Enhanced phthalazinedione chemiluminescence
First Claim
1. In a process for the generation of light chemiluminescently by contacting a chemiluminescence precursor, an oxidant, and an enzyme, the improvement which comprises said chemiluminescense precursor being a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR16## wherein R1 is an amino and R2, R3 and R4 are all H, and wherein R1 is amino and each of R2, R3 and R4 is unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R2 is amino and each of R1, R3 and R4 is H, unsubstituted or substituted C1 -C6 -alkyl, unsubstituted or substituted C2 -C10 alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R1 and R2 together with the ring carbons they are attached to form a fused benz ring substituted with an amino or substituted amino group, and each of R3 and R4 is H, unsubstituted or substituted C1 -C6 -alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 -alkoxy, carboxyl or amino, substituents for said substituted alkyl and alkenyl being selected from the group consisting of chlorine, flourine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol, said substituted amino being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said substituted hydroxy being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said enzyme being a peroxidase enzyme and effecting such cntact in the presence of a nitrogen compound selected from the group consisting of (a) ammonia and its salts, (b) a heterocyclic aromatic, wherein said heterocyclic aromatic is selected from the group consisting of imidazoles and alkyl derivatives thereof, pyridine and alkyl derivatives thereof and thiazine, said thiazine selected from the group consisting of thionine and methylene blue, and (c) a water-soluble organic amine, said water-soluble organic amine selected from the group consisting of alkyl amines, polyamines selected from the group consisting of spermine, spermidine and putrescine, benzylamine, said alkyl amine being of the formula ##STR17## wherein X1, X2 and X3 are the same or different and are unsubstituted alkyl having 1 to 8 carbon atoms or alkyl having 1 to 8 carbon atoms and substituted by a substituent selected from the group consisting of hydroxy, nitro, fluorine, chlorine, bromine, iodine and carboxy.
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Accused Products
Abstract
A chemiluminescence process comprising the contacting of a chemiluminescence precursor, an oxidant, an enzyme and a nitrogen compound selected from the group consisting of ammonia and a water-soluble organic amine. The reaction of such process can be used in detection of nucleic acid hybrids, antibodies, antigens and peroxidase enzymes and in producing light.
45 Citations
20 Claims
- 1. In a process for the generation of light chemiluminescently by contacting a chemiluminescence precursor, an oxidant, and an enzyme, the improvement which comprises said chemiluminescense precursor being a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR16## wherein R1 is an amino and R2, R3 and R4 are all H, and wherein R1 is amino and each of R2, R3 and R4 is unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R2 is amino and each of R1, R3 and R4 is H, unsubstituted or substituted C1 -C6 -alkyl, unsubstituted or substituted C2 -C10 alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R1 and R2 together with the ring carbons they are attached to form a fused benz ring substituted with an amino or substituted amino group, and each of R3 and R4 is H, unsubstituted or substituted C1 -C6 -alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 -alkoxy, carboxyl or amino, substituents for said substituted alkyl and alkenyl being selected from the group consisting of chlorine, flourine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol, said substituted amino being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said substituted hydroxy being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said enzyme being a peroxidase enzyme and effecting such cntact in the presence of a nitrogen compound selected from the group consisting of (a) ammonia and its salts, (b) a heterocyclic aromatic, wherein said heterocyclic aromatic is selected from the group consisting of imidazoles and alkyl derivatives thereof, pyridine and alkyl derivatives thereof and thiazine, said thiazine selected from the group consisting of thionine and methylene blue, and (c) a water-soluble organic amine, said water-soluble organic amine selected from the group consisting of alkyl amines, polyamines selected from the group consisting of spermine, spermidine and putrescine, benzylamine, said alkyl amine being of the formula ##STR17## wherein X1, X2 and X3 are the same or different and are unsubstituted alkyl having 1 to 8 carbon atoms or alkyl having 1 to 8 carbon atoms and substituted by a substituent selected from the group consisting of hydroxy, nitro, fluorine, chlorine, bromine, iodine and carboxy.
- 6. A chemiluminescence assay for the detection of a peroxidase enzyme comprising contacting a sample suspected of containing said peroxidase enzyme with a chemiluminescence precursor, an oxidant and a nitrogen compound selected from the group consisting of ammonia and its salts, a heterocyclic aromatic and a water-soluble organic amine, determining the extend of light emission and relating the amount of light emission to the amount of enzyme in the sample, said chemiluminescence precursor being a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR18## wherein R1 is an amino and R2, R3 and R4 are all H, and wherein R1 is amino and each of R2, R3 and R4 is unsubstituted or substituted C1 -C6 alkyl or unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R2 is amino and each of R1, R3 and R4 is H, unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R1 and R2 together with the ring carbons they are attached to form a fused benz ring substituted with an amino or substituted amino group, and each of R3 and R4 is H, unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl or amino, said substituted alkyl and alkenyl being substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol, said substituted amino being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said substituted hydroxy being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said enzyme being a peroxidase enzyme, said water soluble organic amine selected from the group consisting of alkyl amines, polyamines selected from the group consisting of spermine, spermidine and putrescine, benzylamine, said alkyl amines are of the formula ##STR19## wherein X1, X2, X3 are the same or different and are unsubstituted alkyl having 1 to 8 carbon atoms, or alkyl having 1 to 8 carbon atoms and substituted by a substituent selected from the group consisting of hydroxy, nitro, fluorine, chlorine, bromine, iodine and carboxy and wherein said heterocyclic aromatic is selected from the group consisting of imidazoles and alkyl derivatives thereof, pyridine and alkyl derivatives thereof and thiazine, said thiazine selected from the group consisting of thionine and methylene blue.
- 8. A test kit for the generation of chemiluminescence light comprising a chemiluminescence precursor, an enzyme, an oxidant and a nitrogen compound selected from the group consisting of ammonia and its salts, a heterocyclic aromatic and a water-soluble organic amine, said chemiluminescence precursor being a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR20## wherein R1 is an amino, and R2, R3 and R4 are all H, and wherein R1 is amino and each of R2, R3 and R4 is unsubstituted or substituted C1 -C6 alkyl or unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R2 is amino and each of R1, R3 and R4 is H, unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R1 and R2 are together with the rng carbons they are attached to form a fused benz ring substituted with an amino or substituted amino group, and each of R3 and R4 is H, unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl or amino, said substituted alkyl and alkenyl being substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol, said substituted amino being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said substituted hydroxy being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said enzyme being a peroxidase enzyme, said water soluble organic amine compound selected from the group consisting of alkyl amines, polyamines selected from the group consisting of spermine, spermidine and putrescine, benzylamine, said alkyl amines are of the formula ##STR21## wherein X1, X2, X3 are the same or different and are unsubstituted alkyl having 1 to 8 carbon atoms, or alkyl having 1 to 8 carbon atoms and substituted by a substituent selected from the group consisting of hydroxy, nitro, fluorine, chlorine, bromine, iodine and carboxy, and wherein said heterocyclic aromatic is selected from the group consisting of imidazoles and alkyl derivatives thereof, pyridine and alkyl derivatives thereof and thiazine, said thiazine selected from the group consisting of thionine and methylene blue.
- 10. A chemiluminescence device comprising a vessel and means for combining a nitrogen compound selected from the group consisting of ammonia and it salts, a heterocyclic aromatic, and a water-soluble organic amine, and chemiluminescence reactants including a chemiluminescence precursor, an oxidant, and an enzyme, said chemiluminescence precursor being a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR22## wherein R1 is an amino, and R2, R3 and R4 are all H, and wherein R1 is amino and each of R2, R3 and R4 is unsubstituted or substituted C1 -C6 alkyl or unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R2 is amino or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R2 is amino and each of R1, R3 and R4 is H, unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl, or amino, or R1 and R2 together with the ring carbons they are attached to form a fused benz ring substituted with an amino or substituted amino group, and each of R3 and R4 is H, unsubstituted or substituted C1 -C6 alkyl, unsubstituted or substituted akenyl, unsubstituted or substituted hydroxyl, C1 -C6 alkoxy, carboxyl or amino, said substituted alkyl and alkenyl being substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol, said substituted amino being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said substituted hydroxy being substituted by a substituent selected from the group consisting of C1 to C10 alkyl and C2 to C10 alkenyl, said enzyme being a peroxidase enzyme, said water soluble organic compound selected from the group consisting of alkyl amines, polyamines selected from the group consisting of spermine, spermidine and putrescine, benzylamine, said alkyl amines are of the formula ##STR23## wherein X1, X2, X3 are the same or different and are unsubstituted alkyl having 1 to 8 carbon atoms, or alkyl having 1 to 8 carbon atoms and substituted by a substituent selected from the group consisting of hydroxy, nitro, fluorine, chlorine, bromine, iodine and carboxy, and wherein said heterocyclic aromatic is selected from the group consisting of imidazoles and alkyl derivatives thereof, pyridine and alkyl derivatives thereof and thiazine, said thiazine selected from the group consisting of thionine and methylene blue.
Specification