Fluorescent labeling using microparticles with controllable stokes shift

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 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.

Claim 2

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 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.

Claim 21

A method of detecting multiple different target specific binding pair member in a sample using one fluorescent label, comprising the steps of; 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.

Claim 22

A method of detecting multiple different target specific binding pair member in a sample using one fluorescent label, comprising the steps of; 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.

Claim 26

A method of detecting multiple different target specific binding pair member in a sample using multiple fluorescent labels, comprising the steps of; 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.

Claim 27

A method of detecting multiple different target specific binding pair member in a sample using multiple fluorescent labels, comprising the steps of; 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.

Claim 31

A method of detecting multiple different target specific binding pair members in a sample using mixtures of fluorescent labels, comprising the steps of; 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.

Claim 32

A method of detecting multiple different target specific binding pair members in a sample using mixtures of fluorescent labels, comprising the steps of; 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.