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US 6,265,179 B1Granted
Detection of phosphate using coupled enzymatic reactions
AssigneeMolecular Probes, Inc.
InventorsMingjie Zhou, Richard P. Haugland
Life of this patent
Estimating the term…
Abstract
Inorganic phosphate may be detected and optionally quantified via the coupling of a phosphate-dependent enzymatic reaction with an enzyme system that generates hydrogen peroxide in the presence of a chromogenic or fluorogenic peroxidase substrate. Phosphate consuming or phosphate-producing enzymes or their substrates may also be detected and/or quantified, including pyrophosphatase enzymes or pyrophosphatase.
Claims46
An assay for inorganic phosphate, comprising:
- a) producing a reaction mixture by treating a sample, simultaneously or sequentially, with a phosphorylase enzyme, a phosphorylase enzyme substrate, an oxidase enzyme, a peroxidase enzyme, and a peroxidase enzyme substrate, under conditions such that:
- when inorganic phosphate is present in said reaction mixture, said phosphorylase enzyme converts said inorganic phosphate and said phosphorylase enzyme substrate into one or more phosphorylase products, at least one of which is an oxidase substrate for said oxidase enzyme;
- said oxidase enzyme converts said oxidase substrate into one or more oxidase products, at least one of which is hydrogen peroxide;
- in the presence of hydrogen peroxide, said peroxidase enzyme converts said peroxidase enzyme substrate into a detectable product;
- b) detecting the presence or amount of said detectable product in said reaction mixture;
- and
- c) correlating the presence or amount of said detectable product in said reaction mixture with the presence or amount of inorganic phosphate in said reaction mixture;
- wherein said peroxidase enzyme substrate has the formula
- wherein
- R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- R1 and R6 are H;
- or R1 taken in combination with R2, or R5 taken in combination with R6, or both, form a fused aromatic six membered ring that is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- A and B are independently OH or NR8R9, wherein
- each R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R8 in combination with R9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, sulfonic acid, or a salt of sulfonic acid;
- or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- sulfonic acid, or salt of sulfonic acid;
- X is N—(C═Y)—R10, N—(SO2)—R11, or CHR12;
- wherein
- Y is O or S;
- R10 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R11 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R12 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R12 is a saturated or unsaturated C1-C6 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a Cl-C6 alcohol, a sulfonic acid, a salt of sulfonic acid, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
- or R12 has the formula
- where R13, R14, R15, R16, and R17 are independently H, F, Cl, Br, I, sulfonic acid, salt of sulfonic acid, a carboxylic acid, a salt of carboxylic acid.
26 more elements
An assay for maltose, comprising:
- a) producing a reaction mixture by treating a sample, simultaneously or sequentially, with inorganic phosphate, a maltose phosphorylase enzyme, a glucose oxidase enzyme, a peroxidase enzyme, and a peroxidase enzyme substrate under conditions such that:
- when maltose is present in said reaction mixture, said maltose phosphorylase converts said inorganic phosphate and maltose into glucose and glucose-1-phosphate;
- said glucose oxidase converts said glucose into oxidase products, at least one of which is hydrogen peroxide;
- in the presence of hydrogen peroxide, said peroxidase enzyme converts said peroxidase enzyme substrate into a detectable product;
- b) detecting the presence or amount of said detectable product in said reaction mixture;
- and
- c) correlating the presence or amount of said detectable product in said reaction mixture with the presence or amount of maltose in said reaction mixture;
- wherein said peroxidase enzyme substrate has the formula
- wherein
- R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- R1 and R6 are H;
- or R1 taken in combination with R2, or R5 taken in combination with R6, or both, form a fused aromatic six membered ring that is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- A and B are independently OH or NR8R9, wherein
- each R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R8 in combination with R9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, sulfonic acid, or a salt of sulfonic acid;
- or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- sulfonic acid, or salt of sulfonic acid;
- X is N—(C═Y)—R10, N—(SO2)—R11, or CHR12;
- wherein
- Y is O or S;
- R10 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R11 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R12 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R12 is a saturated or unsaturated C1-C6 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, a sulfonic acid, a salt of sulfonic acid, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
- or R12 has the formula
- where R13, R14, R15, R16, and R17 are independently H, F, Cl, Br, I, sulfonic acid, salt of sulfonic acid, a carboxylic acid, a salt of carboxylic acid.
26 more elements
An assay for a phosphate-producing enzyme, comprising:
- a) producing a reaction mixture by treating a sample, simultaneously or sequentially, with an appropriate substrate for said phosphate-producing enzyme, a phosphorylase enzyme, a phosphorylase enzyme substrate, an oxidase enzyme, a peroxidase enzyme, and a peroxidase enzyme substrate under conditions such that:
- when said phosphate-producing enzyme is present in said reaction mixture, said phosphate-producing enzyme converts said phosphate-producing enzyme substrate into one or more products, at least one of which is inorganic phosphate;
- said phosphorylase enzyme converts said inorganic phosphate and said phosphorylase enzyme substrate into one or more phosphorylase products, at least one of which is an oxidase substrate for said oxidase enzyme;
- said oxidase enzyme converts said oxidase substrate into one or more oxidase products, at least one of which is hydrogen peroxide;
- in the presence of hydrogen peroxide, said peroxidase enzyme converts said peroxidase enzyme substrate into a detectable product;
- b) detecting the presence or amount of said detectable product in said reaction mixture;
- and
- c) correlating the presence or amount of said detectable product in said reaction mixture with the presence or amount of phosphate-producing enzyme in said reaction mixture;
- wherein said peroxidase enzyme substrate has the formula
- wherein
- R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- R1 and R6 are H;
- or R1 taken in combination with R2, or R5 taken in combination with R6, or both, form a fused aromatic six membered ring that is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- A and B are independently OH or NR8R9, wherein
- each R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R8 in combination with R9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, sulfonic acid, or a salt of sulfonic acid;
- or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- sulfonic acid, or salt of sulfonic acid;
- X is N—(C═Y)—R10, N—(SO2)—R11, or CHR12;
- wherein
- Y is O or S;
- R10 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R11 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R12 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R12 is a saturated or unsaturated C1-C6 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, a sulfonic acid, a salt of sulfonic acid, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
- or R12 has the formula
- where R13, R14, R15, R16, and R17 are independently H, F, Cl, Br, I, sulfonic acid, salt of sulfonic acid, a carboxylic acid, a salt of carboxylic acid.
27 more elements
A composition comprising:
- a) a phosphorylase enzyme;
- b) a phosphorylase enzyme substrate;
- c) an oxidase enzyme;
- d) a peroxidase enzyme;
- and
- e) a peroxidase enzyme substrate;
- each selected such that under appropriate conditions including the presence of inorganic phosphate, said phosphorylase enzyme converts said inorganic phosphate and said phosphorylase enzyme substrate into one or more phosphorylase products, at least one of which is an oxidase substrate for said oxidase enzyme;
- said oxidase enzyme converts said oxidase substrate into one or more oxidase products, at least one of which is hydrogen peroxide;
- and
- in the presence of hydrogen peroxide, said peroxidase enzyme converts said peroxidase enzyme substrate into a detectable product;
- wherein said peroxidase enzyme substrate has the formula
- wherein
- R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- R1 and R6 are H;
- or R1 taken in combination with R2, or R5 taken in combination with R6, or both, form a fused aromatic six membered ring that is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- A and B are independently OH or NR8R9, wherein
- each R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R8 in combination with R9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, sulfonic acid, or a salt of sulfonic acid;
- or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- sulfonic acid, or salt of sulfonic acid;
- X is N—(C═Y)—R10, N—(SO2)—R11, or CHR12;
- wherein
- Y is O or S;
- R10 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R11 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R12 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R12 is a saturated or unsaturated C1-C6 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, a sulfonic acid, a salt of sulfonic acid, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
- or R12 has the formula
- where R13, R14, R15, R16, and R17 are independently H, F, Cl, Br, I, sulfonic acid, salt of sulfonic acid, a carboxylic acid, a salt of carboxylic acid.
29 more elements
The kit, comprising:
- a) a phosphorylase enzyme;
- b) a phosphorylase enzyme substrate;
- c) an oxidase enzyme;
- d) a peroxidase enzyme;
- and
- e) a peroxidase enzyme substrate;
- each selected such that under appropriate conditions including the presence of inorganic phosphate, said phosphorylase enzyme converts said inorganic phosphate and said phosphorylase enzyme substrate into one or more phosphorylase products, at least one of which is an oxidase substrate for said oxidase enzyme;
- said oxidase enzyme converts said oxidase substrate into one or more oxidase products, at least one of which is hydrogen peroxide;
- and
- in the presence of hydrogen peroxide, said peroxidase enzyme converts said peroxidase enzyme substrate into a detectable product;
- wherein said peroxidase enzyme substrate has the formula
- wherein
- R2, R3, R4 and R5 are independently H, F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- R1 and R6 are H;
- or R1 taken in combination with R2, or R5 taken in combination with R6, or both, form a fused aromatic six membered ring that is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C18 alkyl, or C1-C18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol, sulfonic acid, or a salt of sulfonic acid;
- A and B are independently OH or NR8R9, wherein
- each R8 and R9 are independently H, C1-C6 alkyl, C1-C6 carboxyalkyl, C1-C6 sulfoalkyl, a salt of C1-C6 carboxyalkyl, or a salt of C1-C6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R8 in combination with R9 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alkyl, sulfonic acid, or a salt of sulfonic acid;
- or R8 in combination with R2, or R9 in combination with R3, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally and independently substituted one or more times by F, Cl, Br, I, CN;
- or C1-C6 alkyl, or C1-C6 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- sulfonic acid, or salt of sulfonic acid;
- X is N—(C═Y)—R10, N—(SO2)—R11, or CHR12;
- wherein
- Y is O or S;
- R10 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R11 is hydrogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkenyl, aryl, amino, alkylamino, or dialkylamino, the alkyl portions of which have 1-6 carbons;
- R12 is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C1-C6 alcohol;
- or R12 is a saturated or unsaturated C1-C6 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C1-C6 alcohol, a sulfonic acid, a salt of sulfonic acid, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;
- or R12 has the formula
- where R13, R14, R15, R16, and R17 are independently H, F, Cl, Br, I, sulfonic acid, salt of sulfonic acid, a carboxylic acid, a salt of carboxylic acid.
29 more elements
Dependencies are inferred from the claim text.