USE OF DIVALENT METALS FOR ENHANCEMENT OF FLUORESCENT SIGNALS

1 . A method of staining a sample, comprising adding to said sample a polymeric dye in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength, wherein the polymeric dye is stored in the presence of a magnesium salt, and wherein the sample comprises a targeting moiety comprising an antibody, wherein 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.

2 . The method of claim 1 , 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′;

Q is, at each occurrence, independently a moiety comprising a reactive group, or protected analogue thereof, capable of forming a covalent bond with an analyte molecule or a targeting moiety;

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 thiophosphoalkylether;

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.

3 . The method of claim 1 , wherein the magnesium salt is magnesium chloride.

4 . The method of claim 1 , wherein the optical response is a fluorescent response.

5 . The method of claim 1 , wherein said sample comprises cells.

6 . The method of claim 2 , wherein the polymeric dye has the following structure (IA):

wherein:

A) z is an integer from 2 to 100; or

B) z is an integer from 3 to 6.

7 . The method of claim 2 , wherein:

A) L 1 has one of the following structures:

B) for at least one occurrence of L 1 , L 1 -M has the following structure:

wherein L 1a and L 1b are each independently optional linkers;

C) for at least one occurrence of L 1 , L 1 -M has the following structure:

wherein L 1a and L 1b are each independently optional linkers,

wherein L 1a and L 1b , when present, are each independently alkylene or heteroalkylene, or

wherein L 1a and L 1b , when present, independently have one of the following structures:

or

D) L 1 is at each occurrence, independently an optional alkylene or heteroalkylene linker.

8 . The method of claim 6 , wherein the compound has the following structure (IB):

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.

9 . The method of claim 2 , wherein R 4 is, at each occurrence, independently OH, O − or OR d , and wherein R 5 is, at each occurrence, oxo.

10 . The method of claim 2 , 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, wherein the activated ester is an N-succinimide ester, imidoester or polyfluorophenyl ester, wherein the azide is an alkyl azide or acyl azide.

11 . The method of claim 2 , 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.

12 . The method of claim 2 , wherein R 1 is, at each occurrence, H.

13 . The method of claim 2 , wherein R 2 and R 3 are each independently OH or —OP(═R a )(R b )R c .

14 . The method of claim 2 , wherein one of R 2 or R 3 is OH or —OP(═R a )(R b )R c , and the other of R 2 or R 3 is Q or a linker comprising a covalent bond to Q.

15 . The method of claim 2 , wherein R 2 and R 3 are each independently —OP(═R a )(R b )R c , wherein R c is OL′.

16 . The method of claim 15 , wherein L′ is a heteroalkylene linker to: Q, the targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (IA).

17 . The method of claim 15 , wherein L′ comprises an alkylene oxide or phosphodiester moiety, or combinations thereof.

18 . The method of claim 15 , wherein L′ has the following structure:

wherein:

m“and n” are independently an integer from 1 to 10;

R e is H, an electron pair or a counter ion;

L″ is R e or a direct bond or linkage to: Q, the targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (IA).

19 . The method of claim 2 , wherein R 2 or R 3 has one of the following structures:

20 . The method of claim 1 , 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:

wherein:

wherein:

wherein:

wherein A is an antibody, and

wherein:

wherein:

wherein m″ is 4 or 10, and

wherein:

wherein m″ is 4 or 10 and A is an antibody, and

wherein:

wherein m″ is 4 or 10, and

wherein:

wherein m″ is 4 or 10, and

wherein:

wherein m″ is 4 or 10, and

wherein:

wherein:

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wherein:

wherein:

wherein m″ is 4 or 10, and

wherein:

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wherein:

wherein:

wherein:

wherein A is an antibody;

wherein:

wherein A is an antibody; or

wherein n is 23,

wherein:

F, F′ and F″ have the following structures, respectively:

dT has the following structure: