Methods and compositions in particle-based detection of target molecules using covalent bond forming reactive pairs
Methods and compositions which can be used to increase the strength and/or probability of forming a binding complex comprising a target molecule and a substrate are disclosed. In one aspect, linking molecules having one or more members of a reactive pair are disclosed which can be used to increase the number of intra-complex binding interactions in a complex comprising a target molecule and a substrate. Intra-complex crosslinking and inter-complex crosslinking can be utilized in connection with these methods and compositions to further strengthen and stabilize the disclosed binding complexes.
1. A method of generating a covalent binding complex comprising a target molecule, said method comprising:
combining in a reaction mixture
a substrate surface, wherein said substrate surface comprises a plurality of target-immobilization probes,
a detectable label, wherein said detectable label comprises a plurality of first members of a reactive pair;
a sample suspected of containing a target molecule; and
a linking molecule, wherein said linking molecule comprises
a plurality of target-specific binding moieties which specifically bind to said target molecule, when present, wherein each of said target-specific binding moieties specifically binds to a different region of said target molecule when present; and
a plurality of second members of said reactive pair,
wherein said combining is under reaction conditions sufficient to provide for specific binding of said target molecule, if present, to one of said plurality of target-immobilization probes, and
wherein said combining is under reaction conditions sufficient to provide for specific binding of said linking molecule to said target molecule, if present; and
subjecting said reaction mixture to conditions suitable for formation of a plurality of covalent bonds between members of said plurality of first members of said reactive pair and members of said plurality of second members of said reactive pair, wherein, when said target molecule is present in said sample, one of said plurality of target-immobilization probes specifically binds to said target molecule, said linking molecule specifically binds to said target molecule and forms a plurality of covalent bonds via reaction of said members of said plurality of first members with said members of said plurality of second members of said reactive pair, thereby forming a complex comprising said substrate surface, said one of said plurality of target-immobilization probes, said target molecule, said linking molecule and said detectable label, and wherein said detectable label is a particle having a diameter of from 0.2 μm to 5 μm and said complex does not comprise more than one of said particle.
2. A method of generating a covalent binding complex comprising a target molecule, said method comprising:
combining in a reaction mixture
a substrate surface, wherein said substrate surface comprises a plurality of target immobilization probes;
a detectable label, wherein said detectable label comprises a plurality of first members of a reactive pair;
a sample suspected of containing a target molecule; and
a plurality of linking molecules, wherein each member of said plurality of linking molecules is independently present in one or more copies, and wherein each member comprises
a target-specific binding moiety which specifically binds to a different region of said target molecule relative to the target-specific binding moieties of the other members of the plurality of linking molecules, when present; and
a second member of said reactive pair,
wherein said combining is under reaction conditions sufficient to provide for specific binding of said target molecule, if present, to one of said plurality of target-immobilization probes, and
wherein said combining is under reaction conditions sufficient to provide for specific binding of said plurality of linking molecules to said target molecule, if present; and
subjecting said reaction mixture to conditions suitable for formation of covalent bonds between first members of said reactive pair and second members of said reactive pair, wherein, when said target molecule is present in said sample, one of said plurality of target-immobilization probes specifically binds to said target molecule, said plurality of linking molecules specifically binds to a plurality of different regions of said target molecule and forms a plurality of covalent bonds via reaction of the first members of said reactive pair with the second members of said reactive pair, thereby forming a complex comprising said substrate surface, said one of said plurality of target immobilization probes, said target molecule, said plurality of linking molecules and said detectable label, and wherein said detectable label is a particle having a diameter of from 0.2 μm to 5 μm and said complex does not comprise more than one of said particle.
3. A reaction mixture comprising:
a substrate surface, wherein said substrate surface comprises a plurality of target-immobilization probes;
a detectable particle having a diameter of from 0.2 μm to 5 μm, wherein said detectable particle comprises a plurality of first members of an azide/alkyne reactive pair;
a sample suspected of containing a target molecule; and
a linking molecule, wherein said linking molecule comprises
a plurality of target-specific binding moieties which specifically bind to said target molecule, when present, wherein each of said target-specific binding moieties specifically binds to a different region of said target molecule when present; and
a plurality of second members of said azide/alkyne reactive pair,
wherein when said target molecule is present in said sample, one of said plurality of target-immobilization probes specifically binds to said target molecule, said linking molecule specifically binds to said target molecule and forms a plurality of covalent bonds via reaction of members of said plurality of first members with members of said plurality of second members of said azide/alkyne reactive pair, thereby forming a complex comprising said substrate surface, said one of said plurality of target-immobilization probes, said target molecule, said linking molecule and said detectable particle, and wherein said complex does not comprise more than one of said detectable particle.
4. The reaction mixture of claim 3 , wherein said detectable particle is a magnetic particle.
5. A reaction mixture comprising:
a substrate surface, wherein said substrate surface comprises a plurality of target-immobilization probes;
a detectable particle having a diameter of from 0.2 μm to 5 μm, wherein said detectable particle comprises a plurality of first members of a reactive pair;
a sample suspected of containing a target molecule; and
a plurality of linking molecules, wherein each member of said plurality of linking molecules is independently present in one or more copies, and wherein each member comprises
a target-specific binding moiety which specifically binds to a different region of said target molecule relative to the target-specific binding moieties of the other members of the plurality of linking molecules, when present; and
a second member of said reactive pair,
wherein when said target molecule is present in said sample, one of said plurality of target-immobilization probes specifically binds to said target molecule, said plurality of linking molecules specifically binds to a plurality of different regions of said target molecule and forms a plurality of covalent bonds via reaction of first members of said reactive pair with second members of said reactive pair, thereby forming a complex comprising said substrate surface, said one of said plurality of target-immobilization probes, said target molecule, said plurality of linking molecules, and said detectable particle, and wherein said complex does not comprise more than one of said detectable particle.
6. The reaction mixture of claim 5 , wherein said detectable particle is a magnetic particle.
7. The method of claim 1 , wherein said detectable label is a magnetic particle.
8. The method of claim 1 , wherein said target molecule is a nucleic acid target molecule.
9. The method of claim 1 , wherein said method further comprises detecting the presence, absence and/or amount of said covalent binding complex.
10. The method of claim 1 , wherein said reactive pair is an azide/alkyne reactive pair.
11. The method of claim 2 , wherein said detectable label is a magnetic particle.
12. The method of claim 2 , wherein said target molecule is a nucleic acid target molecule.
13. The method of claim 2 , wherein said method further comprises detecting the presence, absence and/or amount of said covalent binding complex.
14. The method of claim 2 , wherein said reactive pair is an azide/alkyne reactive pair.
15. The reaction mixture of claim 3 , wherein said target molecule is a nucleic acid target molecule.
16. The reaction mixture of claim 5 , wherein said target molecule is a nucleic acid target molecule.
17. The reaction mixture of claim 5 , wherein said reactive pair is an azide/alkyne reactive pair.