Lifetime-resolved assay procedures
First Claim
1. In a luminescent assay for detection of an analyte in a sample wherein a reaction mixture is formed containing the analyte and a photophore-labelled probe capable of a specific association with the analyte and wherein the presence of the analyte is determined by luminescent emission of a photophore upon excitation of the reaction mixture by a modulated light source, the improvement comprising:
- (1) employing in said reaction mixture a first photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a first emissive lifetime, anda second photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a second emissive lifetime,one of the photophores having a significantly longer emissive lifetime than the other photophore,said one photophore having a significantly longer emissive lifetime being excitable by said modulated light source to an excited state from which energy may be transferred to said other photophore having a significantly shorter emissive lifetime when in sufficient proximity thereto, thereby causing excitation and emisssion of said other photophore,the association of said analyte with said first probe and said second probe resulting in said photophores of said first and second probes being in sufficient proximity to each other to allow the excitation of said one photophore having a significantly longer emissive lifetime by said modulated light source to result in transfer of energy to said other photophore having a significantly shorter emissive lifetime causing excitation of and emission by said other photophore;
(2) exciting the reaction mixture with said modulated light source;
(3) monitoring the reaction mixture to determine the luminescent emission of said other photophore having a significantly shorter emissive lifetime as excited by energy transfer from said one photophore excited by said modulated energy source, at a time beyond the lifetime of emission of said other photophore having a significantly shorter emissive lifetime relative to the time of each excitation of said reaction mixture by said modulated light source and(4) using the determination of luminescent emission of said other photophore obtained by said monitoring step to determine the amount of analyte present in said sample.
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Accused Products
Abstract
Improved luminescent lifetime-resolved association assay techniques for detection of analytes in samples using two photophore-labelled probes, the photophores of which have different emissive lifetimes. One of the photophores is excitable by a modulated energy source to an excited state from which energy may be transferred to the other photophore when in close poximity thereto resulting in excitation and emission of the other photophore. Methods according to the invention involve associating the first photophore-labelled probe with the analyte and associating the second photophore-labelled probe with the analyte or first probe in a reaction mixture bringing the photophores in sufficient proximity to allow energy transfer to occur. The reaction mixture is formed, excited by the modulated energy source and monitored for emission of the photophore excited by energy transfer at a time beyond the emissive lifetime of the shorter-lived photophore.
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Citations
67 Claims
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1. In a luminescent assay for detection of an analyte in a sample wherein a reaction mixture is formed containing the analyte and a photophore-labelled probe capable of a specific association with the analyte and wherein the presence of the analyte is determined by luminescent emission of a photophore upon excitation of the reaction mixture by a modulated light source, the improvement comprising:
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(1) employing in said reaction mixture a first photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a first emissive lifetime, and a second photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a second emissive lifetime, one of the photophores having a significantly longer emissive lifetime than the other photophore, said one photophore having a significantly longer emissive lifetime being excitable by said modulated light source to an excited state from which energy may be transferred to said other photophore having a significantly shorter emissive lifetime when in sufficient proximity thereto, thereby causing excitation and emisssion of said other photophore, the association of said analyte with said first probe and said second probe resulting in said photophores of said first and second probes being in sufficient proximity to each other to allow the excitation of said one photophore having a significantly longer emissive lifetime by said modulated light source to result in transfer of energy to said other photophore having a significantly shorter emissive lifetime causing excitation of and emission by said other photophore; (2) exciting the reaction mixture with said modulated light source; (3) monitoring the reaction mixture to determine the luminescent emission of said other photophore having a significantly shorter emissive lifetime as excited by energy transfer from said one photophore excited by said modulated energy source, at a time beyond the lifetime of emission of said other photophore having a significantly shorter emissive lifetime relative to the time of each excitation of said reaction mixture by said modulated light source and (4) using the determination of luminescent emission of said other photophore obtained by said monitoring step to determine the amount of analyte present in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. In a luminescent assay for detection of an analyte in a sample wherein a reaction mixture is formed containing the analyte and a photophore-labelled probe capable of a specific association with the analyte and wherein the presence of the analyte is determined by luminescent emission of a photophore upon excitation of the reaction mixture by a modulated light source, the improvement comprising:
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(1) employing in said reaction mixture a first photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a first emissive lifetime, and a second photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a second emissive lifetime, one of the two photophores having a significantly shorter emissive lifetime that the other photophore, said one photophore having a significantly shorter emissive lifetime being excitable by said modulated light source to an excited state from which energy may be transferred to said other photophore having a significantly longer emissive lifetime when in sufficient proximity thereto, thereby causing excitation and emission of said other photophore, said other photophore having a significantly longer emissive lifetime being excitable by said energy transfer from said one photophore having a significantly shorter emissive lifetime and said other photophore being not significantly excitable by said modulated light source, the association of said analyte with said first probe and of said second probe resulting in the photophores of said first and second probes being in sufficient proximity to each other to allow excitation of said one photophore having a significantly shorter emissive lifetime by said modulated light source to result in transfer of energy to said other photophore having a significantly longer emissive lifetime causing excitation of and emission by said other photophore; (2) exciting the reaction mixture with said modulated light source; (3) monitoring the reaction mixture to determine the luminescent emission of said other photophore having a significantly longer emissive lifetime as excited by energy transfer from said one photophore having a significantly shorter emissive lifetime excited by said modulated light source, at a time beyond the lifetime of emission of said one photophore having said significantly shorter emissive lifetime relative to the time of each excitation of said reaction mixture by said modulated light source; and (4) using the determination of luminescent emission of said other photophore obtained by said monitoring step to determine the amount of analyte present in the sample. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. In a luminescent assay for detection of an analyte in a sample wherein a reaction mixture is formed containing the analyte and a photophore-labelled probe capable of a specific association with the analyte and wherein the presence of the analyte is determined by luminescent emission of a photophore upon excitation of the reaction mixture by a modulated light source, the improvement comprising;
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(1) employing in said reaction mixture a first photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a first emissive lifetime, and a second photophore-labelled probe capable of a specific association with said first probe in competition with said analyte, the photophore of which has a second emissive lifetime, one of the photophores having a significantly longer emissive lifetime than the other photophore, said one photophore having a significantly longer emissive lifetime being excitable by said modulated light source to an excited state from which energy may be transferred to said other photophore having a significantly shorter emissive lifetime when in sufficient proximity thereto, thereby causing excitation add emission of said other photophore, the association of said first probe and said second probe resulting in said photophores of said first and second probes being in sufficient proximity to each other to allow the excitation of said one photophore having a significantly longer emissive lifetime by said modulated light source to result in transfer of energy to said other photophore having a significantly shorter emissive lifetime causing excitation of an emission by said other photophore, the association of said first probe with said analyte resulting in said photophores of said first and second probes being substantially removed from each other such that excitation of said one photophore having a significantly longer emissive lifetime by said modulated light source does not result in significant transfer of energy to said other photophore having a significantly shorter emissive lifetime, (2) exciting the reaction mixture with said modulated light source; (3) monitoring the reaction mixture to determine the luminescent emission of said other photophore having a significantly shorter emissive lifetime as excited by energy transfer from said one photophore excited by said modulated light source, at a time beyond the lifetime of emission of said other photophore having a significantly shorter emissive lifetime relative to the time of each excitation of said reaction mixture by said modulated light source; and (4) using the determination of luminescent emission of said other photophore obtained by said monitoring step to determine the amount of analyte present in said sample. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)
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52. In a luminescent array for detection of an analyte in a sample wherein a reaction mixture is formed containing the analyte and a photophore-labelled probe capable of a specific association with the analyte and wherein the presence of the analyte is determined by luminescent emission of a photophore upon excitation of the reaction mixture by a modulated light source, the improvement comprising;
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(1) employing in said reaction mixture a first photophore-labelled probe capable of a specific association with said analyte, the photophore of which has a first emissive lifetime, and a second photophore-labelled probe capable of a specific association with said first probe in competition with said analyte, the photophore of which has a second emissive lifetime, one of the photophores having a significantly shorter emissive lifetime than the other photophore, said one photophore having a significantly shorter emissive lifetime being excitable by said modulated light source to an excited state from which energy amy be transferred to said other photophore having a significantly longer emissive lifetime when in sufficient proximity thereto, thereby causing excitation and emission of said other photophore, said other photophore having a significantly longer emissive lifetime being excitable by said energy transfer from said one photophore having a significantly shorter emissive lifetime and said other photophore being not significantly excitable by said modulated light source, the association of said first probe and said second probe resulting in said photophores of said first and second probes being in sufficient proximity to each other to allow the excitation of said one photophore having a significantly shorter emissive lifetime by said modulated light source to result in transfer of energy to said other photophore having a significantly longer emissive lifetime causing excitation of and emission by said other photophore; the association of said first probe with said analyte resulting in said photophores of said first and second probes being substantially removed from each other such that excitation of said one photophore having a significantly shorter emissive lifetime by said modulated light source does not result in significant transfer of energy to said other photophore having a significantly longer emissive lifetime; (2) exciting the reaction mixture with said modulated light source; (3) monitoring the reaction mixture to determine the luminescent emission of said other photophore having a significantly longer emissive lifetime as excited by energy transfer from said one photophore excited by said modulated light source, at a time beyond the lifetime of emission of said one photophore having a significantly shorter emissive lifetime relative to the time of each excitation of said reaction mixture by said modulated light source; and (4) using the determination of luminescent emission of said other photophore obtained by said monitoring step to determine the amount of analyte present in said sample. - View Dependent Claims (53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67)
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Specification