Fluorescent labeling using microparticles with controllable stokes shift
First Claim
1. A method of applying a fluorescent label to a target specific binding pair member in a sample, comprising the steps of;
- a) combining the sample thought to contain the target specific binding pair member with a suspension of one or more microparticle-labeled probes, wherein each microparticle probe comprisesa polymeric microparticle incorporating randomly dispersed therein a series of fluorescent dyes having an initial donor dye with a desired excitation peak and an ultimate acceptor dye with a desired emission peak, where each dye of the series has spectral overlap sufficient to allow for significant energy transfer of excitation energy to occur resulting in an effective Stokes shift; and
a complementary specific binding pair member that binds to the target specific binding pair member and is covalently or non-covalently bound to the microparticle; and
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b) allowing sufficient time for one or more microparticle-labeled probes to form a complex with the target specific binding pair member.
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Accused Products
Abstract
The invention relates to methods for labeling or detecting one or more target materials using surface coated fluorescent microparticles with unique characteristics. The unique microparticles used to practice the invention have at least two components: an external substance or coating that is selective for each target material and an internal mixture of multiple fluorescent dyes. The mixture of dyes is a series of two or more fluorescent dyes having overlapping excitation and emission spectra allowing efficient energy transfer from the excitation wavelength of the first dye in the series, transfer through the dyes in the series and re-emitted as an optical signal at the emission wavelength of last dye in the series, resulting in a desired effective Stokes shift for the microparticle that is controlled through selection of appropriate dyes. The unique microparticles are combined with a sample thought to contain the target material(s), so that the microparticles label the target materials. The sample is then optionally illuminated, resulting in fluorescence of the microparticles that is used to detect one or more target materials.
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Citations
30 Claims
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1. A method of applying a fluorescent label to a target specific binding pair member in a sample, comprising the steps of;
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a) combining the sample thought to contain the target specific binding pair member with a suspension of one or more microparticle-labeled probes, wherein each microparticle probe comprises a polymeric microparticle incorporating randomly dispersed therein a series of fluorescent dyes having an initial donor dye with a desired excitation peak and an ultimate acceptor dye with a desired emission peak, where each dye of the series has spectral overlap sufficient to allow for significant energy transfer of excitation energy to occur resulting in an effective Stokes shift; and a complementary specific binding pair member that binds to the target specific binding pair member and is covalently or non-covalently bound to the microparticle; and
;b) allowing sufficient time for one or more microparticle-labeled probes to form a complex with the target specific binding pair member. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of detecting multiple different target specific binding pair member in a sample using one fluorescent label, comprising the steps of;
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a) preparing a sample thought to contain up to n different target specific binding pair members, where n is an integer greater than 1; b) preparing a suspension of microparticle-labeled probes, wherein said microparticle-labeled probes comprise polymeric microparticles incorporating randomly dispersed therein a series of fluorescent dyes having an initial donor dye with a desired excitation peak and an ultimate acceptor dye with a desired emission peak, where each dye in the series has spectral overlap sufficient to allow for significant energy transfer of excitation energy to occur resulting in an effective Stokes shift; and up to n complementary specific binding pair members that bind to the different target specific binding pair members, where each complementary specific binding pair member is covalently or non-covalently bound to the microparticle;
such that the microparticle-labeled probes are capable of forming a complex with all target specific binding pair members in the sample;c) combining the suspension with the sample to allow the microparticle-labeled probes to form a complex with the target specific binding pair members; d) illuminating the sample with means for exciting fluorescence in the micro-particle-labeled probes; and e) detecting the fluorescence of the microparticle-labeled probes that have formed a complex with the target specific binding pair members. - View Dependent Claims (21, 22, 23)
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24. A method of detecting multiple different target specific binding pair member in a sample using multiple fluorescent labels, comprising the steps of;
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a) preparing a sample thought to contain up to n different target specific binding pair members, where n is an integer greater than 1; b) preparing a suspensions of n different microparticle-labeled probes, wherein at least one microparticle-labeled probe comprises a polymeric microparticle incorporating randomly dispersed therein a series of fluorescent dyes having an initial donor dye with a desired excitation peak and an ultimate acceptor dye with a desired emission peak, where each dye in the series has spectral overlap sufficient to allow for significant energy transfer of excitation energy to occur resulting in an effective Stokes shift; and a complementary specific binding pair member that binds to a corresponding target specific binding pair member, where the complementary specific binding pair member is covalently or non-covalently bound to the microparticle;
where each different microparticle-labeled probe has detectably distinct spectral properties and forms a complex with a different corresponding target specific binding pair member;c) combining the suspensions, collectively or sequentially, with the sample to allow each different microparticle-labeled probe to form a complex with a different corresponding target specific binding pair member; d) illuminating the sample, simultaneously or sequentially, with means for exciting fluorescence in the microparticle-labeled probes; and e) detecting the fluorescence of the microparticle-labeled probes that have formed a complex with the target specific binding pair members. - View Dependent Claims (25, 26, 27)
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28. A method of detecting multiple different target specific binding pair members in a sample using mixtures of fluorescent labels, comprising the steps of;
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a) preparing a sample thought to contain up to n different target specific binding pair members, where n is an integer greater than 2; b) preparing n different mixtures of m different microparticle-labeled probes, where m is an integer greater than 1 and less than n, and where at least one microparticle-labeled probe comprises a polymeric microparticle incorporating randomly dispersed therein a series of fluorescent dyes having an initial donor dye with a desired excitation peak and an ultimate acceptor dye with a desired emission peak, where each dye in the series has spectral overlap sufficient to allow for significant energy transfer of excitation energy to occur resulting in an effective Stokes shift; and a complementary specific binding pair member that binds to a corresponding target specific binding pair member, where the complementary specific binding pair member is covalently or non-covalently bound to the microparticle;
where each different microparticle-labeled probe has detectably distinct spectral properties and where each mixture has a characteristic percentage of each m microparticle-labeled probes that gives the mixture detectably distinct spectral properties, wherein the microparticle-labeled probes in any given mixture have the same complementary specific binding pair member;c) combining the mixtures, collectively or sequentially, with the sample to allow each microparticle-labeled probe to form a complex with its corresponding target specific binding pair member; d) illuminating the sample, simultaneously or sequentially, with means for exciting fluorescence in the microparticle-labeled probes; and e) detecting the fluorescence of the microparticle-labeled probes that have formed a complex with their target specific binding pair members. - View Dependent Claims (29, 30)
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Specification