CA2740747A1 - Charged conjugated polyelectrolytes with aptamer-functionalized silica nanoparticles - Google Patents

Abstract

A method of detecting a target in a sample is disclosed, comprising functionalizing a solid support with a ligand; incubating the ligand-functionalized solid support with a sample; incubating the sample with a charged conjugated polyelectrolyte (CPE) or charged conjugated oligoelectrolyte (COE); and detecting the fluorescence of the solid support, thereby detecting the target.

Description

4459 1012-001 CA - Application.docx PATENT APPLICATION
AOCRLS/1b(jt) Docket No. 4459.1012-001 May 19, 2011 CHARGED CONJUGATED POLYELECTROLYTES WITH
APTAMER-FUNCTIONALIZED SILICA NANOPARTICLES
BACKGROUND OF THE INVENTION

Protein detection and quantification are of vital importance in both basic discovery research and clinical diagnosis. Enzyme immunosorbent assay (ELISA) is a widely used immunoassay and requires antibodies to be immobilized on a substrate to capture antigens and the secondary antibodies. Despite its high sensitivity, ELISA
requires tedious protein modification and is limited by the availability of commercial antibodies. Although alternative assays have been developed for protein detection using aptamers as the recognition elements, most of these assays require the modification of aptamers with fluorescent dyes or other reporter groups, which are expensive and can impair the original affinity and specificity of the aptamer toward target proteins.
Furthermore, the fluorescence signal of these dyes can be greatly affected by proteins in biological media.
Therefore, there remains a need for CPE-based assays that can be used for real-sample detection (i.e., target detection in mixed protein samples and/or in biological media).
SUMMARY OF THE INVENTION
One embodiment of the invention is a compound of Structural Formula (I), wherein the values and alternative values for the variables are as defined in the Detailed Description of the Invention.
Another embodiment of the invention is a method of detecting a target in a sample, comprising functionalizing a solid support with a ligand; incubating the ligand-functionalized solid support with a sample; incubating the sample with a charged 1092269.1 4459.1012-000 conjugated polyelectrolyte (CPE) or charged conjugated oligoelectrolyte (COE);
and detecting the fluorescence of the solid support, thereby detecting the target.
Yet another embodiment of the invention is a method of detecting a target in a sample, comprising functionalizing a surface of a solid support with a charged ligand, thereby creating a charge on the surface of the solid support; incubating the ligand-functionalized solid support with a sample, whereupon binding of the target, the charge on the surface of the solid support switches; incubating the sample with a conjugated polyelectrolyte (CPE) or a conjugated oligoelectrolyte (COE) that has a complementary charge to the charge of the target-bound surface; and detecting the fluorescence of the solid support, thereby detecting the target.
The compounds of the invention possess high photoluminescence quantum yields in biological media, low cytotoxicity, and excellent environmental stability and photostability, and can be used in biosensor and bioimaging applications.

BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings.
FIG. 1 is a schematic illustration of CPE-based, label-free protein detection.
FIG. 2 is an absorbance spectrum of PFVSO3 in water at [RU] = 4 M
(excitation at 428 nm).

FIG. 3 is a graph depicting the photoluminescence intensity (triangle) and percentage of unbound lysozyme (square) as a function of surface density of aptamers on silica nanoparticle (NP) surface.
FIG. 4 is a photoluminescence (PL) spectrum of polymer-stained NPs incubated with (a) 20 g/mL lysozyme; (b) a mixture of 20 gg/mL each for BSA, thrombin, and trypsin; or (c) a mixture of (a) and (b) followed by subsequent staining with PFVSO3Na in 15 mM PBS at pH = 7.4 (excitation at 428 nm).

FIG. 5 is a PL spectra of polymer-stained NPs incubated with increasing concentrations of lysozyme in 15 mM PBS at pH = 7.4 (excitation at 428 nm).
1092269.1 4459.1012-000 FIG. 6 is the calibration curves for lysozyme detection plotted as PL
intensity as a function of lysozyme concentration (each data point represents the average value of six independent experiments with error bars indicated).
FIG. 7 depicts the synthetic route to P2.
FIG. 8 depicts the synthetic route to P4.1.
DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.
The invention generally relates to a heterogeneous assay that uses charged CPEs or charged COEs with biofunctionalized nanoparticles (NPs) for label-free and, optionally, naked-eye detection of proteins.
As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a biomolecule" can include a plurality of biomolecules. Further, the plurality can comprise more than one of the same biomolecule or a plurality of different biomolecules.
As used herein, "conjugated polyelectrolyte," "conjugated oligoelectrolyte,"
"CPE" and "COE" refer to fluorescent macromolecules with electron-delocalized backbones and water-soluble side chains. CPEs and COEs combine the light-harvesting properties of conjugated polymers with the electrostatic behavior of electrolytes, providing unique opportunities for construction of sensory and imaging materials.
As used herein, "oligo" refers to a monomer unit repeating ten or less times in the chain. For example, "oligo(ethylene oxide)" refers to an ethylene oxide repeat unit [e.g., -(CH2CH2O)õ ], wherein n is 1-10; 2-10; 2-5; 5-10; 2-8; 2-6; or 3-6.
As used herein, "poly" refers to a monomer unit repeating ten or more times in the chain. For example, "poly(ethylene oxide)" refers to an ethylene oxide repeat unit [e.g., -(CH2CH2O)õ], wherein n is greater than 10. Specifically, n is 10-100, 10-200;
10-50; or 50-100.

1092269.1 4459.1012-000 In some embodiments of the invention, the CPEs and COEs are functionalized with polyhedral oligomeric silsesquioxanes (POSS). As used herein, "polyhedral oligomeric silsesquioxanes" or "POSS" are a category of polycyclic compounds, which consist of a silicon/oxygen cage surrounded by tunable organic substitution groups.
Due to the nano-scaled dimension and facile modification of substitution groups, POSS
serve as organic-inorganic nanobuilding blocks for the construction of fluorescent nanomaterials. Functionalization with POSS can minimize self-quenching of CPEs and COEs, which can be desirable for optical applications.

A first embodiment of the invention a CPE or COE represented by Structural Formula (I):

R' R3 M
(I), or a salt thereof, wherein:

R and R2 are each independently -(OCH2CH2)pOCH3 or -(CH2CH2O)pCH3, wherein p is an integer between 1 and 100, inclusive;
R' and R3 are each independently hydrogen or a charged side group;
in is an integer between 2 and 50, inclusive; and T and T' are each independently a terminating group.
In a first aspect of the first embodiment, the CPE or COE is represented by Structural Formula (I), or a salt thereof, with the proviso that the CPE or COE is not represented by the following structural formula:

1092269.1 4459.1012-000 NaO3S SO3Na T- T' OO O
O

wherein the values and alternative values for the remaining variables are as described in the first embodiment.
In a second aspect of the first embodiment, R and R2 are each -(OCH2CH2)pOCH3 or -(CH2CH2O)pCH3. Specifically, R and R2 are each -(CH2CH2O)pCH3. More specifically, p is an integer between 1 and 50, inclusive, between, 1 and 25, inclusive, between 1 and 10, inclusive, or between 1 and 5, inclusive. The values and alternative values for the remaining variables are as described in the first embodiment, or first aspect thereof.
In a third aspect of the first embodiment, R' and R3 are each independently a charged side group, wherein the values and alternative values for the remaining variables are as described in the first embodiment, or first or second aspects thereof.
In a fourth aspect of the first embodiment, R' and R3 are each a charged side group. Specifically, R' and R3 are each an anionic side group. Alternatively, R' and R3 are each a cationic side group. The values and alternative values for the remaining variables are as described in the first embodiment, or first through third aspects thereof.
In a fifth aspect of the first embodiment, in is an integer between 2 and 10, inclusive, or 20 and 30, inclusive. Specifically, in is an integer between 2 and 10, inclusive. Alternatively, in is an integer between 20 and 30, inclusive. The values and alternative values for the remaining variables are as described in the first embodiment, or first through fourth aspects thereof.

1092269.1 4459.1012-000 In some embodiments of the invention, the charged side group can be a cationic alkyl side group, a cationic oligo(ethylene oxide) side group or a cationic poly(ethylene oxide) side group. As used herein, "a cationic alkyl side group" is a (C 1-C
15)alkyl that includes a moiety, such as an amine, that confers a positive charge. As used herein, "cationic oligo(ethylene oxide) side group" and "cationic poly(ethylene oxide) side group" refer to a polymer of ethylene oxide that includes a moiety, such as an amine, that confers a positive charge. The amine can be a primary, a secondary, a tertiary or a quaternary amine. Specifically, the amine is a quaternary amine.
Alternatively, the amine is a protonated amine.
In some embodiments of the invention, the charged side group can be an anionic alkyl side group, an anionic oligo(ethylene oxide) side group or an anionic poly(ethylene oxide) side group. As used herein, "anionic alkyl side group"
refers to a (C 1-C 15)alkyl that includes a moiety, such as a phosphonate, a sulfonate or a carboxylate, that confers a negative charge. As used herein, "anionic oligo(ethylene oxide) side group" and "anionic poly(ethylene oxide) side group" refer to a polymer of ethylene oxide that includes a moiety, such as a phosphonate, a sulfonate or a carboxylate, that confers a negative charge.
In some embodiments of the invention, the charged side groups are selected from the group consisting of -(CH2)õN(R2)3X, -(OCH2CH2)õ N(R2)3X, -(CH2CH2O)gCH2CH2N(R2)3X, -(CH2)õ X', -(OCH2CH2)õ X', -(OCH2CH2)nOX', -(CH2CH2O)õX' and -(CH2CH2O)gCH2CH2X', wherein R2 is (C 1-C6)alkyl, n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, X is an anionic counterion and X' is -CO2Y, -SO3Y or -P03Y2, wherein Y is hydrogen or a cationic counterion.
In some embodiments of the invention, the charged side groups are selected from the group consisting of -(CH2),,N(R2)3X, -(OCH2CH2)õ N(R2)3X and -(CH2CH2O)gCH2CH2N(R2)3X, wherein R2 is (Cl-C6)alkyl, n is an integer between and 13, inclusive, q is an integer between 1 and 12, inclusive, and X is an anionic counterion. Specifically, R2 is methyl or ethyl.

1092269.1 4459.1012-000 In some embodiments of the invention, the charged side groups are selected from the group consisting of -(CH2)õX', -(OCH2CH2)õX', -(OCH2CH2)õ OX', -(CH2CH2O)õX' and -(CH2CH2O)gCH2CH2X', wherein n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, and Xis -CO2Y, -SO3Y
or -PO3Y2, wherein Y is hydrogen or a cationic counterion. Specifically, X is -SO3Y or -P03Y2. More specifically, Xis -SO3Y. Alternatively, Y is a cationic counterion.
In a second embodiment of the invention, the CPE or COE is represented by Structural Formula (II):

R' R3 H3C~0 O)CH3 P P M
(II), or a salt thereof, wherein the values and alternative values for the variables are as described in the first embodiment, or aspects thereof.
In a first aspect of the second embodiment, p is 3, wherein the values and alternative values for the variables are as described in the first embodiment, or aspects thereof or the second embodiment.
In a second aspect of the second embodiment, R' and R3 are each an anionic alkyl side group, wherein the values and alternative values for the variables are as described in the first embodiment, or aspects thereof, or the second embodiment, or first aspect thereof.
In a third aspect of the second embodiment, p is 3 and R' and R3 are each -(CH2)nSO3Y, wherein n is 4 and Y is sodium, wherein the values and alternative values for the variables are as described in the first embodiment, or aspects thereof or the second embodiment, or first or second aspects thereof.

1092269.1 4459.1012-000 In a fourth aspect of the second embodiment, the CPE or COE is represented by Structural Formula (II), or a salt thereof, with the proviso that the CPE or COE is not represented by the following structural formula:

Na03S S3Na T- T' % O

wherein the values and alternative values for the remaining variables are as described in the first embodiment, or aspects thereof, or the second embodiment, or first through third aspects thereof.

In a third embodiment of the invention, the CPE or COE is functionalized with POSS and is represented by the following structural formula:
R R

Linker Ar A A
0-Si-0-S1 A. ' 0. 0 O J R R
Si-. SI~A
O(A~~ l A= Linker ArO-Si10/ OSi\ CPO
3i\O.-Si A A R R

A: Conjugated Oligelectrolyte Linker .. A r =

R: Cationic Side Group or a salt thereof, wherein:

Ar is an optionally substituted aromatic group;
1092269.1 4459.1012-000 Linker is a single bond, double bond, triple bond or -CR'2-; wherein each R' is independently hydrogen, halogen, hydroxy, amino, (C 1 -C6)alkyl, (CI-C6)alkenyl, (C1-C6)alkynyl, or (C1-C6)alkoxy; wherein the alkyl, alkenyl, alkynyl or alkoxy may be optionally substituted with halogen, hydroxy, (C1-C4)alkoxy or amino;
each R is independently hydrogen, a cationic alkyl side group or a cationic oligo or poly(ethylene oxide) group.
In a first aspect of the third embodiment, Linker is a single bond, double bond, triple bond, -CH2- or -CH2CH2-, wherein the values and alternative values for the variables are as described in the third embodiment or in the fourth embodiment, or aspects thereof.

In a second aspect of the third embodiment, the values and alternative values for the variables are as described in the fourth embodiment or aspects thereof.
In a fourth embodiment, the CPE or COE is functionalized with POSS and is represented by the following structural formula:
A
A~,Si ' R'- Ar' - Linker ~' Ar 0, -0-SI

A- ' O-SI-OrSi ASi o .~~Si A A= R'-` Ar' Linker Ar A: Conjugated Oligelectrolyte R-Ar - Linker_ Ar ora salt thereof, wherein:

Ar each is independently selected from:

R R N,R OR R
\ / or RO R

1092269.1 4459.1012-000 each Ar is independently an optionally substituted aromatic group;
each R is independently a cationic, anionic, or neutral alkyl group or a cationic, anionic, or neutral oligo or poly(ethylene oxide) group;
each Linker is a single bond, double bond, triple bond, -CH2- or -CH2CH2-; and each R' is independently a terminating group.
In a first aspect of the fourth embodiment, Ar is fluorene, benzene, biphenyl, pyridine, bipyridinium, triphenylamine, anthracene, thiophene, carbazole, or benzothiadiazole. Optional substituents include those defined by R. The values and alternative values for the remaining variables are as described in the third embodiment, or aspects thereof, or in the fourth embodiment.
In a second aspect of the fourth embodiment, each R is independently selected from the group consisting of hydrogen, -(CH2)nNMe3X; -(CH2)õNEt3X;
-(CH2CH2O)gCH2CH2NMe3X and -(CH2CH2O)gCH2CH2NEt3X, wherein X is an anionic counterion, n is an integer between 2 and 13, inclusive, and q is an integer between 1 and 12, inclusive. Specifically, each R is independently selected from the group consisting of hydrogen, -(CH2)nNMe3X and -(CH2CH2O)gCH2CH2NMe3X, wherein X is an anionic counterion, n is an integer between 2 and 13, inclusive, and q is an integer between 1 and 12, inclusive. The values and alternative values for the remaining variables are as described in the third embodiment, or aspects thereof, or in the fourth embodiment, or first aspect thereof.
In a third aspect of the fourth embodiment, the POSS-functionalized CPE or COE is represented by the following structural formula:

1092269.1 4459.1012-000 R
R
R R
\ I ~ /
R \ I / R
R R R
\ ~ R I i R
si-10-' R R
R R ip!p g.0 Sip Si-0/ OSi R
_-Si R
R R
R R
R
R R
i R
R
\ R Q
R = {CFh)s (CH3)3Br In a fourth aspect of the fourth embodiment, the POSS-functionalized CPE or COE is represented by the following structural formula:

/I
R RR \ /
N
R S
R - N-S ,N \ N R R
Ni /S
I \ i S. ~ Oi-O_Si\
N\ N Sid O-S\
O \ 1 N
-Si- Si N-S
Si\O-Si'O
R R S-N N~
N~ \ N. S-N
R
N

R R
R R
R = -(CH2)6N(CH3)3Br O O
1092269.1 4459.1012-000 In a fifth aspect of the fourth embodiment, R is an anionic group selected from -(CH2)õX', -(OCH2CH2)õX', -(OCH2CH2)nOX', -(CH2CH2O)õX' and -(CH2CH2O)gCH2CH2X', wherein Xis selected from -SO3Y, -P03Y2, and -CO2Y, n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, and Y is a cationic counterion. The values and alternative values for the remaining variables is as described in the fourth embodiment, or aspects thereof, or the fifth embodiment, or the first through fourth aspects thereof.
In a fifth embodiment of the invention, the CPE or COE is a hyperbranched CPE
(HCPE). Specifically, the HCPE is represented by structural formula (III):

T' Ar T
M
Q-~
T,. R' R3 (III), or a salt thereof, wherein:
R' and R3 are each independently hydrogen or a charged side group;
in is an integer between 2 and 50, inclusive;
Ar is an optionally substituted monocyclic or polycyclic aromatic ring system or an optionally substituted monocyclic or polycyclic heteroaromatic ring system; and T, T' and T" are each independently a terminating group.
As used herein, "hyperbranched conjugated polyelectrolyte" or "HCPE" refers to a CPE which has a densely branched structure and a large number of end groups.
In a first aspect of the fifth embodiment, Ar is fluorene, benzene, biphenyl, thiophene, benzothiadiazole, 4,7-di(thien-5'-yl)-2,1,3-benzothiadiazole, pyridine, bipyridinium, triphenylamine, anthracene or carbazole. Specifically, Ar is benzothiadiazole. The values and alternative values for the remaining variables are as described in the first or second embodiments, or aspects thereof, or the fifth embodiment.

1092269.1 4459.1012-000 In a second aspect of the fifth embodiment, T, T' and T" are each -CCH, wherein the values and alternative values for the remaining variables are as described in the first or second embodiments, or aspects thereof, or the fifth embodiment, or the first aspect thereof.

In a third aspect of the fifth embodiment, Wand R3 are each a charged side group. Specifically, the charged side groups are selected from the group consisting of -(CH2)õN(R2)3X, -(OCH2CH2)õN(R2)3X and -(CH2CH2O)gCH2CH2N(R2)3X, wherein R2 is (C 1-C6)alkyl, n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, and X is an anionic counterion. The values and alternative values for the remaining variables are as described in the first or second embodiments, or aspects thereof, or the fifth embodiment t, or the first or second aspects thereof.
In a fourth aspect of the fifth embodiment, the HCPE is represented by the following structural formula:

T' N\ S, N

T R'= R3 = (CH2)6N(CH3)3Br M
T"
R' R3 wherein the values and alternative values for the remaining variables are as described in the first or second embodiments, or aspects thereof, or the fifth embodiment, or the first through third aspects thereof.
In a fifth aspect of the fifth embodiment, in is an integer between 2 and 30, inclusive, wherein the values and alternative values for the remaining variables are as described in the first or second embodiments, or aspects thereof, or the fifth embodiment, or the first through fourth aspects thereof.
In a sixth aspect of the fifth embodiment, the values and alternative values for the variables are as described in the first, second or sixth embodiments, or aspects thereof.

1092269.1 4459.1012-000 A sixth embodiment of the invention is a molecular brush represented by structural formula (IV):

T - \ / \Ar T
R' R3 M
(IV), or a salt thereof; wherein:
R' and R3 are each independently hydrogen or a charged side group;
m is an integer between 2 and 50, inclusive;
Ar is an optionally substituted monocyclic or polycyclic aromatic ring system or an optionally substituted monocyclic or polycyclic heteroaromatic ring system; and T and T' are each independently a terminating group.
As used herein, "molecular brush" refers to a CPE or COE with densely grafted side chains on a linear polymeric backbone.
In a first aspect of the sixth embodiment of the invention, the values and alternative values for the variables are as defined in the first, second or fifth embodiments, or aspects thereof.
In a second aspect of the sixth embodiment, T and T are each independently hydrogen, halo, -CH=CH2 or -CH2CH3, wherein the values and alternative values for the variables are as defined in the first, second or fifth embodiments, or aspects thereof, or the sixth embodiment, or the first aspect thereof.
In a third aspect of the sixth embodiment, the CPE or COE is represented by the following structural formula:

1092269.1 4459.1012-000 N\S, N

T T' R'= R3 = (CH2)6N(CH3)3Br R' R3 wherein the values and alternative values for the variables are as defined in the first, second or fifth embodiments, or aspects thereof, or the sixth embodiment, or the first or second aspects thereof.
In a fourth aspect of the sixth embodiment, Ar is an optionally substituted monocyclic or polycyclic (C6-C12)aromatic ring system or an optionally substituted monocyclic or polycyclic (C6-C 12)heteroaromatic ring system, wherein the values and alternative values for the remaining variables are as described in the first, second, or fifth embodiments, or aspects thereof, of the sixth embodiment, or the first through third aspects thereof.

In a fifth aspect of the sixth embodiment, the CPE or COE is not represented by the following structural formula:

Na03S S3Na Of 0 01O

wherein the values and alternative values for the remaining variables are as described in the first, second, or fifth embodiments, or aspects thereof, of the sixth embodiment, or the first through fourth aspects thereof.

1092269.1 4459.1012-000 In a sixth aspect of the sixth embodiment, in is an integer between 2 and 10, inclusive, or 20 and 30, inclusive, wherein the values and alternative values for the remaining variables are as described in the first, second, or fifth embodiments, or aspects thereof, or the sixth embodiment, or the first through fifth aspects thereof.
As used herein, "terminating group" refers to the functional group left at each end of a polymer upon termination of the polymerization reaction. Non-limiting examples of terminating groups include hydrogen, halo, -CH=CH2, -CCH and -CH2CH3.

"Alkyl" means an optionally substituted saturated aliphatic branched or straight-chain monovalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C1-C6) alkyl" means a radical having from 1-6 carbon atoms in a linear or branched arrangement. "(C1-C6)alkyl" includes, for example, methyl, ethyl, propyl, iso-propyl, n-butyl, tent-butyl, pentyl and hexyl.

"Alkenyl" refers to a straight or branched aliphatic group with at least one double bond. Typically, alkenyl groups have from 2 to 12 carbon atoms, from 2 to 8, from 2 to 6, or from 2 to 4 carbon atoms. Examples of alkenyl groups include ethenyl (-CH=CH2), n-2-propenyl (allyl, -CH2CH=CH2), pentenyl, hexenyl, and the like.
"Alkynyl" refers to a straight or branched aliphatic group having at least 1 site of alkynyl unsaturation. Typically, alkynyl groups contain 2 to 12, 2 to 8, 2 to 6 or 2 to 4 carbon atoms. Examples of alkynyl groups include ethynyl (-C=CH), propargyl (-CH2C=CH), pentynyl, hexynyl, and the like.

As used herein, "halogen" refers to fluorine, chlorine, bromine or iodine.
"Halogen" and "halo" are used interchangeably herein.
"Alkoxy" means an alkyl radical attached through an oxygen linking atom.
"(C1-C3)alkoxy" includes methoxy, ethoxy and propoxy.

"Aryl" or "aromatic" means an aromatic monocyclic or polycyclic (e.g., bicyclic or tricyclic) carbocyclic ring system. Thus, "(C5-C14)aryl" is a (5-14)-membered monocylic or bicyclic system. Aryl systems include, but are not limited to, phenyl, naphthalenyl, fluorenyl, indenyl, azulenyl, and anthracenyl.

1092269.1 4459.1012-000 "Hetero" refers to the replacement of at least one carbon atom in a ring system with at least one heteroatom selected from N, S and 0. "Hetero" also refers to the replacement of at least one carbon atom in an acyclic system. A hetero ring system or a hetero acyclic system may have, for example, 1, 2 or 3 carbon atoms replaced by a heteroatom.
"Heteroaryl" means a monovalent heteroaromatic monocyclic or polycyclic (e.g., bicylic or tricyclic) ring radical. A heteroaryl contains 1, 2, 3 or 4 heteroatoms independently selected from N, 0 and S. Thus, "(C5-C14)heteroaryl" refers to a (5-14)-membered ring system, wherein at least one carbon atom has been replaced with at least one heteroatom selected from N, S and 0. Heteroaryls include, but are not limited to furan, oxazole, thiophene, 1,2,3-triazole, 1,2,4-triazine, 1,2,4-triazole, 1,2,5-thiadiazole 1,1-dioxide, 1,2,5-thiadiazole 1-oxide, 1,2,5-thiadiazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole, 1,3,5-triazine, imidazole, isothiazole, isoxazole, pyrazole, pyridazine, pyridine, pyridine-N-oxide, pyrazine, pyrimidine, pyrrole, tetrazole, and thiazole.
"Bicycloheteroaryl," as used herein, refers to bicyclic heteroaryl rings, such ase bicyclo[4.4.0] and bicyclo[4.3.0] fused ring systems containing at least one aromatic ring and 1 to 4 heteroatoms independently selected from N, 0 and S. In some embodiments of the invention, the first ring is a monocyclic heterocyclyl (such as dioxolane) and the second ring is a monocyclic aryl (such as phenyl) or a monocyclic heteroaryl (such as pyridine). Examples of bicyclic heteroaryl rings include, but are not limited to, indole, quinoline, quinazoline, benzothiophene, benzofuran, 2,3-dihydrobenzofuran, benzodioxole, benzimidazole, indazole, benzisoxazole, benzoxazole and benzothiazole.
Each aryl and heteroaryl is optionally and independently substituted.
Exemplary substituents include halogen, (C1-C3)alkoxy, (C1-C3)alkylthio, hydroxy, (C5-C14)aryl, (C5-C14)heteroaryl, (C3-C1s)cycloalkyl, (C3-C15)heterocyclyl, amino, (C1-Cs)alkylamino, (C1-C5)dialkylamino, thio, oxo, (C1-C5)alkyl, (C5-C14)aryl(C1-C5)alkyl, (C5-C14)heteroaryl(C1-Cs)alkyl, nitro, cyano, sulfonato, phosphonato, carboxylate, hydroxyl(C1-C5)alkyl and halo(C1-C5)alkyl.

1092269.1 4459.1012-000 "Anionic counterion," as used herein, refers to a negatively charged ion.
Examples of anionic counterions include, but are not limited to, halide, trifluoroacetate, acetate, benzenesulfonate, benzoate, perchlorate, sulfonate, bicarbonate, carbonate, citrate, mesylate, methylsulfate, nitrate, phosphate/diphosphate, sulfate, trifluoromethanesulfonate, tetrafluoroborate, ammonium hexafluorophosphate and tetrakis[3,5,-bis(trifluoromethyl)phenyl]borate. Specifically, the anionic counterion is halide, tetrafluoroborate, trifluoromethanesulfonate, ammonium hexafluorophosphate or tetrakis[3,5,-bis(trifluoromethyl)phenyl]borate. More specifically, the halide is bromide or iodide. Yet more specifically, the halide is bromide.
"Cationic counterion," as used herein, refers to a positively charged ion.
Specifically, the cationic counterion is sodium, lithium or potassium. More specifically, the cationic counterion is sodium or potassium.
One embodiment of the invention is illustrated in FIG. 1. FIG. 1 depicts the functionalization of NPs [e.g., silica NPs, polystyrene NPs, poly(methylmethacrylate) NPs], with a ligand, such as an aptamer, to yield ligand-functionalized NPs.
These ligand-functionalized NPs can be further treated with a blocking agent, such as ethanolamine, to generate blocked NPs. Upon incubation with a sample containing a target, such as a protein (e.g., lysozyme), the blocked NPs specifically bind the target.
Binding of the target switches the charge of the NPs. For example, if the NPs were initially negatively-charged, upon binding of the target, the NPs will be positively-charged. A fluorescent CPE that has a complementary charge to the target can be added to the NP-treated sample to yield CPE/target/ligand complexes on the surface of the NP, giving rise to fluorescent NPs after removal of excess CPE, which can be accomplished, for example, by a wash-centrifugation-redispersion process. Since no binding takes place between the ligand and non-specific proteins, the surface charge on the ligand-functionalized NPs that are not bound to the target remains the same as that of the CPE.
The CPE is thus electrostatically repelled from NPs not bound to the target and, as a result, NPs not bound to the target remain non-fluorescent. By taking advantage of the 1092269.1 4459.1012-000 recognition-induced switching of surface charge, label-free, naked-eye protein detection can be realized.

"Biomolecule," as used herein, refers to a natural or synthetic molecule for use in biological systems. Examples of biomolecules include, but are not limited to, proteins, peptides, enzyme substrates, pharmaceuticals, ligands, hormones, antibodies, antigens, haptens, carbohydrates, oligosaccharides, polysaccharides, nucleic acids, aptamer, fragments of DNA, fragments of RNA and mixtures thereof.

"Ligand," as used herein, refers to a molecule that specifically binds to a biomolecule, such as a target. Examples of ligands include, but are not limited to, aptamers [e.g., anti-lysozyme aptamer (5'-NH2-ATC TAC GAA TTC ATC AGG GCT
AAA GAG TGC AGA GTT ACT TAG; SEQ. ID. NO. 1), anti-thrombin aptamer (5'-NH2-GGT TGG TGT GGT TGG; SEQ. ID. NO. 2)] and antibodies (e.g., anti-thrombin).

Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. More specifically, aptamers can be classified as: DNA or RNA
aptamers, consisting of (usually short) strands of oligonucleotides or peptide aptamers, consisting of a short variable peptide domain, attached at both ends to a protein scaffold. An aptamer to be immobilized on the solid support is selected based upon its ability to bind the biological molecule of interest.
"Target," as used herein, refers to a biomolecule that specifically binds to another biomolecule. Examples of targets include, but are not limited to, a protein, a peptide, an enzyme, an oligosaccharide, a polysaccharide, a fragment of DNA
and a fragment of RNA. In some embodiments of the invention, target proteins (e.g., lysozyme, thrombin) bind ligands (e.g., anti-lysozyme aptamer, anti-thrombin aptamer).
As used herein, "functionalized" refers both to (1) the covalent attachment of a ligand to a nanoparticle, as might be achieved, for example, by chemical reaction, and to (2) the noncovalent attachment of a ligand to a nanoparticle, as might be achieved, for example, by surface adsorption. In some embodiments, a surface of a solid support (e.g., NP) is functionalized with a ligand.

1092269. I

4459.1012-000 The compounds according to the present invention may be in free form or in the form of salts. These salts may be obtained by reacting the respective compounds with acids and bases. Examples of such salts include but are not limited to hydrochloride, hydrobromide, hydroiodide, hydrofluoride, nitrate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, phosphate, acid phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, isonicotinate, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, lactate, salicylate, citrate, tartrate, oxalate, malonate, suberate, sebacate, mandelate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, phenylacetate, malate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)] salts.
Certain compounds of the invention can form salts with various amino acids.
Suitable base salts include, but are not limited to, aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine salts.

CPEs undergo a photophysical property change upon interaction with proteins.
For example, the emission intensity, emission maximum, and/or the absorption maximum, as well as the associated fluorescence and absorbance profiles, can change upon interaction with proteins. (See (a) Ambade, A. V., et al., S. Polym. Int.
2007, 56, 474-481. (b) Ho, H. A., et al., Ace. Chem. Res. 2008, 41, 168-178. (c) Li, K.;
Liu, B.
Polym. Chem. 2010, 1, 252-259.) Water solubility of CPEs is achieved through introduction of charged hydrophilic functionalities to the macromolecular backbone. Good water solubility minimizes polymer interchain aggregation, which leads to less fluorescence quenching and greater fluorescence intensity in aqueous solution. (See (a) Khan, A., et al., Chem.
Commun. 2005, 584-586. (b) Lee, K. W., et al.,Chem. Commun. 2006, 1983-1985;
the entire teachings of which are incorporated herein by reference). In addition, good 1092269.1 4459.1012-000 polymer water solubility can minimize nonspecific interactions between CPEs and the nanoparticles, thereby decreasing any background signal.
One embodiment of the present invention is a method of detecting a target in a sample, comprising: functionalizing a solid support with a ligand; incubating the ligand-functionalized solid support with a sample; incubating the sample with a CPE
or COE;
and detecting the fluorescence of the solid support, thereby detecting the target.
Specifically, the CPE or COE is a charged CPE or COE.
A sample can be, for example, a cellular lysate, a biomolecule, a cell, a mixture of biomolecules, or a mixture thereof. A sample can be in the form of a solution in buffer, for example, and can include biological media.
As used herein, "incubating the sample with a CPE or COE" means the sample and the CPE or COE are present in the same container or in the same solution and may come into contact. Incubating the sample with the CPE or COE includes adding the CPE or COE, either in suspension or as a solid, to the sample.
In some embodiments, the method further includes isolating the solid support from the sample. In other embodiments, the method further includes isolating the solid support from the sample and washing the solid support. Isolating the solid support from the sample and/or washing the solid support can occur before detecting the fluorescence of the solid support.

Suitable solid supports include nanoparticles (NPs) or solid-state substrates (e.g., paper, glass, quartz). Silica NPs, in particular, can be easily functionalized, are chemically inert, and are easily separable from biological media. The chemical modification of silica NPs can be accomplished chemically using reactive functional groups (e.g., cyanuric chloride, aldehyde, and NHS ester) (see, for example, Steinberg, G., et al., Biopolymers 2004, 73, 597-605; Kato, N.; Caruso, F. J. Phys. Chem.
B 2005, 109, 19604-19612; and Liang, Y, et al., Talanta 2007, 72, 443-449, the entire teachings of each are incorporated herein by reference). Meanwhile, the high density of silica (1.96 g/cm3) facilitates easy separation of NPs from biological media via centrifugation-washing-redispersing circles. Such a method can help to eliminate 1092269.1 4459.1012-000 nonspecific proteins, while retaining the bound target, and can promote the trace detection of a target in biological samples. In addition, silica NPs of 100 nm in diameter are transparent in dilute solutions, and their optical properties do not interfere with those of fluorescent dyes or CPEs.
Aptamer-functionalized silica NPs can be an effective platform for selectively capturing a target, such as lysozyme or thrombin, and effectively isolating the target via centrifugation-washing-redispersing circles. Lysozyme binding to aptamer-functionalized silica NPs switches the surface charges of Apt-NP from negative to partially positive, which subsequently allows for CPE binding, which can be detected as blue-green fluorescence by, for example, the naked eye or a fluorescence spectrometer.
Moreover, the linear intensity increase of polymer emission as a function of lysozyme concentration allows the accurate quantification of lysozyme in the concentration range of 0 to approximately 22.5 M with a limit of detection of approximately 0.36 g/mL.
The high quantum yield and good water solubility of CPEs also enables naked-eye lysozyme detection with picomole sensitivity.
In a specific embodiment, aptamer-functionalized silica nanoparticles (NPs) have been synthesized to capture lysozyme, resulting in a switching of the surface charge from negative to partially positive. The aptamer/protein binding event can be monitored by fluorescence spectroscopy. Upon its addition, PFVSO3 binds to and "stains" the protein/aptamer/NP complexes via an electrostatic interaction.
The blue-green fluorescence of PFVSO3 can be observed in the presence of lysozyme by the naked eye, while no fluorescence is obtained for NPs treated with a non-specific mixture of proteins.
One embodiment of the invention is a method of detecting a target in a sample, comprising functionalizing a surface of a solid support with a charged ligand, thereby creating a charge (e.g., a positive or negative charge) on the surface of the solid support;
incubating the ligand-functionalized solid support with a sample, whereupon binding of the target, the charge on the surface of the solid support switches (e.g., from positive to negative or from negative to positive); incubating the sample with a conjugated 1092269.1 4459.1012-000 polyelectrolyte (CPE) or a conjugated oligoelectrolyte (COE) that has a complementary charge to the charge of the target-bound surface (i.e., if the target-bound surface is negatively charged, the CPE or COE is positively charged and visa versa); and detecting the fluorescence of the sample, thereby detecting the target.
In some embodiments, the ligand is a charged ligand. As used herein, "charged ligand" refers to a ligand having a net positive or net negative charge under the conditions of the assay. Typically, the conditions are neutral conditions or neutral pH.
Proteins, CPEs and COEs can also be described as "charged" if they have a net positive or net negative charge under the conditions of the assay.
In a specific embodiment, the biological molecule to be detected is lysozyme, which has an isoelectric point (pI) of 11.0, and is, therefore, positively charged at neutral pH. Lysozyme is a ubiquitous protein serving as the "body's own antibiotic" by cleaving acetyl groups in the polysaccharide walls of many bacteria.
Therefore, the lysozyme level in blood is regarded as the clinical index for many diseases such as HIV, myeloid leukemia, etc. (see (a) Vocadlo, D. J., et al., Nature 2001, 412, 835-838. (b) Lee-Huang, S. et al., Proc. Natl. Acad. Sci. U &A. 1999, 96, 2678-268 1, the teachings of each are herein incorporated by reference).
One embodiment of the invention is a label-free, naked-eye lysozyme detection method using aptamer-functionalized silica NPs as the recognition element to capture a target and an anionic conjugated polymer as "a polymeric stain" to transduce a signal.
EXEMPLIFICATION
Example 1. Label-free, Naked-eye detection of Lysozyme Using CPEs Antilysozyme aptamer (5'-NH2-ATC TAC GAA TTC ATC AGG GCT AAA
GAG TGC AGA GTT ACT TAG SEQ ID NO.: 1) was ordered from Sigma-Genosys.
Hen egg white lysozyme, BSA, and human trypsin were ordered from Sigma-Aldrich.
Human R-thrombin was ordered from HTI.

Instrumentation. The NMR spectra were collected on a Bruker ACF400 (400 MHz). The absorption spectra of aptamer and lysozyme were measured using a UV-vis 1092269.1 4459.1012-000 spectrometer (Shimadzu, UV- 1700, Japan). The photoluminescence spectra were recorded on a fluorometer (Perkin-Elmer, LS-55) equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 PMT, using 90 angle detection for solution samples. The size of silica NPs was calculated using a field emission scanning electron microscope (FE-SEM JEOLJSM-6700 F) after coating a thin Pt layer via a platinum coater. The zeta-potential of the NPs was measured using a zeta-potential analyzer (ZetaPlus, Brookhaven Instruments Corp.) at room temperature.
Synthesis and Characterization of PFVSO3. 2,7-Dibromo-9,9-bis(2-(2-(2-methoxyethoxy)-ethoxy)ethyl)fluorene was synthesized according to our previous report. (See, for example, (a) Pu, K. Y., et al., Adv. Funct. Mater. 2008, 18, 1321-1328;
(b) Wang, F. K.; Bazan, G. C., J. Am. Chem. Soc. 2006,128,15786-15792; (c) Pu, K.
Y., et al., Chem. Mater. 2009, 21, 3816-3822, the entire teachings of which are incorporated herein by reference.) 9,9-Bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-2,7-divinylfluorene (1). 2,7-dibromo-9,9-bis(2-(2-(2-methoxyethoxy)-,ethoxy)ethyl)fluorene (1.23 g, 2.0 mmol), tributylvinyltin (1.33 g, 4.2 mmol), PdC12(PPh3)2 (56 mg, 0.09 mmol), 2,6-di-tert-butylphenol (8 mg, 38 mmol), and toluene (20 mL) were mixed in a 50-mL flask.
The reaction mixture was stirred and heated at 100 C for 24 hours under nitrogen.
After cooling to room temperature, the mixture was diluted with ether, treated with an aqueous solution of HF (approximately 10%), and stirred for 12 hours. The mixed solution was then filtered to remove the solids, and the filtrate was dried over anhydrous MgSO4. The solvent was removed under reduced pressure, and the residue was chromatographed on silica gel using hexanes/ethyl acetate (1:1) as eluent to give 1 (0.70 g, 68%) as a blue liquid. 1HNMR (500 MHz, CDC13, 6 ppm):7.60(d, 2H, J=7.8 Hz), 7.44 (s, 2 H), 7.39 (d, 2 H, J=7.7 Hz), 6.78 (dd, 2 H, J=10.9 Hz, J=17.6 Hz), 5.80 (d, 2 H, J=17.5Hz),5.27(d, 1092269.1 4459.1012-000 2H, J=10.9 Hz),3.51 (dd,4H, J = 3.4 Hz, J = 5.9 Hz), 3.46 (dd, 4 H, J = 3.3 Hz, J =6.0 Hz), 3.39 (t, 4 H, J = 3.2 Hz), 3.33 (s, 6 H), 3.21 (t, 4 H, J= =3.3 Hz), 2.76 (t, 4 H, J =
Hz), 2.40 (t, 4 H, J =5.17 Hz). 13CNMR (125 MHz, CDC13, S ppm): 149.50, 139.96, 137.00, 136.83, 125.82, 120.69, 119.85, 113.54, 71.83, 70.43, 70.39, 69.96, 66.98, 58.96, 50.96, 39.75.
2,7-Dibromo-9,9-bis(4-sulfonatobutyl)fluorene disodium (2). 2,7-Dibromofluorene (4 g, 12 mmol) and tetrabutylammoium bromide (80 mg) were dissolved in a mixture of a 50 wt % aqueous solution of sodium hydroxide (8 mL) and dimethyl sulfoxide (DMSO) (60 mL). A solution of 1,4-butane sultone (4 g, 29 mmol) in DMSO (20 mL) was added dropwise into the mixture under nitrogen. After stirring at room temperature for 4 hours, the reaction mixture was precipitated into acetone to afford the crude product. The product was collected by filtration, washed with ethanol, recrystallized twice from acetone/water, and dried under vacuum at 60 C for 24 hours to yield 2 as white needle crystals (4.3 g, 58.6%). 'H NMR (500 MHz, CD3OD, 6 ppm):
7.68 (d, J=8.11 Hz, 2 H), 7.63 (d, 2 H, J = 1.45 Hz), 7.52 (dd, 2 H, J = 1.42, 8.08 Hz), 2.68-2.47 (m, 4 H), 2.22-2.00 (m, 4 H), 1.62 (td, 4 H, J =7.83, J=7.83, J=15.65 Hz,), 0.67 (td, 4 H, J= 7.83, J=7.83, J=15.65 Hz). 13C NMR (125 MHz, CD3OD, 6 ppm):
153.39, 140.68, 131.61, 127.38, 122.74, 122.52, 52.37, 40.76, 26.19, 24.25. MS
(MALDI-TOF): m/z 619.89 [M-Na]. (See, for example, Huang, F., et al., Polymer 2005, 46, 12010-12015, the entire teachings of which are incorporated herein by reference.) Poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)fluorenevinylene-alt-9,9-bis(4-sulfonatobutyl)fluorenevinylene Sodium Salt] (PFVSO3). 1 (216 mg, 0.423 mmol), 2 (271 mg, 0.423 mmol), Pd(OAc)2 (4.0 mg, 0.018 mmol), and P(o-tolyl)3 (30 mg, 0.098 mmol) were placed in a round-bottomed flask. A mixture of DMF (3.0 mL), H2O (1.0 mL), and triethylamine (1.5 mL) was added to the flask, and the reaction vessel was degassed. The mixture was vigorously stirred at 110 C for 12 hours. The mixture was filtered through a 0.22 m syringe driven filter unit, and the filtrate was poured into acetone. The precipitate was collected and washed with acetone and then 1092269.1 4459.1012-000 dried under vacuum for 24 hours to afford PFVSO3 (328 mg, 78%, Mn=15000) as yellow fibers. 'H NMR (500 MHz, CD3OD, 6 ppm): 7.87-7.51(m, 12 H), 7.38 (br, 4 H), 3.54-3.39 (m, 12 H), 3.36 (br, 4 H), 3.27-3.13 (m, 6 H), 2.90 (br, 4 H), 2.57 (br, 8 H), 2.20 (br, 4 H), 1.63 (br, 4 H), 0.76 (br, 4H). 13C NMR (125 MHz, CD3OD, 6 ppm):
150.90, 149.97, 140.69, 140.00, 137.01, 128.63, 128.25, 126.13, 125.81, 120.86, 120.45, 119.71. 119.58, 71.45, 69.95, 69.91, 69.85, 69.82, 69.50, 57.74, 54.67, 51.18, 42.01, 39.20, 25.00.
Comparison of the integrated areas between the peak at 5.95 ppm and the peak at 0.76 ppm revealed that the number-average degree of polymerization (DP) of PFVSO3 is approximately 15. Thus, the number-average molecular weight is approximately 15,000.
The water solubility of PFVSO3 is approximately 20 mg/mL at 24 C.
The absorbance and photoluminescence (PL) spectra of PFVSO3 in water are depicted in FIG. 2. The polymer concentration based on repeat unit (RU) is 4 M.
PFVSO3 has an absorption maximum at 428 nm and a shoulder peak at 455 nm, while its emission maximum is at 475 nm. While not wishing to be bound by any particular theory, the blue-green emission of PFVSO3 is attributed to the introduction of CdC bond to the polymer backbone, which elongates the effective conjugated length relative to that of polyfluorene. The PL quantum yield of PFVSO3 in water is 0.56 and was measured using quinine sulfate in O.1M H2SO4 (quantum yield = 0.55) as the reference.
The high water solubility provided by the terminal sulfonate groups and the ethylene oxide side chains is thought to be responsible for the high quantum yield of PFVSO3 in aqueous solution. (See Mikroyannidis, J. A.; Barberis, V. P. J. Polym. Sci., Part A:
Polym. Chem.
2007, 45, 1481-1491.) Preparation of anti-Lysozyme Aptamer-Functionalized Silica NPs. The bare silica NPs were synthesized according to a modified Stober method, which yielded uniform NPs with a diameter of approximately 100 nm. (See Stober, W., et al., J.
Colloid Interface Sci. 1968, 26,62-69, the entire teachings of which are incorporated herein by reference.) On the basis of the NP size and the density of silica (1.96 g cm 3), it can be estimated that 1.0 mg of the synthesized NPs contained approximately 1 x 1012 1092269.1 4459.1012-000 NPs. Modification of the silica NP surface involved two steps. (See Wang, Y.
S.; Liu, B.
Anal. Chem. 2007, 79, 7214-7220, the entire teachings of which are incorporated herein by reference). First, the silica NP was reacted with 3-aminopropyltriethoxysilane (APTES) to generate amino groups on the NP surface. Then, the amino-functionalized NPs were treated with 2,4,6-trichloro-1,3,5-triazine to produce a triazine-covered surface for subsequent aptamer immobilization. After chemical modification, the triazine-functionalized silica NPs (1 mg) were dispersed in immobilization buffer (20.1 mM
boric acid, 1.4 mM sodium tetraborate decahydrate, 1.2 M NaCl pH 8.5, 25 L).
In heterogeneous assays, the kinetic and thermodynamic binding process of the analyte can be significantly influenced by the density of the recognition element on the solid support. (See, for example, (a) Peterson, A. W., et al., Nucleic Acids Res. 2001, 29, 5163-5168; (b) Gong, P.; Levicky, R., Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5306; (c) Herne., T. M.; Tarlov., M. J., J. Am. Chem. Soc. 1997, 119, 8916-8920, the entire teachings of which are incorporated herein by reference.) Previous studies have shown that aptamer-target binding can be inhibited by densely-packed aptamers on gold rod electrodes due to cross-hybridization of individual aptamer sequences (See, for example, White, R. J., et al., Langmuir 2008, 24, 10513-10518, the entire teachings of which are incorporated herein by reference.) To study the effect of aptamer density on lysozyme detection, different concentrations of aptamers, ranging from 2 to 36 gM were incubated with silica NPs (1 mg) to prepare Apt-NPs with different aptamer densities on the NP surface. The surface density, expressed as "number of aptamers per NP", was determined by the ratio of the total number of immobilized aptamers to the total number of silica NPs in solution.
Various aliquots of NH2-aptamer solution (100 M) from 0.5 to 9 L were subsequently added into the NP suspension and incubated at room temperature for 14 hours.
The NP
suspension was centrifuged, and the supernatant was collected for absorbance measurements. The aptamer-immobilized NPs were washed with immobilization buffer.
The number of immobilized aptamer molecules on the silica NPs was calculated from the absorbance difference at 260 nm between the aptamer solution before 1092269.1 4459.1012-000 immobilization and the supernatant after immobilization and NP removal. The surface density was calculated to be in a range of 30 Apt/NP to 510 Apt/NP.

To minimize nonspecific absorption of proteins on NPs, ethanolamine was used to block the free triazine sites on the NP surface after aptamer immobilization. (See, for example, Wang, Y. S.; Liu, B. Chem. Commun. 2007, 34, 3553-3555; Frederix, F., et al., Biochem. Biophys. Methods 2004, 58, 67-74; the teachings of which are incorporated herein by reference.) Blocking was carried out by redispersing the Apt-NPs (1 mg) in blocking buffer (4 M ethanolamine, 20 mM Tris-HCI, 100 mM NaC1, 5 mM MgC12, pH
= 8.5, 200 L) and incubating the resulting mixture for 1 hour at room temperature. The NP suspension was then centrifuged and washed with washing buffer (20 mM Tris-HCI, 100 mM NaCl, 5 mM MgCl2, pH = 8.5).
Optimization of Assay. Aptamer-functionalized NPs (2 mg) with different probe densities were incubated with the same concentration of lysozyme (20 g/mL), then washed. The lysozyme bound aptamer-NPs (lysozyme/Apt-NPs) were subsequently treated with 10 M PFVSO3 based on repeat unit (RU) for 5 minutes, which was followed by washing to remove excess polymer. The PL intensity of the final NP
suspension was plotted as a function of aptamer surface density, and the results are shown in FIG. 3. The PL intensity significantly decreases with increased surface aptamer density, which could be ascribed to insufficient binding of lysozyme to aptamer at elevated surface density. (See Cheng, A. K. H., et al., Anal. Chem. 2007, 79, 5158-5164, the entire teachings of which are incorporated herein by reference). At low surface density, aptamers have more space which favors their G-quartet folding structure for lysozyme binding. However, in the case of high surface density, steric/conformational effects could hamper the specific binding between lysozyme and the aptamer. To further confirm this hypothesis, the adsorbed lysozyme was monitored according to the UV difference at 280 nm between the same lysozyme solution before incubation and the supernatant solution after incubation with different Apt-NPs and NP
removal. As shown in FIG. 3, the percentage of unbound lysozyme increases with increased aptamer density on NPs, which verifies that more lysozyme molecules are 1092269.1 4459.1012-000 captured by Apt-NPs at a low surface density. The optimum surface density was approximately 60 aptamers per NP (60 Apt-NP), where the polymer stained Apt-NP
PL
intensity reached the maximum, which is beneficial for effective lysozyme quantification.
To understand the surface charge change upon aptamer/lysozyme/PFVSO3 interaction, the zeta-potentials of 60 Apt-NP, lysozyme/Apt-NPs (2 mg of 60 Apt-NP
upon incubation with 20 gg/mL of lysozyme, followed by washing with washing buffer and redispersion), and PFVSO3/lysozyme/Apt-NP (the obtained lysozyme/Apt-NPs upon further treatment with 1 gM PFVSO3 followed by washing with water and redispersion) were measured. 60 Apt-NP possess a negative zeta-potential value of -39.34 1.55 mV, due to the large amount of negatively-charged aptamers on NP
surface. The capture of lysozyme shifts the zeta potential from -39.35 to -14.96 f 0.88 mV, due to the presence of positively charged lysozyme molecules on NP
surface.
Staining with PFVSO3 results in an increase in zeta-potential from -14.96 to -35.75 +
1.44 mV due to self-assembly between PFVSO3 and lysozyme on NPs. This data confirms that the NP surface charge changes in the recognition event, which plays a vital role in lysozyme detection.
Lysozyme Detection Using Blocked Apt-NPs. Various volumes of lysozyme (1.5 mg/mL) were added to the Apt-NPs (0.2 mg) in lysozyme reaction buffer (20 mM
Tris-HCI, 100 mM NaCl, 5 mM MgC12, pH = 8.5, 100 L) to yield final lysozyme concentrations from 0 to 37.5 g/mL. The resulting mixtures were incubated for minutes at room temperature. Free lysozyme was removed and the NPs were washed with washing buffer three times. The lysozyme-associated NPs were redispersed in Milli-Q TM (Millipore Corp.) water (100 .tL), and PFVSO3 (100 M, 1 L) was added.
The mixture was incubated for 5 minutes. Excess PFVSO3 was washed away by a centrifugation-washing-redispersion process with washing buffer (100 mL, 3 times).
The collected NPs were redispersed in 15 mM PBS buffer (pH = 7.4) for fluorescence measurements.

1092269.1 4459.1012-000 Parallel experiments were conducted using a mixture of BSA (20 g/mL), thrombin (20 g/mL), and trypsin (20 gg/mL) to examine the assay specificity.
BSA, human thrombin, and trypsin have pI values of 4.7, 7.0-7.6, and 10.5, respectively, with net negative, neutral, and positive charges on the protein surface under the experimental conditions. The 60 Apt-NP (0.2 mg) was incubated with lysozyme (20 g/mL) as well as a mixture of interference proteins (20 gg/mL BSA, 20 gg/mL
thrombin, and 20 gg/mL trypsin) in binding buffer (20 mM Tris-HCI, 100 mM
NaCl, 5 mM MgC12, pH = 8.5), followed by polymer staining ([RU] =1 M) for 5 minutes and NP washing with washing buffer (20 mM Tris-HCI, 100 mM NaCl, 5 mM MgC12, pH
= 8.5). The PL spectra of the redispersed NPs are shown in FIG. 4. Intense polymer emission at 475 nm is only witnessed in the presence of lysozyme due to the recognition-induced switching of lysozyme/Apt-NP charge, followed by PFVSO3 self-assembly due to electrostatic interaction. No polymer fluorescence was observed in the presence of interference proteins. The nonspecific absorption of foreign proteins (e.g., positively charged trypsin) was largely avoided by blocking the NPs with ethaholamine and washing the NPs.

As such, PFVSO3 hardly stains negatively charged Apt-NPs due to electrostatic repulsion in our experimental conditions and NPs remain nonfluorescent. In addition, the fluorescent signal from 60 Apt-NPs upon incubation with the mixture of lysozyme and interference proteins (20 pg/mL each) after washing is shown in curve c of FIG. 4.
The polymer signal obtained from lysozyme in protein mixtures is almost the same as that from the pure lysozyme. The specific recognition of lysozyme in protein mixtures not only indicates the effectiveness of aptamer-protein binding but also highlights the intelligent target capture and interference isolation of the silica NP sensing platform.
To demonstrate lysozyme quantification, different concentrations of lysozyme (ranging from 0 to 37.5 g/mL) were incubated with 60 Apt-NP suspension for 30 minutes. The lysozyme/Apt-NPs were then stained with 1 M PFVSO3 for 5 minutes, then washed. The PL spectra of polymer-stained NPs are shown in FIG. 5. The PL
intensities of the NPs progressively increase with increased lysozyme concentrations.
1092269.1 4459.1012-000 This is due to increased positive charge on the Apt-NP surface in the presence of higher lysozyme concentrations, which enables increased numbers of negatively charged PFVSO3 to self-assemble on the NPs. In addition, the fluorescence of the NP
suspension upon treatment with lysozyme and PFVSO3 can be monitored by the naked eye. The intensity of the blue-green fluorescence of PFVSO3 gradually increases in the presence of increased concentrations of lysozyme, which allows clear naked-eye discrimination of lysozyme with a limit of detection (LOD) as low as 1.5 gg/mL
(10 pmol).
The calibration curve for lysozyme detection is shown in FIG. 6. The PL
intensity of the NP suspension increases linearly with lysozyme concentration and finally saturates at a lysozyme concentration of approximately 22.5 gg/mL. The LOD is estimated to be 0.36 gg/mL (2.4 pmol, based on R y from six independent measurements) using a standard fluorometer, which is more sensitive to aptamer-based electrochemical and fluorescent arrays and is similar to that obtained from a standard of ELISA. (See Vidal, M. L., et al., Agric. Food Chem. 2005, 53, 2379- 2385, the entire teachings of which are incorporated herein by reference). However, the strategy of using Apt-NP as a platform for lysozyme detection reduces the bonding affinity (Kd) of the aptamer to its target. The apparent Kd in our assay is approximately 9 g/ mL (approximately 625 nM), which is estimated from the lysozyme concentration that induces half-maximum signal in FIG. 6. Similar to that of aptamer-immobilized gold assays, this Kd value is 20-fold larger compared to that measured in solution (31 nM). (See, for example, Cox, J. C.;
Ellington, A. D., Bioorg. Med. Chem. 2001, 9, 2525-2531, the entire teachings are incorporated herein by reference). The large Kd on the NP surface is detrimental to assay sensitivity, could be the result of. (1) the steric hindrance induced by the folded aptamer upon binding to lysozyme which prevents the adjacent aptamers from folding into G-quartet structure; (2) the binding of lysozyme on the Apt-NP surface hampers subsequent aptamer/lysozyme binding due to electrostatic repulsion.

Although quite a few strategies have been reported for lysozyme detection, very few allow label-free and visible detection and quantification of lysozyme in real time.
1092269.1 4459.1012-000 Example 2. Synthesis of POSSFF and POSSFBT
Synthesis of 2-(9, 9-bis(6-bromohexyl)fluoren-2 yl)-4, 4, 5, 5-tetramethyl-1, 3,2-dioxaborolane (1). 2-Bromo-9,9-bis- (6-bromohexyl)fluorene (4.54 g, 7.95 mmol),bis(pi nacolatodiboron) (3.02 g, 11.93 mmol), and potassium acetate (2.94 g,29.82 mmol) were placed in a 100-mL round bottom flask. Anhydrous dioxane (80 mL) and [PdC12(dppf)]
(0.20 g,0.24 mmol) were added to the flask and the reaction vessel was degassed. The mixture was stirred at 80 C for 12 h under nitrogen. After the mixture had been cooled to room temperature, dioxane was removed by rotary evaporation. The residue was extracted with dichloromethane, and the organic phase was washed with water and brine, and dried over magnesium sulfate. The solvent was removed and the residue was purified by silica gel column chromatography (dichloromethane/hexane=1:2) to afford 2.
Synthesis of 2, 7-dibromo-9, 9-bis(6-bromohexyl)fluorene (2). 2,7-Dibromofluorene (1.23 g, 5 mmol) was added to a mixture of aqueous potassium hydroxide (100 mL, 50 w%), tetrabutylammonium bromide (0.330 g, 1 mmol), and 1,2-bis(2-bromoethoxy)ethane (13.9 g, 50 mmol) at 75 C. After 15 min, the mixture was cooled to room temperature. After extraction with CH2C12, the combined organic layers were washed successively with water, aqueous HCl (1 M), water, and brine and then dried over Na2SO4. After removal of the solvent and the excess 1,2-bis(2-bromoethoxy)ethane, the residue was purified by silica gel column chromatography using hexane and dichloromethane (1:2) as the eluent, and recrystallized from ethanol and CH2C12 (5:1) to afford M2 as white needle crystals (1.50 g, 48.0%).
Synthesis of 2-(7-bromo-9, 9-bis(6-bromohexyl)fluorenyl)-9,9-bis(6-bromohexyl)fluorene (3). 1 (2.84 g, 4.60 mmol), 2 (4.5 g, 6.9 mmol), Pd(PPh3)4 (53 mg, 0.046 mmol), potassium carbonate (4.43, 32.0 mmol) were placed in a 100 mL
round bottom flask. A mixture of water (12 mL) and toluene (30 mL) was added to the flask and the reaction vessel was degassed. The mixture was vigorously stirred at 90 C for 2 days. After it was cooled to room temperature, dichloromethane was added to the reaction mixture. The organic portion was separated and washed with brine before drying over anhydrous MgSO4. The solvent was evaporated off, and the solid residues 1092269.1 4459.1012-000 were purified by column chromatography on silica gel using dichloromethane/hexane (1:5) as eluent to afford 3.
Synthesis of 2-(7-bromo-bis(6-N,N,N-trimethylammonium)hexyl)fluorenyl)-bis(6-N, N, N-trimethylammonium) hexyl)fluorene (4). Condensed trimethylamine (-5 mL) was added dropwise to a solution of 3 (1 g, 0.94 mmol) in THE (10 mL) at -78 C.
The mixture was allowed to warm to room temperature. The precipitate was redissolved by the addition of water (10 mL). After the mixture was cooled to -78 C, additional trimethylamine (-3 mL) was added. The mixture was stirred at room temperature for 24 h. After removal of the solvent, acetone was added to precipitate 4 (1.2 mg, 98%) as white powders.
Synthesis of 4-(9,9-bis(6-bromohexyl)-9H fluoren-2 yl)-7-bromobenzothiadiazole (7). 2-(9,9-bis(6-bromohexyl)- fluoren-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (6) (2.84 g, 4.60 mmol), 4,7-dibromobenzothiadiazole (2.16 g, 7.36 mmol), Pd(PPh3)4 (53 mg, 0.046 mmol), potassium carbonate (4.43, 32.0 mmol) were placed in a 100 mL round bottom flask. A mixture of water (12 mL) and toluene (30 mL) added to the flask and the reaction vessel was degassed. The mixture was vigorously stirred at 90 C for 2 days. After it was cooled to room temperature, dichloromethane was added to the reaction mixture. The organic portion was separated and washed with brine before drying over anhydrous MgSO4. The solvent was evaporated off, and the solid residues were purified by column chromatography on silica gel using dichloromethane/hexane (1:5) as eluent to afford as grassy liquid.

(500 MHz, CD3OD, 6 ppm): 8.0-7.87 (m, 3 H), 7.85 (d, 1 H, J = 7.84), 7.77 (d, 1 H, J =
7.26), 7.66 (d, 1 H, J = 7.57), 7.45-7.30 (m, 3 H), 3.27 (t, 4 H, J = 6.84, 6.84), 2.14-1.97 (m, 4 H), 1.74-1.62 (m, 4 H), 1.32-1.18 (m, 4 H), 1.17-1.04 (m, 4 H), 0.83-0.66 (m, 4 H). 13C NMR (125 MHz, CD3OD, 6 ppm): 154.00, 153.35, 152.83, 150.90, 141.76, 140.50, 135.37, 134.49, 132.31, 128.24, 128.05, 127.58, 127.08, 123.79, 122.91, 120.13, 119.89, 112.81, 55.16, 40.12, 33.92, 32.60, 29.04, 27.73, 23.61. MS (MALDI-TOF):
m/z 707.37 [M]+.

1092269.1 4459.1012-000 Synthesis of 4-(9, 9-bis(6-N, N,N-trimethylammonium)hexyl)fluorenyl)-7-bromobenzothiadiazole (8). Synthesis of Condensed trimethylamine ('5 mL) was added dropwise to a solution of 2 (1 g, 0.94 mmol) in THE (10 mL) at -78 C. The mixture was allowed to warm to room temperature. The precipitate was redissolved by the addition of water (10 mL). After the mixture was cooled to -78 C, additional trimethylamine (-3 mL) was added. The mixture was stirred at room temperature for 24 h. After removal of the solvent, acetone was added to precipitate 3 (1.4 mg, 99%) as yellow powders. 1H
NMR (500 MHz, CD3OD, 6 ppm): 8.38-8.26 (m, 2 H), 8.26-8.19 (m, 1 H), 8.19-8.12 (m, 1), 8.12-8.00 (m, 2 H), 7.79-7.56 (m, 3 H), 3.53-3.42 (m, 4 H), 3.09 (3, 18 H), 2.55-2.42 (m, 4 H), 1.95-1.72 (m, 4 H), 1.53-1.31 (m, 8 H), 1.12-0.78 (m, 4H). (13C
NMR
(125 MHz, CD3OD, 6 ppm): 155.28, 154.50, 152.26, 152.055, 143.31, 142.18, 136.97, 135.38, 134.03, 129.73, 128.93, 128.46, 125.18, 124.33, 121.35, 121.05, 113.78, 67.81, 55.58, 53.68, 41.19, 30.35, 26.98, 24.92, 23.75.
Synthesis ofPOSSFF. Octavinyl POSS (5) (11.4 mg, 0.018 mmol), 4 (187 mg, 0.144 mmol), Pd(OAc)2 (3.2 mg, 14.4 mol), and P(o-tolyl)3 (24 mg, 78.4 mol) were placed in a 25 mL round bottom flask. A mixture of DMF (1 mL), and triethylamine (0.5 mL) was added to the flask and the reaction vessel was degassed. The mixture was vigorously stirred at 100 C for 36 h. It was then filtered and the filtrate was poured into acetone. The precipitate was collected and washed with acetone, and was redissolved in water. The solution was filtered through a 0.22 m syringe driven filter to give limpid solution. Finally, the product was purified by dialysis against Milli-Q water using a 3.5 kDa molecular weight cutoff dialysis membrane for 5 days. After freeze-drying, POSSFF (74 mg, 45%) was obtained as light yellow powders.
Synthesis of POSSFBT. Octavinyl POSS (5) (11.4 mg, 0.018 mmol), 8 (119 mg, 0.144 mmol), Pd(OAc)2 (3.2 mg, 14.4 gmol), and P(o-tolyl)3 (24 mg, 78.4 mol) were placed in a 25 mL round bottom flask. A mixture of DMF (1 mL), and triethylamine (0.5 mL) was added to the flask and the reaction vessel was degassed. The mixture was vigorously stirred at 110 C for 36 h. It was then filtered and the filtrate was poured into acetone. The precipitate was collected and washed with acetone, and was redissolved in 1092269.1 4459.1012-000 water. The solution was filtered through a 0.22 m syringe driven filter to give limpid solution. Finally, the product was purified by dialysis against Milli-Q water using a 3.5 kDa molecular weight cutoff dialysis membrane for 5 days. After freeze-drying, POSSBT (96 mg, 73%) was obtained as yellow fibers. 'H NMR (500 MHz, CD3OD, 6 ppm): 8.47 (s, 1 H), 8.43 (d, 2 H), 8.31 (d, 1 H), 8.25 (d, 2 H), 7.74-7.76 (m, 2 H), 7.83-7.74 (m, 1 H), 7.74-7.63 (m, 2 H), 3.54-3.38 (m, 4 H), 3.09 (s, 18 H), 2.57-2.39 (m, 4 H), 1.95-1.80 (m, 4 H), 1.54-1.40 (m, 8 H), 1.13-0.95 (m, 4 H).
This unimolecular nanoparticle has a good water-solubility (-23 mg/mL at 24 C), as a result of its high charge density on its nanoglobular surface. The morphology and size of POSSFBT were studied by high-resolution transmission electron microscopy (HR- TEM). Spherical nanoparticles with an average diameter of 3.3 + 0.5 nm were observed, which coincides well with the single-molecular size of POSSFBT.
POSS compounds containing catonic, anionic or neutral R groups on either Ar or Ar' can be synthesized by the similar method as that used to synthesize POSSFF
and POSSFBT.
Example 3. Synthesis of P2.

The synthesis of P2 is depicted in FIG. 7.

Synthesis of 4-(9, 9'-Bis(6-bromohexyl)fluorenyl)-7-bromobenzothiadiazole (2).
2-(9,9-Bis(6-bromohexyl)fluorenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1) (2.84 g, 4.60 mmol), 4,7-dibromobenzothiadiazole (2.16 g, 7.36 mmol), Pd(PPh3)4 (53 mg, 0.046 mmol), potassium carbonate (4.43 g, 32.0 mmol) were placed in a 100 mL round bottom flask. A mixture of water (12 mL) and toluene (30 mL) were added to the flask and the reaction vessel was degassed. The mixture was vigorously stirred at 90 C for 2 days.
After it was cooled to room temperature, dichloromethane was added to the reaction mixture. The organic portion was separated and washed with brine before drying over anhydrous MgSO4. The solvent was evaporated off, and the solid residues were purified by column chromatography on silica gel using dichloromethane/hexane (1 : 5) as eluent to afford 2 as grassy yellow liquid (2 g, 62%). 'H NMR (500 MHz, CD3C1, 6 ppm): 8.0-7.87 (m, 3 H), 7.85 (d, 1 H, J= 7.84 Hz), 7.77 (d, 1 H, J= 7.26 Hz), 7.66 (d, 1 H, J=
1092269.1 4459.1012-000 7.57 Hz), 7.45-7.30 (m, 3 H), 3.27 (t, 4 H, J= 6.84 Hz), 2.14-1.97 (m, 4 H), 1.74-1.62 (m, 4 H), 1.32-1.18 (m, 4 H), 1.17-1.04 (m, 4 H), 0.83-0.66 (m, 4 H). 13C NMR
(125 MHz, CD3Cl, 6 ppm): 154.00, 153.35, 152.83, 150.90, 141.76, 140.50, 135.37, 134.49, 132.31, 128.24, 128.05, 127.58, 127.08, 123.79, 122.91, 120.13, 119.89, 112.81, 55.16, 40.12, 33.92, 32.60, 29.04, 27.73, 23.61. MS (MALDI-TOF): m/z 707.37 [M]+.
Synthesis of 4-Bromo- 7-(7-bromo-9,9 '-bis(6-bromohexyl)fluorenyl) benzothiadiazole (3). 2 (0.80 g, 1.14 mmol) was dissolved in dichloromethane (20 mL) and cooled in an ice bath. Bromine liquid (0.45 g, 2.72 mmol) was then added slowly.
After stirring at 45 C for 12 h, the reaction was quenched with sodium sulfite solution.
Dichloromethane was added, and the organic portion was separated and washed with brine before drying over anhydrous MgSO4. The solvent was evaporated off, and the solid residues were purified by column chromatography on silica gel using dichloromethane/hexane (1 : 5) as eluent to afford 3 as yellow crystals (0.81 g, 90%). 'H
NMR (500 MHz, CD3C1, 6 ppm): 7.95 (d, 1H, J= 7.75 Hz), 7.91 (dd, 1 H, J= 1.33, 7.89 Hz), 7.88 (s, 1 H), 7.81 (d, 1 H, J= 7.88 Hz), 7.64 (dd, 2 H, J= 8.12, 13.86 Hz), 7.50 (m, 2 H), 3.28 (t, 4 H, J= 6.70 Hz), 2.0 (m, 4 H), 1.67 (m, 4 H), 1.23 (m, 4 H), 1.11 (m, 4 H), 0.73 (td, 4 H, J= 7.74, 15.61 Hz). 13C NMR (125 MHz, CD3C1, 6 ppm):
153.98, 153.14, 150.46, 140.60, 139.54, 135.86, 134.20, 132.29, 130.30, 128.46, 128.13, 126.23, 123.83, 121.50, 120.04, 113.04, 55.51, 40.05, 33.96, 32.61, 29.00, 27.74, 23.60. MS
(MALDI-TOF): m/z 785.44 [M]+.
Synthesis of 4-(9,9 '-Bis(6-bromohexyl)- 7-((trimethylsilyl)ethynyl)fluorenyl)-((trimethylsilyl)ethynyl)benzothiadiazole (4). A solution of trimethylsilyl acetylene (1.08 g, 1.55 mL, 11.0 mmol, d = 0.695 g/mL) in diisopropylamine ((iPr)2NH) (20.0 mL) was slowly added to a solution of 3 (3.9 g, 5.0 mmol), (Ph3P)2PdC12 (0.175 g, 0.25 mmol), and Cul (0.047 g, 0.25 mmol) in (iPr)2NH (50.0 mL) under nitrogen at room temperature. The reaction mixture was then stirred at 70 C for 8 h. The solvent was removed under reduced pressure, and the residue was chromatographed on silica gel using hexane as eluent to give 4 (2.8 g, 65%) as yellow crystals. 1H NMR (500 MHz, CD3C1, 6 ppm): 7.94 (m, 2 H), 7.87 (d, 1 H, J= 7.39 Hz), 7.81 (d, 1 H, J= 7.85 Hz), 1092269.1 4459.1012-000 7.73 (d, I H, J= 7.28 Hz), 7.69 (d, 1 H, J= 7.82 Hz), 7.50 (d, 1 H, J= 7.86 Hz), 7.47 (s, 1 H), 3.26 (t, 4 H, J = 6.79 Hz), 2.00 (m, 4 H), 1.66 (m, 4 H), 1.21 (m, 4 H), 1.09 (m, 4 H), 0.70 (td, 4 H, J= 7.70, 15.16 Hz), 0.36 (s, 9 H), 0.30 (s, 9 H). 13C NMR
(125 MHz, CD3C1, d ppm): 155.41, 153.20, 151.10, 150.87, 141.01, 140.91, 136.19, 135.16, 133.82, 131.43, 128.51, 127.27, 126.27, 123.86, 123.85, 121.85, 120.23, 119.85, 115.58, 106.05, 101.84, 100.52, 94.46, 55.27, 40.09, 33.90, 32.64, 29.00, 27.76, 23.57, 0.10, 0.04. MS (MALDI-TOF): m/z 819.70 [M]+.
Synthesis of 4-(9, 9'-Bis(6-bromohexyl)-7-ethynyljluorenyl)-7-ethynylbenzothiadiazole (5). A KOH aqueous solution (3.0 mL, 20.0%) was diluted with methanol (15.0 mL) and added to a stirred solution of 4 (2.1 g, 2.5 mmol) in THE
(20.0 mL). The mixture was stirred at room temperature for 6 h and extracted with hexane. The organic fraction was washed with water and dried over sodium sulfate. The crude product was chromatographed on silica gel using hexane as the eluent.
Recrystallization of the product from methanol gave 5 (1.6 g, 92%) as yellow crystals.
1H NMR (500 MHz, CD3C1, 6 ppm): 7.98 (dd, 1 H, J= 1.47, 7.87 Hz), 7.94 (s, 1 H), 7.91 (d, 1 H, J = 7.34 Hz), 7.84 (d, 1 H, J = 7.90 Hz,), 7.76 (d, 1 H, J =
7.47 Hz,), 7.72 (d, 1 H, J= 7.80 Hz), 7.53 (dd, 1 H, J= 1.10, 7.63 Hz,), 7.50 (s, 1 H), 3.64 (s, 1 H), 3.27 (t, 1 H, J= 6.74, 6.74 Hz), 3.17 (s, 1 H), 2.03 (m, 4 H), 1.66 (m, 4 H), 1.22 (m, 4 H), 1.10 (m, 4 H), 0.71 (td, 4 H, J= 7.72, 15.20 Hz). 13C NMR (125 MHz, CD3C1, ppm): 155.61, 153.16, 151.15, 150.97, 141.24, 140.97, 136.15, 135.69, 133.98, 131.46, 128.55, 127.25, 126.55, 123.91, 120.84, 120.31, 120.07, 114.48, 84.52, 83.70, 79.55, 77.47, 55.27, 40.06, 33.88, 32.61, 29.02, 27.75, 23.60. MS (MALDI-TOF): m/z 673.01 [M]+.

Synthesis of Neutral Hyperbranched Conjugated Polymer (P0). A Schlenk tube charged with 5 (100 mg, 0.15 mmol) was degassed with three vacuum-nitrogen cycles.
A solution of cyclopentadienylcobaltdicarbonyl (CpCo(CO)2) in anhydrous toluene (1.5 mL, 0.01 M) was then added to the tube, and the system was further frozen, evacuated, and thawed three times to remove oxygen. The mixture was vigorously stirred at under irradiation with a 200 W Hg lamp (operating at 100 V) placed close to the tube for 1092269.1 4459.1012-000 8 h. After the mixture was cooled to room temperature, it was dropped into methanol (100 mL) through a cotton filter. The precipitate was collected and redissolved in tetrahydrofuran. The resultant solution was filtered through 0.22 m filter, and poured into hexane to further precipitate the product. After dried in vacuum at 40 C, PO was obtained as brown powders (65 mg, 65%). 'H NMR (500 MHz, CDC13, 6 ppm): 8.50-7.30 (m, 8 H), 7.20 (br, 1 H), 3.67 (s, 0.20 H), 3.30 (br, 4 H), 3.20 (s, 0.20 H), 2.0 (br, 4 H), 1.70 (br, 4 H), 1.42-1.06 (m, 8 H), 0.77 (br, 4 H). 13C NMR (125 MHz, CDC13, 6 ppm): 155.41, 154.34, 153.73, 153.06, 151.10, 150.97, 150.91, 150.08, 141.43, 140.50, 137.87, 134.02, 131.45, 129.04, 128.53, 128.23, 126.54, 125.30, 123.97, 120.68, 120.30, 119.98, 84.60, 83.30, 80.88, 77.92, 55.27, 40.10, 33.91, 32.64, 29.06, 27.77, 23.65. Mn =
6700, MH,IMõ = 1.8.

Synthesis of Cationic HCPE (PI). Trimethylamine (2 mL) was added dropwise to a solution of PO (50 mg) in THE (10 mL) at -78 C. The mixture was stirred for 12 h, and then allowed to warm to room temperature. The precipitate was redissolved by the addition of methanol (8 mL). After the mixture was cooled to -78 C, additional trimethylamine (2 mL) was added, and the mixture was stirred at room temperature for 24 h. After removal of the solvent, acetone was added to precipitate P1 as brown powders (55 mg, 95%). 'H NMR (500 MHz, CD3OD, 6 ppm): 8.77-7.35 (m, 9 H), 3.63 (s, 0.20 H), 3.28 (br, 4 H), 3.05 (s, 18 H), 2.05 (br, 4 H), 1.58 (br, 4 H), 1.20 (br, 8 H), 0.77 (br, 4 H). 13C NMR (125 MHz, CD3OD, 6 ppm): 155.49, 154.10, 150.97, 141.91, 141.37, 140.70, 138.15, 134.00, 133.43, 131.07, 130.22, 128.54, 128.32, 126.23, 125.97, 123.89, 121.27, 121.13, 119.92, 87.08, 80.08, 66.31, 55.21, 52.20, 39.52, 28.73, 25.38, 23.29, 22.17.
Synthesis of Core-Shell HCPE (P2). P1 (30 mg, 0.05 mmoL alkyne) and N3-PEG-NH2 (140 mg, 0.25 mmoL) were dissolved in DMF (5 mL). The mixture was degassed, and then N,N,N',N",N"'-pentametyldiethylenetriamine (PMDETA) (12 mg, 0.0825 mmoL) and CuBr (11.8 mg, 0.0825 mmoL) were added. After reaction at 65 C
under nitrogen for 24 h, the reaction mixture was cooled to room temperature and filtered through 0.22 pm syringe driven filter. The filtrate was precipitated into diethyl 1092269.1 4459.1012-000 ether to give red powders. The crude product was redissolved in water and further purified by dialysis against Mill-Q water using a 3.5 kDa molecular weight cutoff dialysis membrane for 3 days. After freeze-drying, P2 (45 mg, 78%) was obtained as brown fibers. 'H NMR (500 MHz, d(-DMS ), 6 ppm): 8.60-7.05 (m, 10.8 H), 4.56-3.40 (m, 145 H), 3.00-2.65 (m, 8 H), 2.47-1.70 (m, 22 H), 1.66-0.78 (m, 12 H), 0.56 (br, 4 H).
Example 4. Synthesis of Molecular Brush (P4.1).

Molecular brushes are unique macromolecules with densely grafted side chains on a linear polymeric backbone. Although several "grafting from" methods including nitroxyl radical mediated polymerization (NRMP) and atom transfer radical polymerization (ATRP) have been utilized to synthesize neutral conjugated polymer based molecular brushes, the resultant polymers share the drawbacks of incapability of further biofunctionalization. In comparison, "grafting onto" strategy is more versatile as it offers a facile way to modify the brush prior to attachment onto the backbone, while the brush density is strongly limited by the grafting chemical reaction used.
Fortunately, the Huisgen 1,3-dipolar cycloaddition reaction between organic azides and alkynes, known as click chemistry, recently emerged as an advanced chemistry technology, allowing post-polymerization with nearly quantitative yield, mild reaction condition, and broad tolerance towards various functional groups. In light of these considerations, the "grafting onto" strategy based on click chemistry is adopted to synthesize the surface-amenable CPE-g-PEG molecular brush.
The synthetic route toward the CPE-g-PEG molecular brush and its folic acid (FA)-functionalized derivative is shown in FIG. 8. 9,9-Bis(6'-bromohexyl)-2,7-diviny1fluorene (2.1), was synthesized in 78% yield by heating the mixture of 2,7-dibromo-9,9-bis(6'-bromohexyl)-fluorene (1.1) and tributylvinyltin in toluene at 100 C
for 24 h using PdC12(PPh3)2/2,6-di-tert-butylphenol as catalyst. Treatment of 2.1 with dimethylamine in THE afforded the divinyl monomer, 9,9-bis(6'-(N,N-dimethylamino)hexyl)-2,7-divinylfluorene (3.1). After successful determination of the chemical structure of 3.1 by NMR and mass spectrum, it was polymerized with 4,7-1092269.1 4459.1012-000 dibromobenzothiadiazole (4.1) via a Pd(OAc)2/P(o-tolyl)3 catalyzed Heck coupling reaction in the mixture of DMF/TEA (2 : 1) at 100 C to afford the neutral polymer, poly[9,9-bis(6'-(N,N-dimethylamino)hexyl))fluorenyldivinylene-alt-4,7-(2',1',3',-benzothiadiazole) dibromide] (P1.1). Quaternization of P1.1 with 4-bromobut-1-yne in the mixture of THF/DMF/DMSO at 55 C gave the clickable cationic polymer, poly[9,9-bis(N-(but-3'-ynyl)-N,N-dimethylamino)hexyl))fluorenyldivinylene-alt-4,7-(2',1',3',-benzothiadiazole) dibromide] (P2.1). This polymer precursor has alkyne groups at the end of the side chains, which allows for subsequent click reaction with azide compounds. The click reaction was carried out in DMF between P2.1 and azide-functionalized monodispersed PEG-NH2 (N3-PEG-NH2) at 65 C using N,N,N',N",N"'-pentametyldiethylenetriamine (PMDETA) and CuBr as the catalyst, leading to the CPE-g-PEG (P3.1). Finally, coupling reaction between the amine groups of P3.1 and y-carboxylic acid of FA using dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) as the catalyst in DMSO gave the FA-functionalized CPE-g-PEG (P4.1). The cationic polymers P2.1, P3.1 and P4.1 were purified by micro-filtration, precipitation, and finally dialysis against Mill-Q water using a 3.5 kDa molecular weight cutoff dialysis membrane for 3 days.
The chemical structures of these polymers were determined by 1H NMR spectra.
As compared to P1.1, a new resonance peak at 3.08 ppm appears in the 'H NMR
spectrum of P2.1, which is assigned to the alkyne protons. The integral ratio of the peak at 3.08 ppm to that at 2.64 ppm (corresponding to the methylene protons near the 9-position of fluorene) is close to 0.48, indicating that the degree of quaternization is --96%. The successful click reaction is verified by the presence of a single resonance peak at 8.00 ppm in the 'H NMR spectrum of P3.1, which corresponds to the proton next to the nitrogen atom of the triazole group. Comparison of the integrated areas between the multiple peaks ranging from 4.56 to 3.40 ppm (assigned to the methylene protons of PEG) and the peak at 0.56 ppm (assigned to the methylene protons secondly close to the 9-position of fluorene) reveals a high PEG graft efficiency of -90%, which is attributed to the high activity of the click reaction using PMDETA/CuBr as the 1092269.1 4459.1012-000 catalyst. After FA functionalization of P3.1, the 1H NMR spectrum becomes more complicated for P4.1. Nevertheless, the characteristic proton resonance peak of FA
located at 8.66 ppm is separated from those of the conjugated backbone.
Thereby, the molar percentage of FA in P4.1 is calculated to be -60%.
Synthesis of 9,9-Bis(6'-(N,N-dimethylamino)hexyl)-2, 7-divinylfluorene (3.1):
Dimethylamine solution (5 mL, 5.6 M in absolute ethanol) was added dropwise to a solution of 2.1 (500 mg, 0.92 mmol) in THE (8 mL) at room temperature. After stirring for 12 h, additional dimethylamine solution (3 mL) was added, and the mixture was stirred at room temperature for 12 h. The solvent was then removed under reduced pressure, and the residual was washed by hexane and methanol to afford 3.1 (370 mg, 85%) as white powders. 'H NMR (500 MHz, CDC13, S ppm): 7.61 (d, 2 H, J= 7.78 Hz), 7.39 (d, 2 H, J= 7.12 Hz), 7.35 (s, 2 H), 6.79 (dd, 2 H, J= 10.85, 17.54 Hz), 5.82 (d, 2 H, J= 17.54 Hz), 5.27 (d, 2 H, J= 10.85 Hz), 2.14 (s, 12 H), 2.10 (m, 4 H), 1.96 (m, 4 H), 1.27 (m, 4 H), 1.08 (m, 8 H), 0.65 (m, 4 H). 13C NMR (125 MHz, CDC13, 6 ppm):
151.25, 140.72, 137.40, 136.51, 125.30, 120.48, 119.72, 113.04, 59.79, 54.88, 45.46, 40.33, 29.90, 27.59, 27.07, 23.68. EIMS (m/z): 472.30 (M).

Synthesis ofPoly[9,9-bis(6'-(~A;N-climethylamino)hexyl))fluorenyldivinylene-alt-4, 7-(2 ,1, 3 , -benzothiadiazole) dibromide] (P1.1): A Schlenk tube was charged with 3.1 (100 mg, 0.212 mmol), 4.1 (62 mg, 0.212 mmol), Pd(OAc)2 (2 mg, 9 mmol), and P(o-tolyl)3 (15 mg, 49 mol) before it was sealed with a rubber septum. The Schlenk tube was degassed with three vacuum-argon cycles to remove air. Then, DMF (1.6 mL) and triethylamine (0.8 mL) was added to the Schlenk tube and the mixture was frozen, evacuated, and thawed three times to further remove air. The polymerization was carried out at 100 C under vigorous stir for 12 h. It was then filtered through 0.22 gm syringe driven filter and the filtrate was poured into diethyl ether. The precipitate was collected and washed with methanol and acetone, and then dried under vacuum for 24 h to afford P1.1 (108 mg, 81%) as red fibers. 'H NMR (500 MHz, CDC13, 6 ppm): 8.14 (br 4 H), 7.93-7.36 (m, 8 H), 2.30 (br, 4 H), 2.13 (s, 12 H), 2.00 (br, 4 H), 1.30 (br, 4 H), 1.12 (br, 8 H), 0.73 (br, 4 H). 13C NMR (125 MHz, CDC13, d ppm): 154.05, 151.68, 141.21, 1092269.1 4459.1012-000 136.71, 133.89, 129.43, 127.04, 126.43, 123.92, 121.25, 120.18, 59.77, 55.17, 45.41, 40.51, 29.98, 27.59, 27.17, 23.82. Mõ = 9500, MWIMõ = 2.1.

Synthesis ofPoly[9,9-bis(N-(but-3' ynyl)-N,N-dimethylainilio)hexyl))fluorenyldivinylene-alt-4, 7-(2 ;1, 3, -benzothiadiazole) dibromide]
(P2.1): 4-Bromobut-l-yne (2 mL) was added to P1.1 (50 mg) in THE (5 mL) and DMF
(5 mL), and the mixture was stirred at 55 C for 2 h. Then, DMSO (5 mL) was added to dissolve the precipitate. After reaction for 48 h, THE and methanol was removed under reduced pressure. The residual solution was then poured into acetone to give the crude product as dark red powders. The product was further purified by dialysis against Mill-Q
water using a 3.5 kDa molecular weight cutoff dialysis membrane for 3 days.
After freeze-drying, P2.1 (56 mg, 78%) was obtained as red fibers. 'H NMR (500 MHz, d7-DMF, 6 ppm): 8.53 (br, 4 H), 8.36-8.18 (m, 6 H), 7.99 (br, 2 H), 3.65 (br, 3.84 H), 3.42 (br, 4 H), 3.20 (br, 3.84 H), 3.08 (t, 1.92 H), 2.95 (br, 12 H), 2.44 (br, 4 H), 1.79 (br, 4 H), 1.32 (br, 8 H), 0.88 (br, 4 H).

Synthesis of PFVBT-g-PFG (P3.1): P2.1 (30 mg, 0.05 mmoL alkyne) and N3-PEG-NH2 (140 mg, 0.25 mmoL) were dissolved in DMF (5 mL). The mixture was degassed, and then N,N,N',N",N"'-pentametyldiethylenetriamine (PMDETA) (12 mg, 0.0825 mmoL) and CuBr (11.8 mg, 0.0825 mmoL) were added. After reaction at 65 C
under nitrogen for 24 h, the reaction mixture was cooled to room temperature and filtered through 0.22 m syringe driven filter. The filtrate was precipitated into diethyl ether to give red powders. The crude product was redissolved in water and further purified by dialysis against Mill-Q water using a 3.5 kDa molecular weight cutoff dialysis membrane for 3 days. After freeze-drying, P3.1 (45 mg, 78%) was obtained as red powders. 1H NMR (500 MHz, d6-DMSO, 6 ppm): 8.60-7.05 (m, 10.8 H), 4.56-3.40 (m, -145 H), 3.00-2.65 (m, 8 H), 2.47-1.70 (m, -22 H), 1.66-0.78 (m, 12 H), 0.56 (br, 4 H).

Synthesis of PFVBT-g-PEG-FA (P4.1): The carboxylic acid group of FA (16.5 mg, 0.0335 mmol) dissolved in DMSO (0.8 mL) was pre-activated with DCC (8.25 mg, 1092269.1 4459.1012-000 0.04 mmol) and NHS (7.5 mg, 0.065 mmol) at room temperature. In the reaction, dicyclohexylurea was formed and removed by filtration. Although FA has a- and y-carboxylic acid groups, y-carboxylic acid was primarily activated in the DCC/NHS
reaction due to its higher reactivity. P3.1 (12 mg, 0.02 mmoL -NH2) was added to the NHS-activated FA solution. The reaction was kept at room temperature for 48 h.
The product was further purified by dialysis against Mill-Q water using a 3.5 kDa molecular weight cutoff dialysis membrane for 3 days. After freeze-drying, P4.1 (22 mg, 72%) was obtained as red powders. 'H NMR (500 MHz, d6-DMSO, 6 ppm): 8.66 (s, 1.2), 8.13-6.56 (m, 13 H), 5.57 (br, 2.4), 4.50-2.60 (m, 157 H), 2.3-1.44 (m, 27 H), 1.36-0.93 (m, 12 H), 0.76 (br, 4 H).

The relevant teachings of all patents, published applications and publications cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

1092269.1

Claims (28)

1. A compound represented by structural formula (I):
(I), or a salt thereof, wherein:

R and R2 are each independently -(OCH2CH2)p OCH3 or -(CH2CH2O)p CH3, wherein p is an integer between 1 and 100, inclusive;
R' and R3 are each independently hydrogen or a charged side group;
m is an integer between 2 and 50, inclusive; and T and T' are each independently a terminating group.

2. The compound of Claim 1, or a salt thereof, wherein the charged side group is an anionic or cationic alkyl side group, an anionic or cationic oligo(ethylene oxide) side group or an anionic or cationic poly(ethylene oxide) side group.

3. The compound of Claim 1, or a salt thereof, wherein the charged side group is selected from the group consisting of -(CH2)n N(R2)3X, -(OCH2CH2)n N(R2)3X, -(CH2CH2O)q CH2CH2N(R2)3X, -(CH2)n X', -(OCH2CH2)n X', -(OCH2CH2)n OX', -(CH2CH2O)n X' and -(CH2CH2O)q CH2CH2X', wherein R2 is (C1-C6)alkyl, n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, X is an anionic counterion and X' is -CO2Y, -SO3Y or -PO3Y2, wherein Y is hydrogen or a cationic counterion.

4. The compound of Claim 1, or a salt thereof, wherein:
R and R2 are each -(OCH2CH2)p OCH3 or -(CH2CH2O)p CH3; and R' and R3 are each hydrogen or a charged side group.

5. The compound of Claim 1, or a salt thereof, wherein R' and R3 are each independently a charged side group.

6. The compound of Claim 5, or a salt thereof, wherein R' and R3 are each a charged side group.

7. The compound of Claim 1, wherein the compound is represented by Structural Formula (II):

(II), or a salt thereof.

8. The compound of Claim 1, wherein the compound is represented by the following structural formula:

9. A method of detecting a target in a sample, comprising:
functionalizing a solid support with a ligand;
incubating the ligand-functionalized solid support with a sample;
incubating the sample with a charged conjugated polyelectrolyte (CPE) or conjugated oligoelectrolyte (COE); and detecting the fluorescence of the solid support, thereby detecting the target.

10. The method of Claim 9, wherein the solid support is a nanoparticle.

11. The method of Claim 10, wherein the nanoparticle is a silica nanoparticle.

12. The method of Claim 9, wherein the ligand is an aptamer having the sequence of SEQ ID NO.: 1.

13. The method of Claim 9, wherein the CPE or COE is a compound represented by Structural Formula (I):

(I), or a salt thereof, wherein:

R and R2 are each independently -(OCH2CH2)p OCH3 or -(CH2CH2O)p CH3, wherein p is an integer between 1 and 100, inclusive;
R' and R3 are each independently hydrogen or a charged side group;
m is an integer between 2 and 50, inclusive; and T and T' are each independently a terminating group.

14. The method of Claim 13, wherein the charged side group is selected from the group consisting of -(CH2)n N(R2)3X, -(OCH2CH2)n N(R2)3X, -(CH2CH2O)q CH2CH2N(R2)3X, -(CH2)n X', -(OCH2CH2)n X', -(OCH2CH2)n OX', -(CH2CH2O)n X' and -(CH2CH2O)q CH2CH2X', wherein R2 is (C1-C6)alkyl, n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, X is an anionic counterion and X' is -CO2Y, -SO3Y or -PO3Y2, wherein Y is hydrogen or a cationic counterion.

15. The method of Claim 13, wherein the compound is represented by Structural Formula (II):

(II), or a salt thereof.

16. The method of Claim 13, wherein the compound is represented by the following structural formula:

17. The method of Claim 9, wherein the CPE or COE is a compound represented by the following structural formula:

, or a salt thereof, wherein:
Ar is an optionally substituted aromatic group;
Linker is a single bond, double bond, triple bond or -CR12-; wherein each R1 is independently hydrogen, halogen, hydroxy, amino, C1 -C6alkyl, C1-C6alkenyl, C1-C6alkynyl, or C1-C6alkoxy; wherein the alkyl, alkenyl, alkynyl or alkoxy may be optionally substituted with halogen, hydroxy, C1-C4alkoxy or amino; and each R is independently hydrogen, a cationic alkyl side group or a cationic oligo or poly(ethylene oxide) group.

18. The method of Claim 17, wherein the compound is represented by the following structural formula:

19. The method of Claim 17, wherein the compound is represented by the following structural formula:

20. The method of Claim 9, wherein the CPE or COE is a compound represented by the following structural formula:

, or a salt thereof; wherein:
each is independently selected from:

each Ar is independently an optionally substituted aromatic group;
each R is independently a cationic, anionic, or neutral alkyl group or a cationic, anionic, or neutral oligo or poly(ethylene oxide) group;
each Linker is a single bond, double bond, triple bond, -CH2- or -CH2CH2-; and each R' is independently a terminating group.

21. The method of Claim 20, wherein each R is selected from -(CH2)n X', -(OCH2CH2)n X', -(OCH2CH2)n OX', -(CH2CH2O)n X' and -(CH2CH2O)y CH2CH2X', wherein X' is selected from -SO3Y, -PO3Y2, and -CO2Y, n is an integer between 2 and 13, inclusive, q is an integer between 1 and 12, inclusive, and Y is sodium or potassium.

22. The method of Claim 9, wherein the target is lysozyme.

23. The method of Claim 9, wherein the CPE or COE is represented by Structural Formula (III):

or a salt thereof; wherein:
R' and R3 are each independently hydrogen or a charged side group;
m is an integer between 2 and 50, inclusive;

Ar is an optionally substituted monocyclic or polycyclic aromatic ring system or an optionally substituted monocyclic or polycyclic heteroaromatic ring system; and T, T' and T" are each independently a terminating group.

24. The method of Claim 23, wherein Ar is fluorene, benzene, biphenyl, thiophene, benzothiadiazole, 4,7-di(thien-5'-yl)-2,1,3-benzothiadiazole, pyridine, bipyridinium, triphenylamine, anthracene or carbazole.

25. The method of Claim 23, wherein the CPE or COE is represented by the following structural formula:

R'= R3 = (CH2)6N(CH3)3Br

26. The method of Claim 9, wherein the CPE or COE is represented by Strucutral Formula (IV):

, or a salt thereof; wherein:
R' and R3 are each independently hydrogen or a charged side group;
m is an integer between 2 and 50, inclusive;
Ar is an optionally substituted monocyclic or polycyclic aromatic ring system or an optionally substituted monocyclic or polycyclic heteroaromatic ring system; and T and T' are each independently a terminating group.

27. The method of Claim 26, with the proviso that the CPE or COE is not represented by the following structural formula:

28. A method of detecting a target in a sample, comprising:
functionalizing a surface of a solid support with a charged ligand, thereby creating a charge on the surface of the solid support;
incubating the ligand-functionalized solid support with a sample, whereupon binding of the target, the charge on the surface of the solid support switches;
incubating the sample with a charged conjugated polyelectrolyte (CPE) or a charged conjugated oligoelectrolyte (COE), wherein the charge of the CPE or the COE is complementary to the charge on the surface of the target-bound surface; and detecting the fluorescence of the solid support, thereby detecting the target.