosuffate, N-methylphenazine methosulfate, methylene blue, or anthroquinones suhstituted with a member selected from the group consisting of methyl-, chloro-, bromo-, amino-. hydroxy-, nitro, methoxy or sulfuric acid.

Claim 13

The assay system of claim 4 wherein the metal salt which enhances the catalytic activity of the catalyst is present and is manganous sulfate, or a water soluble salt of platinum or palladium.

Claim 14

The assay system of claim 1 wherein the chromogen is azinobis- (3-ethylbenz-thiazoline sulfonic acid) (ABTS), orthodiansidine or 4-aminoantipyrine, or it is 4-aminoantipyrine and phenol.

Claim 15

The assay system of claim 1 wherein when a visible color change occurs indicative of the reductant being depleted and of the threshold concentration of hydrogen peroxide, there is generated less than one equivalent of oxidized chromogen per equivalent of hydrogen peroxide.

Claim 16

The assay system of claim 1 which comprises an oxidase which corresponds to a specific organic substrate. the concentration of which is to be measured, and/or an organic substrate corresponding to the oxidase, the concentration of which organic substrate is to be measured.

Claim 17

The assay system of claim 21 wherein the hydrogen peroxide is generated by a reaction between the organic substrate and the oxidase which corresponds to said organic substrate.

Claim 18

The assay system of claim 22 wherein, the organic substrate is selected from the group consisting of cholesterol, glucose, lactic acid, bilirubin or a compound which reacts to generate said substrate in situ, and the oxidase which corresponds to said organic substrate.

Claim 19

The assay system of claim 1 wherein the first reductant is present in different pre-selected concentrations, each concentration corresponding to a concentration of hydrogen peroxide to be determined, wherein of the different first reductant concentrations, some will be depleted and some will not be depleted as being shown by the development of a color and being indicative of the determined threshold concentration of hydrogen peroxide.

Claim 20

The assay system of claim 24 wherein the first reductant is a water soluble alkali metal salt of ferrocyanide and the chromogen is ABTS. The assay system of claim 25 which comprises an oxidase which corresponds to a specific organic substrate, the concentration of which is to be measured. The assay system of claim 26 which comprises an organic substrate corresponding to the oxidase, the The assay system of claim 27 wherein the hydrogen peroxide is generated by a reaction between the organic substrate and the oxidase which corresponds to said organic substrate. The assay system of claim 28 which comprises an organic substrate selected from the group consisting of cholesterol, glucose, lactic acid, bilirubin or a compound which reacts to generate said substrate in situ, and the oxidase which corresponds to said organic substrate.

Claim 21

A diagnostic colorimetric device which is designed to measure the concentration of hydrogen peroxide by developing a color signal when the hydrogen peroxide is above a threshold concentration and by the absence of a color signal when the concentration is below the threshold, which device comprises a physical support means for a chromogen capable of reducing hydrogen peroxide and capable of changing color in a visible range to yield an oxidized chromogen of a different color, for an air-stable first reductant of hydrogen peroxide that has a larger reduction potential than that of the chromogen and for an electron carrier catalyst, which catalyst is capable of transferring electrons from the first reductant to said hydrogen peroxide or said oxidized chromogen. The device of claim 30 wherein the physical support means is a water absorbing material inert to the reactants. The device of claim 31 wherein the water absorbing material inert to the reductant is a hybrid ceramic, polymer material. The device of claim 32 wherein the hybrid ceramic,polymer material is polyvinyl chloride sheet that contains embedded silica particles. The device of claim 33 wherein the polyvinyl chloride sheet is fastened to a plastic carrier. The device of claim 31 wherein the water absorbing material inert to the reactants is situated inside of a controlled volume capillary. The device of claim 35 wherein the water absorbing material inert to the reactants is selected from the group consisting of chromogenic paper, a gel, or a synthetic resin. The device of claim 36 wherein the water absorbing gel is selected from the group consisting of gelatin, agarose, agar, polyvinyl alcohol, polyvinyl pyrrolidone, alginate, carrageenan, dextran, xanthan gum, or mixtures thereof. The device of claim 37 wherein the water absorbing gel is a multilayer dry gel. The device of claim 38 wherein the multilayer dry gel comprises four layers consisting of (1) an oxidase corresponding to a specific organic substrate, the concentration of which is to be measured, (2) a separation layer, (3) first reductant and chromogen, and (4) another separation layer. The device of claim 39 wherein the multilayer dry gel comprises a fifth wicking layer. The device of claim 39 wherein the first reductant is ferrocyanide and the chromogen is ABTS. The device of claim 41 wherein the oxidase is selected from the group consisting of glucose oxidase, lactate oxidase or bilirubin oxidase. The device of claim 41 wherein the oxidase is cholesterol oxidase. The device of claim 43 which comprises in addition cholesterol esterase. The device of claim 40 wherein the wicking layer contains microcrystalline cellulose. The device of claim 35 wherein at least one surface of the controlled volume capillary consists of material selected from the group consisting of teflon, nylon, polyethylene, polypropylene. polyalkylene acetate or poiycarbonate. The device of claim 46 wherein the concentration of organic substrate to be measured exceeds the aqueous solubility of oxygen, the material is microporous. The device of claim 30 wherein the concentration of first reductant to hydrogen peroxide at the threshold is either 1 to 1 or 2 to 1, depending on the electron-donating ability of the first reductant. The device of claim 30 wherein the catalyst which is capable of transferring electrons from the first reductant to hydrogen peroxide or oxidized chromogen is diaphorase. The device of claim 30 which comprises in addition, a metal salt which enhances the catalytic activity of the catalyst. A diagnostic colorimetric device which is designed to measure the concentration of hydrogen peroxide by developing a color signal when the hydrogen peroxide is above a threshold concentration and by the absence of a color signal when the concentration of hydrogen peroxide is below the threshold, which device comprises a physical support means for chromogen capable of reducing hydrogen peroxide and capable of changing color in a visible range to yield an oxidized chromogen of a different color, for an air-stable first reductant of hydrogen peroxide that has a larger reduction potential than that of the chromogen, and for an electron carrier catalyst. which catalyst is capable of transferring electrons from the first reductant to said hydrogen peroxide or said oxidized chromogen, in which device the physical support means has a plurality of regions. each region having a different concentration of first reductant, which concentration in the respective regions is in a linear relationship, in which device the visible color change takes place in one region. which is indicative and corresponds to the concentration of hydrogen peroxide. The device of claim 50 wherein the first reductant is ferrocyanide and the chromogen is ABTS. The device of claim 51 which comprises in addition, an oxidase which corresponds to a specific substrate, the concentration of which is to be measured. The device of claim 53, which comprises an organic substrate corresponding to the oxidase. the concentration of which organic substrate is to be measured. The device of claim 54 wherein the substrate is selected from the group consisting of cholesterol, glucose, lactic acid, bilirubin, or a compound which reacts to generate said substrate in situ. A diagnostic colorimetric device which is designed to measure the concentration of hydrogen peroxide by developing a color signal when the hydrogen peroxide is above a threshold concentration and by the absence of a color signal when the concentration of hydrogen peroxide is below the threshold, which device comprises a physical support means for a chromogen capable of reducing hydrogen peroxide and capable of changing color in a visible range to yield an oxidized chromogen of a different color, for an air-stable first reductant of hydrogen peroxide that has a larger reduction potential than that of the chromogen, and for an electron carrier catalyst, which catalyst is capable of transferring electrons from the first reductant to said hydrogen peroxide or said oxidized chromogen, in which device the physical support means has a plurality of regions, each region having a different concentration of first reductant, which concentration in the respective regions is in a linear relationship, in which device the visible color change takes place in one region, which is indicative and corresponds to the concentration of hydrogen peroxide. and which device includes an oxidase which corresponds to an organic substrate, the concentration of which is to be measured, the reaction between the oxidase and the corresponding organic substrate producing hydrogen peroxide. The device of claim 56 which comprises an organic substrate corresponding to the oxidase, the concen- tratioan of which organic substrate is to be measured. The assay system of claim 1 wherein the first reductant is essentially all depleted and oxidized, the chromogen is oxidized, having generated a visible color and water, which visible color is different from the color of the system in the absence of the oxidized chromogen and which visible color is indicative of the concentration of hydrogen peroxide above the threshold. The diagnostic colorimetric device of claim 30 wherein the organic compound the concentration of which has been measured is in the oxidized state, the first reductant is depleted, and the chromogen is in a oxidized and colored state, which color is indicative of the presence of hydrogen peroxide above the threshold concentration.

Claim 22

An analytical colorimetric digital method of measuring the concentration of hydrogen peroxide generated by an organic substrate the concentration of which is to be measured by developing a color signal when the hydrogen peroxide is above a threshold concentration and by the absence of a color signal when the concentration of hydrogen peroxide is below the threshold, which method comprises, adding to the mixture which comprises a chromogen which is a reductant of hydrogen peroxide and which is capable of changing color in a visible range to yield an oxidized chromogen of a different color, a first reductant of greater reduction potential than the chromogen, which first reductant prevents accummulation of oxidized chromogen and thereby a visible color change until the amount of first reductant is essentially depleted at which threshold a color is developed when hydrogen peroxide is present and in the absence of hydrogen peroxide, no color is developed, an electron-carrier catalyst, which catalyst is capable of transferring electrons from the first reductant to said hydrogen peroxide or said chromogen, and a specific oxidase to a substrate, the concentration of which is to be measured, a substrate the concentration of which is to be measured, reacting the substrate and corresponding oxidase, thereby generating hydrogen peroxide and allowing the reaction to proceed until the first reductant is depleted, at which point a visible color is generated, which is indicative of the concentration of substrate.

Claim 23

An analytical colorimetric digital method of measuring the concentration of hydrogen peroxide generated by an organic substrate the concentration of which is to be measured by developing a color signal when the hydrogen peroxide is above a threshold concentration and by the absence of a color signal when the concentration of hydrogen peroxide is below the threshold, which method comprises, adding to the mixture which comprises a chromogen which is a reductant of hydrogen peroxide and which is capable of changing color in a visible range to yield an oxidized chromogen of a different color, a first reductant of greater reduction potential than the chromogen, which first reductant prevents accummulation of oxidized chromogen and thereby a visible color change until the amount of first reductant is essentially depleted at which threshold a color is developed when hydrogen peroxide is present and in the absence of hydrogen peroxide, no color is developed, an electron-carrier catalyst, which catalyst is capable of transferring electrons from the first reductant to said hydrogen peroxide or said chromogen, and a specific oxidase to a substrate, the concentration of which is to be measured, a substrate the concentration of which is to be measured, reacting the substrate and corresponding oxidase, thereby generating hydrogen peroxide and allowing the reaction to proceed until the first reductant is depleted, at which point a visible color is generated, which is indicative of the concentration of substrate.