Use of divalent metals for enhancement of fluorescent signals
Compositions comprising fluorescent or colored dyes and methods of using the same in combination with divalent metal salts to analyze samples are disclosed.
1. A method for forming a covalent conjugate of a polymeric dye and a targeting moiety, the method comprising preparing a mixture comprising the polymeric dye, the targeting moiety, and a magnesium salt, and allowing the mixture to age for a time and at a temperature sufficient to form the covalent conjugate, wherein:
a) the polymeric dye comprises:
i) two or more fluorescent or colored moieties;
ii) at least one negatively charged phosphate group; and
iii) a reactive group Q capable of forming a covalent bond with a complementary reactive group Q′ on the targeting moiety,
wherein each of the two or more fluorescent or colored moieties are joined to an adjacent fluorescent or colored moiety via a linker comprising the at least one negatively charged phosphate group; and
b) the targeting moiety has affinity for a target analyte and comprises the complementary reactive group Q′.
2. A method for detecting a target analyte, the method comprising:
a) associating a covalent conjugate with the target analyte to form an analyte-targeting moiety complex in the presence of a magnesium salt, the covalent conjugate comprising:
i) a targeting moiety that has affinity for the target analyte and comprises a covalent bond to a polymeric dye;
ii) the polymeric dye comprising:
A) two or more fluorescent or colored moieties;
B) at least one negatively charged phosphate group; and
C) a covalent bond to the targeting moiety,
wherein each of the two or more fluorescent or colored moieties are joined to an adjacent fluorescent or colored moiety via a linker comprising the at least one negatively charged phosphate group; and
b) detecting a fluorescent or colored signal from the analyte-targeting moiety complex.
3. The method of claim 2 , further comprising treating the analyte-targeting moiety complex with a wash solution comprising a magnesium salt.
4. A method for detecting a target analyte, the method comprising:
a) associating a covalent conjugate with the target analyte to form an analyte-targeting moiety complex, the covalent conjugate comprising:
i) a targeting moiety that has affinity for the target analyte and comprises a covalent bond to a polymeric dye; and
ii) the polymeric dye comprising:
A) two or more fluorescent or colored moieties;
B) at least one negatively charged phosphate group; and
C) a covalent bond to the targeting moiety,
wherein each of the two or more fluorescent or colored moieties are joined to an adjacent fluorescent or colored moiety via a linker comprising the at least one negatively charged phosphate group;
b) treating:
i) the covalent conjugate and the target analyte with a magnesium salt during the associating the covalent conjugate and the target analyte; and/or
ii) the analyte-targeting moiety complex with a wash solution comprising a magnesium salt after the associating the covalent conjugate and the target analyte; and
c) detecting a fluorescent or colored signal from the analyte-targeting moiety complex.
5. The method of claim 2 , further comprising substantially removing all of the magnesium salt from the analyte-targeting moiety complex before detecting the fluorescent or colored signal.
6. The method of claim 5 , wherein the magnesium salt is removed from a buffer comprising the analyte-targeting moiety complex.
7. The method of claim 2 , further comprising forming the covalent conjugate.
8. The method of claim 7 , wherein the polymeric dye and a magnesium salt have been admixed to form a composition comprising the polymeric dye and the magnesium salt prior to forming the covalent conjugate.
9. The method of claim 8 , further comprising aging the composition comprising the polymeric dye and the magnesium salt prior to forming the covalent conjugate.
10. The method of claim 7 , wherein magnesium salts are substantially absent while forming the covalent conjugate.
11. A composition comprising a covalent conjugate and a magnesium salt, the covalent conjugate comprising:
i) a targeting moiety comprising a covalent bond to a polymeric dye;
ii) the polymeric dye comprising:
a) two or more fluorescent or colored moieties;
b) at least one negatively charged phosphate group; and
c) a covalent bond to the targeting moiety,
wherein each of the two or more fluorescent or colored moieties are joined to an adjacent fluorescent or colored moiety via a linker comprising the at least one negatively charged phosphate group.
12. The method of claim 2 , wherein the magnesium salt is magnesium chloride.
13. The method of claim 2 , further comprising purifying the covalent conjugate.
14. The method or composition of claim 13 , wherein purifying comprises substantially removing all of the magnesium salt from the covalent conjugate.
15. The method of claim 2 , wherein the covalent conjugate has a fluorescent extinction coefficient at least 1.1 times higher than a corresponding conjugate prepared in the absence of the magnesium salt.
16. The method of claim 2 , wherein the targeting moiety is an antibody.
17. The method of claim 2 , wherein the polymeric dye has the following structure (I):
or a stereoisomer, salt or tautomer thereof, wherein:
M is, at each occurrence, independently a moiety comprising the fluorescent or colored moiety;
L 1 is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups;
L 2 and L 3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;
L 4 is, at each occurrence, independently an alkylene or alkylene oxide linker;
R 1 is, at each occurrence, independently H, alkyl or alkoxy;
R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, —OP(═R a )(R b )R c , Q, or a protected form thereof, or L′;
R 4 is, at each occurrence, independently OH, SH, O − , S − , OR d or SR d ;
R 5 is, at each occurrence, independently oxo, thioxo or absent;
R a is O or S;
R b is OH, SH, O − , S − , OR d or SR d ;
R c is OH, SH, O − , S − , OR d , OL′, SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkyl ether;
R d is a counter ion;
L′ is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to the targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (I);
m is, at each occurrence, independently an integer of zero or greater, provided that at least one occurrence of m is an integer of one or greater, such that the compound includes at least one L 4 ; and
n is an integer of one or greater.
18. The method claim 17 , wherein the polymeric dye has the following structure (IA):
wherein z is an integer from 2 to 100.
19. The method of claim 18 , wherein the compound has the following structure (TB):
wherein:
x 1 , x 2 , x 3 and x 4 are, at each occurrence, independently an integer from 0 to 6; and
z is an integer from 2 to 100.
20. The method of claim 17 , wherein L′ comprises an alkylene oxide or phosphodiester moiety, or combinations thereof.
21. The method of claim 17 , wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional group.
22. The method of claim 17 , wherein M is, at each occurrence, independently a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, bis-fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9, 10-ethynylanthracene or ter-naphthyl moiety.
23. The method of claim 17 , wherein the polymeric dye has one of the following structures:
wherein:
z is an integer from 3 to 6,
m is an integer from 2 to 5, and
n is an integer from 1 to 10;
wherein:
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wherein A is an antibody, and
wherein:
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wherein m″ is 4 or 10, and
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wherein m″ is 4 or 10 and A is an antibody, and
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wherein m″ is 4 or 10, and
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wherein m″ is 4 or 10, and
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wherein m″ is 4 or 10, and
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wherein m″ is 4 or 10, and
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wherein A is an antibody;
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wherein A is an antibody; or
wherein n is 23, wherein:
F, F′ and F″ have the following structures, respectively:
and
dT has the following structure: