Radiative and non-radiative energy transfer and absorbance modulated fluorescence detection methods and sensors
First Claim
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1. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:
- obtaining a discrete absorber ligand having a definable light absorption spectrum, and absorber ligand being in definable relationship with the analyte of interest in the sample;
admixing a fluorophore with said absorber ligand, said fluorophore having a definable light absorption spectrum which does not overlap the light absorption spectrum of said absorber ligand yet having a definable light emission spectrum which does overlap the light absorption spectrum of said absorber ligand, said fluorophore being able(a) to absorb exciting light energy of a first wavelength,(b) to transfer a part of said fluorophore absorbed energy to said absorber ligand for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers, and(c) to emit the other part of said fluorophore absorbed energy as emitted light of a second wavelength;
introducing exciting light energy of the first wavelength to said admixture sufficient to cause said fluorophore to absorb said exciting light energy and to emit light of the second wavelength, a portion of said fluorophore absorbed energy being transferred to and absorbed by such ligand as is present in said admixture; and
detecting said light of the second wavelength emitted from said admixture after absorption of transferred energy by said ligand, said detected emitted light being a measure of the analyte of interest in the sample.
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Abstract
A variety of methods and apparatus for the detection of an analyte of interest in a fluid sample is provided which relies upon the interaction of a fluorophore and a chromophoric light absorbing compound for qualitative and quantitative results. The methods preferably employ fiber optic sensors in combination with fluorophores and/or proto-absorber substances in mobile and immobilized modes of use. The methods and apparatus rely upon the ability of the light absorbing compositions to absorb energy which is transferred either radiatively or non-radiatively by the fluorophore when in an excited state.
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Citations
24 Claims
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1. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:
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obtaining a discrete absorber ligand having a definable light absorption spectrum, and absorber ligand being in definable relationship with the analyte of interest in the sample; admixing a fluorophore with said absorber ligand, said fluorophore having a definable light absorption spectrum which does not overlap the light absorption spectrum of said absorber ligand yet having a definable light emission spectrum which does overlap the light absorption spectrum of said absorber ligand, said fluorophore being able (a) to absorb exciting light energy of a first wavelength, (b) to transfer a part of said fluorophore absorbed energy to said absorber ligand for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers, and (c) to emit the other part of said fluorophore absorbed energy as emitted light of a second wavelength; introducing exciting light energy of the first wavelength to said admixture sufficient to cause said fluorophore to absorb said exciting light energy and to emit light of the second wavelength, a portion of said fluorophore absorbed energy being transferred to and absorbed by such ligand as is present in said admixture; and detecting said light of the second wavelength emitted from said admixture after absorption of transferred energy by said ligand, said detected emitted light being a measure of the analyte of interest in the sample. - View Dependent Claims (7, 9, 10, 11, 12, 13, 14)
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2. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:
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obtaining a discrete ligand whose presence is in definable relationship with the analyte of interest in the sample; combining said ligand with a specifically binding proto-absorber to form an absorption complex having a definable light absorption spectrum, at least a portion of said proto-absorber binding with such ligand as is present to form said absorption complex while the remainder of said proto-absorber remains unbound; admixing a fluorophore with said absorption complex, said fluorophore having a definable light absorption spectrum which does not overlap the individual light absorption spectrum of said absorption complex and said unbound proto-absorber yet having a definable light emission spectrum which does overlap the light absorption spectrum of only said absorption complex, said fluorophore being able (a) to absorb exciting light energy of a first wavelength, (b) to transfer a part of said fluorophore absorbed energy to said absorption complex for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers, and (c) to emit the other part of said fluorophore absorbed energy as emitted light of a second wavelength; introducing exciting light energy of the first wavelength to said admixture sufficient to cause said fluorophore to absorb said exciting light energy and to emit light of the second wavelength, a portion of said fluorophore absorbed energy being transferred to and absorbed by such absorption complex as is present in said admixture; and detecting said light of the second wavelength emitted from said admixture after absorption of transferred energy by said absorption complex, said detected emitted light being a measure of the analyte of interest in the sample. - View Dependent Claims (8, 15)
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3. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:
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obtaining a discrete ligand whose presence is in definable relationship with the analyte of interest in the sample; combining said ligand with a specifically binding proto-absorber having a definable light absorption spectrum, at least a portion of said proto-absorber binding with such ligand as is present to form a complex while the remainder of said proto-absorber remains unbound; admixing a fluorophore with said unbound proto-absorber, said fluorophore having a definable light absorption spectrum which does not overlap the individual light absorption spectrum of said formed complex and said unbound proto-absorber yet having a definable light emission spectrum which does overlap the light absorption spectrum of only said unbound protoabsorber, said fluorophore being able (a) to absorb exciting light energy of a first wavelength, (b) to transfer a part of said fluorophore absorbed energy to said unbound proto-absorber for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers, and (c) to emit the other part of said fluorophore absorbed energy as emitted light of a second wavelength;
introducing exciting light energy of the first wavelength to said admixture sufficient to cause said fluorophore to absorb said exciting light energy and to emit light of the second wavelength, a portion of said fluorophore absorbed energy being transferred to and absorbed by such unbound proto-absorber as is present in said admixture; anddetecting said light of the second wavelength emitted from said admixture after absorption of transferred energy by said unbound proto-absorber, said detected emitted light being a measure of the analyte of interest in the sample.
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4. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:
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obtaininig a discrete absorber ligand having a definable light absorption spectrum, said absorber ligand being in definable relationship with the analyte of interest in the sample; admixing a plurality of different fluorophores with said absorber ligand, none of said different fluorophores having a definable light absorption spectrum which overlaps the light absorption spectrum of said absorber ligand while at least one of said fluorophores has a definable light emission spectrum which does overlap the light absorption spectrum of said absorber ligand, wherein (a) at least one fluorophore is able to absorb exciting light energy of a first wavelength and to transfer at least a part of said fluorophore absorbed energy to another fluorophore for absorption, (b) at least one other fluorophore is able to absorb energy transferred from another fluorophore and to emit at least part of said absorbed energy as emitted light of a second wavelength, and (c) at least one of said fluorophores transfers at least a part of said fluorophore absorbed energy to said absorber ligand for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers; introducing exciting light energy of the first wavelength to said admixture sufficient to cause at least one of said fluorophores to absorb said exciting light energy and at least one other of said fluorophores to emit light of the second wavelength, portions of said fluorophore absorbed energy being transferred to and absorbed by at least one of said fluorophores and such absorber ligand as is present in said admixture; and detecting said light of the second wavelength emitted from said admixture after partial absorption of transferred energy by said absorber ligand, said detected emitted light being a measure of the analyte of interest in the sample.
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5. A method for detecting any analyte of interest in a fluid sample, said method comprising the steps of:
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obtaining a discrete ligand whose presence is in definable relationship with the analyte of interest in the sample; combining said ligand with a specifically binding proto-absorber to form an absorption complex having a definable light absorption spectrum, at least a portion of said proto-absorber binding with such ligand as is present to form said absorption complex while the remainder of said proto-absorber remains unbound; admixing a plurality of different fluorophores with said absorption complex, none of said different fluorophores having a definable light absorption spectrum which overlaps the individual light absorption spectrum of said absorption complex and said unbound proto-absorber while at least one of said fluorophores has a definable light emission spectrum which does overlap the light absorption spectrum of only said absorption complex, wherein (a) at least one fluorophore is able to absorb exciting light energy of a first wavelength and to transfer at least a part of said fluorophore absorbed energy to another fluorophore for absorption, (b) at least one other fluorophore is able to absorb energy transferred from another fluorophore and to emit at least part of said absorbed energy as emitted light of a second wavelength, and (c) at least one of said fluorophores transfers at least a part of said fluorophore absorbed energy to said absorption complex for absorption, said transfer being selected from the group consisting radiative and non-radiative energy transfers; introducing exciting light energy of the first wavelength to said admixture sufficient to cause at least one of said fluorophores to absorb said exciting light energy and at least one other of said fluorophores to emit light of the second wavelength, portions of said fluorophore absorbed energy being transferred to and absorbed by at least one of said fluorophores and such absorption complex as is present in said admixture; and detecting said light of the second wavelength emitted from said admixture after partial absorption of transferred energy by said absorption complex, said detected emitted light being a measure of the analyte of interest in the sample.
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6. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of:
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obtaining a discrete ligand whose presence is in definable relationship with the analyte of interest in the sample; combining said ligand with a specifically binding proto-absorber having a definable light absorption spectrum, at least a portion of said proto-absorber binding with such ligand as is present to form a complex while the remainder of said proto-absorber remains unbound; admixing a plurality of different fluorophores with said unbound proto-absorber, none of said different fluorophores having a definable light absorption spectrum which overlaps the individual light absorption spectrum of said formed complex and said unbound proto-absorber while at least one of said fluorophores has a definable light emission spectrum which does overlap the light absorption spectrum of only said unbound proto-absorber, wherein (a) at least one fluorophore is able to absorb exciting light energy of a first wavelength and to transfer at least a part of said fluorophore absorbed energy to another fluorophore for absorption, (b) at least one other fluorophore is able to absorb energy transferred from another fluorophore and to emit at least part of said absorbed energy as emitted light of a second wavelength, and (c) at least one of said fluorophores transfers at least a part of said fluorophore absorbed energy to said unbound proto-absorber for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers; introducing exciting light energy of the first wavelength to said admixture sufficient to cause at least one of said fluorophores to absorb said exciting light energy and at least one other of said fluorophores to emit light of the second wavelength, portions of said fluorophore absorbed energy being transferred to and absorbed by at least one of said fluorophores and such unbound proto-absorber as is present in said admixture; and detecting said light of the second wavelength emitted from said admixture after partial absorption of transferred energy by said unbound proto-absorber, said detected emitted light being a measure of the analyte of interest in the sample.
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16. An integral fiber optic sensor useful in an apparatus for detecting an analyte of interest in a fluid sample, the presence of the analyte being in a definable relationship with a discrete absorber ligand in the fluid, said sensor comprising:
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a fiber optic strand for introducing exciting light energy of a first wavelength and for detecting emitted light of a second wavelength; a porous matrix disposed at one end of said fiber optic strand; and a fluorophore immobilized via said porous matrix for admixture with the absorber ligand in the fluid, said fluorophore having a definable light absorption spectrum which does not overlap the light absorption spectrum of said absorber ligand yet having a definable light emission spectrum which does overlap the light absorption spectrum of said absorber ligand, said immobilized fluorophore being able (a) to absorb exciting light energy of a first wavelength, (b) to transfer a part of said fluorophore absorbed energy to the absorber ligand for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers, and (c) to emit the other part of said fluorophore absorbed - View Dependent Claims (22, 24)
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- 17. energy as emitted light of a second wavelength.
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18. An integral fiber optic sensor useful in an apparatus for detecting an analyte of interest in a fluid sample, the presence of the analyte being in a definable relationship with a discrete ligand in the fluid, said sensor comprising:
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a fiber optic strand for introducing exciting light energy of a first wavelength to a fluorophore and for detecting emitted light of a second wavelength emitted by a fluorophore, the fluorophore having a definable light absorption spectrum and a definable light emission spectrum; a porous matrix disposed at one end of said fiber optic strand; and a specifically binding proto-absorber having a definable light absorption spectrum immobilized via said porous matrix on said fiber optic strand, at least a portion of said immobilized proto-absorber being able to bind with such ligand as is present in the fluid to form an immobilized complex having a definable light absorption spectrum while the remainder of said immobilized proto-absorber remains unbound, only one between said unbound immobilized proto-absorber and said immobilized complex having an individual light absorption spectrum which overlaps the light emission spectrum of the fluorophore.
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19. An integral fiber optic sensor useful in an apparatus for detecting an analyte of interest in a fluid sample, the presence of the analyte being in a definable relationship with a discrete ligand in the fluid, said sensor comprising:
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a fiber optic strand for introducing exciting light energy of a first wavelength and for detecting emitted light of a second wavelength emitted by a fluorophore; a porous matrix disposed at one end of said fiber optic strand; and a specifically binding proto-absorber having a definable light absorption spectrum immobilized via said porous matrix on said fiber optic strand, at least a portion of said immobilized proto-absorber being able to bind with such ligand as is present in the fluid to form an immobilized complex having a definable light absorption spectrum while the remainder of said immobilized proto-absorber remains unbound; and a fluorophore immobilized via said porous matrix on said fiber optic strand, said immobilized fluorophore having a definable light absorption spectrum which does not overlap the individual light absorption spectrum of said unbound immobilized proto-absorber and said immobilized complex while having a definable light emission spectrum which does not overlap the light absorption spectrum of only one between said unbound immobilized proto-absorber and said immobilized complex, said immobilized fluorophore being able (a) to absorb exciting light energy of a first wavelength, (b) to transfer a part of said fluorophore absorbed energy to only one between said immobilized complex and said unbound proto-absorber for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers, and (c) to emit the other part of said fluorophore absorbed energy as emitted light of a second wavelength.
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20. An integral fiber optic sensor useful in an apparatus for detecting an analyte of interest in a fluid sample, the presence of the analyte being in a definable relationship witth a discrete absorber ligand having a definable light absorption spectrum in the fluid, said sensor comprising:
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a fiber optic strand for introducing exciting light energy of a first wavelength and for detecting emitted light of a second wavelength; a porous matrix disposed at one end of said fiber optic strand; and a plurality of different fluorophores immobilized via said porous matrix on said fiber optic strand, none of said immobilized fluorophores having a definable light absorption spectrum which overlaps the light absorption spectrum of the absorber ligand while at least one immobilized fluorophore has a definable light emission spectrum which does overlap the light absorption spectrum of the absorber ligand, wherein (a) at least one immobilized fluorophore is able to absorb exciting light energy of a first wavelength and to transfer at least a part of said fluorophore absorbed energy to another fluorophore for absorption, (b) at least one other immobilized fluorophore is able to absorb energy transferred from another fluorophore and to emit at least part of said abosrbed energy as emitted light of a second wavelength, and (c) at least one fluorophore transfers at least a part of said fluorophore absorbed energy to said absorber ligand for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers.
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21. An integral fiber optic sensor useful in an apparatus for detecting an analyte of interest in a fluid sample, the presence of the analyte being in a definable relationship with a discrete ligand in the fluid, said sensor comprising:
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a fiber optic strand for introducing exciting light energy of a first wavelength and for detecting emitted light of a second wavelength; a porous matrix disposed at one end of said fiber optic strand; a specifically binding proto-absorber having a definable light absorption spectrum immobilized via said porous matrix on said fiber optic strand, at least a portion of said immobilized proto-absorber being able to bind with such ligand as is present in the fluid to form an immobilized complex having a definable light absorption spectrum while the remainder of said immobilized proto-absorber remains unbound, and a plurality of different fluorophores immobilized via said porous matrix on said fiber optic strand, none of said immobilized fluorophores having a definable light absorption spectrum which overlaps the individual light absorption spectrum of said unbound immobilized proto-absorber and said immobilized complex while at least on immobilized fluorophore has a definable light emission spectrum which does overlap the light absorption spectrum of only one between said unbound immobilized proto-absorber and said immobilized complex, wherein (a) at least one immobilized fluorophore is able to absorb exciting light energy of a first wavelength and to transfer at least a part of said fluorophore absorbed energy to another fluorophore for absorption, (b) at least one other immobilized fluorophore is able to absorb energy transferred from another fluorophore and to emit at least part of said absorbed energy as emitted light of a second wavelength, and (c) at least one fluorophore transfers at least a part of said fluorophore absorbed energy to only one between said immobilized complex and said unbound immobilized proto-absorber for absorption, said transfer being selected from the group consisting of radiative and non-radiative energy transfers.
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Specification