WO2018039585A1

WO2018039585A1 - Redox mediator-functionalized water-soluble polymer

Info

Publication number: WO2018039585A1
Application number: PCT/US2017/048655
Authority: WO
Inventors: Cuihua Xue
Original assignee: Hitachi Chemical Co Ltd; Hitachi Chemical Co America Ltd
Current assignee: Resonac Corp; Resonac America Inc
Priority date: 2016-08-25
Filing date: 2017-08-25
Publication date: 2018-03-01
Anticipated expiration: 2019-02-25

Abstract

Described herein are a series of water-soluble conjugated polymers having redox mediators and functional groups on flexible side chains. The polymers may include a backbone having a conjugated polymer; a first side chain attached to the backbone, the first side chain having a ferrocene group or a derivative thereof; a second side chain attached to the backbone, the second side chain having an organic acid, a salt of an organic acid, or quaternary ammonium; and at least one of the first and second side chains having at least one of a carbon atom, a nitrogen atom, an oxygen atom, and a sulfur atom. Because of their redox activity, water solubility and ease of further coupling with versatile functional species, the polymers discussed in this application can be used as sensor interface materials for various biosensor developments.

Description

REDOX MEDIATOR-FUNCTIONALIZED WATER-SOLUBLE POLYMER

FIELD

[0001] The present invention relates to, in general, water-soluble conjugated polymers having redox mediators and functional groups on flexible side chains, methods of manufacturing such water-soluble conjugated polymers, and applications thereof.

BACKGROUND

[0002] Redox mediators that assist in transferring electrons are a necessary component in the fabrication of electrochemical sensors and bio-sensors. Mediators are often small molecules that leach easily from the sensor interface, which affects the stability and durability of the sensors and also has potential harmful effects if the sensor (e.g., a wearable sensor) makes contact with the body. Polymers are macromolecules and covalent attachment of mediators onto the polymers prevents leaching of the mediators.

[0003] Ferrocene is a redox mediator with reversible redox properties and polythiophenes are well-known conducting polymers. Both ferrocene and

polythiophenes have been used separately or together in many sensing applications. When ferrocene-polythiophenes are used, however, the ferrocene-polythiophene films are formed directly on the electrode surface by electrochemically co-polymerizing small thiophenes and ferrocene-containing thiophenes, and the ferrocene-polythiophenes are not isolated materials. Such ferrocene-polythiophenes are limited when used in sensor mass production due to the lack of control of the ferrocene number in the sensor film. Also the ferrocene-polythiophenes are mostly hydrophobic, which lacks compatibility with biological species for biosensor development.

[0004] Therefore, there is a need for versatile, isolated ferrocene-functionalized polythiophene materials with good water-solubility for compatibility with biological species, easy sensor fabrication, and easy surface ferrocene control while preventing leaching of the ferrocene from the sensor.

[0005] In this specification where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.

[0006] While certain aspects of conventional technologies have been discussed to facilitate disclosure of the invention, Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass or include one or more of the conventional technical aspects discussed herein.

SUMMARY

[0007] The present invention may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies, or provide benefits and advantages, in a number of technical areas. Therefore the invention should not necessarily be construed as being limited to addressing only the particular problems or deficiencies discussed herein.

[0008] To address the above-discussed drawbacks of small molecule ferrocene- based redox mediators, the inventor of this application developed a series of ferrocene- functionalized water-soluble polythiophenes that function well as redox mediator macromolecular materials, while effectively preventing leaching of the ferrocenes from the sensor functioning surface. The materials show good redox activity and are very easy to process with various hydrophilic reagents/biological species in water or buffers. The number of ferrocenes in the sensor interface can be easily controlled by controlling the concentration of the ferrocene-polythiophene to optimize sensor performance. Also, the redox mediator macromolecular materials have potential reactive sites for further modification with other functional species, which may lead to further fabrication of versatile sensor platforms, and can be used in many other applications like medical detections, targeting etc. Furthermore, due to the redox activity, the materials can be used as semiconducting materials in organic photovoltaic cells, organic light-emitting diodes, field-effect transistors, organic semiconductors, electronic optical sensors and other opto-electronic devices, and the like.

[0009] According to one aspect, the present invention provides a polymer comprising at least one repeating unit selected from repeating unit (A), repeating unit (B) and repeating unit (C).

[0010] According to a another aspect, the present invention provides a polymer comprising at least one repeating unit (A) and at least one repeating unit (B) or a polymer comprising at least one repeating unit (A) and at least one repeating unit (C). [0011] According to another alternative aspect, the present invention provides a polymer comprising at least one repeating unit (A), at least one repeating unit (B) and at least one repeating unit (C).

[0012] Repeating unit (A) is one or more monomers represented by the following

[0013] Repeating unit (B) is one or more monomers represented by the following

[0014] Repeating unit (C) is one or more monomers represented by the following

[0015] In the formulae above, X is a single bond, C1 -C20 alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl,

[0016] n is 0, 1 or an integer greater than 1 , [0017] M is

substituted ferrocene or ferrocene derivatives,

[0018] R is OH, Br, I, CI, SH, COOH, SO₃H, NH₂, Ν⁺(R₁)₃, YRiCOOH, YRTSOSH, maleimide, NHS ester, YR^, COONa, SO₃Na, COOK, SO₃K, YR^OONa, YR^OsNa, YR^OOK or YR^OsK;

[0019] Y is C, O, S or N; R-i is alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, and can be the same or different; and

[0020] L is selected from carbohydrates, proteins, peptides, DNA, RNA, antibodies, antigens, enzymes, bacteria, redox molecules, host molecules, guest molecules, haptens, lipids, microbes, sugars and aptamers, maleimide and NHS ester.

[0021] According to a further exemplary aspect, the present invention provides a polymer comprising: a structural unit (A), a structural unit (Β'), and a structural unit (C). The polymer comprises 20-40 mol% of structural unit (A), 20-40 mol% of structural unit (Β'), and 20-40 mol% of structural unit (C). The structural unit (A), structural unit (Β'), and structural unit (C) have a composition represented by the following formulas, respectively:

[0022] wherein: X = S or N;

[0023] Y = ferrocene or its derivatives; [0024] Z = a carboxylic acid group, a carboxylate group, a sulfonic group or a sulfonate group;

[0025]

[0026] R² = a divalent organic group;

[0027]

[0028] R⁴ = a divalent organic group; and

[0029] m = 1 or 2, n = 1 or 2, p = 2 to 20, and q = 2 to 20.

[0030] The divalent organic group referenced above may be a 1 -10C alkylene group, which may contain one or more hetero atoms or one or more unsaturated bonds,

[0031] p may further optionally be of 2 to 5, and/or q may optionally be 2 to 5.

[0032] The polymer may comprise 25-35 mol% of structural unit (Α'), 25-35 mol% of structural unit (Β'), and 25-35 mol% of structural unit (C), according to optional embodiments.

[0033] According to yet another alternative aspect, the present invention provides a polymer comprising: a backbone having a conjugated polymer; a first side chain attached to the backbone, the first side chain having a ferrocene group or a derivative thereof; a second side chain attached to the backbone, the second side chain having an organic acid, a salt of an organic acid, or quaternary ammonium; and at least one of the first and second side chains having at least one of a carbon atom, a nitrogen atom, an oxygen atom, and a sulfur atom. [0034] The conjugated polymer may optionally comprise at least one of: a polythiophene, a polyaniline, a polyacetylene, a poly(p-phenylene), a polypyrrole or derivatives thereof.

[0035] The first chain can optionally include 5 to 40 atoms between the ferrocene group or derivative thereof and the conjugated polymer backbone. The first chain may further optionally comprise 10 to 30 atoms between the ferrocene group or derivative thereof and the conjugated polymer backbone.

[0036] The first side chain can optionally include 5 to 40 atoms between the organic acid, salt of an organic acid or quaternary ammonium and the conjugated polymer backbone. The first side chain may further optionally comprise 10 to 30 atoms between the organic acid, salt of an organic acid or quaternary ammonium and the conjugated polymer backbone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Fig. 1 A is an initial cyclic voltammetry curve for 2.5 mg of Polymer 1 in 1 ml_ of 0.1 M PBS buffer, measured using a glassy carbon electrode.

[0038] Fig. 1 B are cyclic voltammetry curves for a Polymer 1 film formed on a glassy carbon electrode in 0.1 M PBS buffer, where the two curves correspond to a CV scan before sonication (solid line) and a CV scan after 10 minutes of sonication (broken line) in the PBS buffer.

[0039] Fig. 2 is a cyclic voltammetry curve for 1 .5 mg of Polymer 2 in 1 mL of 0.1 M KCIO₄. [0040] Fig. 3A is an initial cyclic voltammetry curve for Polymer 3 in PBS buffer (2.5 mg/ml), measured using a glassy carbon electrode.

[0041] Fig. 3B is a cyclic voltammetry curve for a Polymer 3 film formed on a glassy carbon electrode in 0.1 M PBS buffer.

[0042] Fig. 4 is a cyclic voltammetry curve for 2 mg of Polymer 4 in 10 mL of 0.1 M PBS buffer, measured using a glassy carbon electrode.

[0043] Fig.5 is a cyclic voltammetry (CV) curve of Polymer 5 in 0.1 M PBS buffer (~2mg/1 ml_) using glassy carbon electrode, which shows its redox activity in a low potential range (0-0.2V).

DETAILED DESCRIPTION

[0044] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Additionally, the use of "or" is intended to include "and/or", unless the context clearly indicates otherwise.

[0045] A used herein, the term "redox mediator" refers to a chemical moiety capable of undergoing oxidation or reduction through electron transfer with an electrode and with a redox enzyme.

[0046] As used herein, the term " redox mediator macromolecular materials" refers to a polymer modified with a redox mediator.

[0047] As used herein, the term "reactive site" refers to a functional group capable of reacting with another functional group. [0048] An exemplary embodiment of the invention described in the application is a series of water-soluble conjugated polymers having redox mediators and functional groups on flexible side chains.

[0049] According to one aspect, the present invention provides a polymer comprising at least one repeating unit selected from repeating unit (A), repeating unit (B) and repeating unit (C).

[0050] According to another aspect, the present invention provides a polymer comprising at least one repeating unit (A) and at least one repeating unit (B) or a polymer comprising at least one repeating unit (A) and at least one repeating unit (C).

[0051] According to yet another aspect, the present invention provides a polymer comprising at least one repeating unit (A), at least one repeating unit (B) and at least one repeating unit (C). The repeating units may be conjugated via a single, double or triple bond.

[0052] Repeating unit (A) is one or more monomers selected from Table 1 ;

repeating unit (B) is one or more monomers selected from Table 2; and repeating unit (C) is one or more monomers selected from Table 3.

[0053] TABLE 1

X = a single bond, C1 -C20 alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl; n = 0, 1 or an integer greater than 1 ;

M =

substituted ferrocene or ferrocene derivatives;

R = OH, Br, I, CI, SH, COOH, SO₃H, NH₂, Ν⁺(R₁)₃, YRiCOOH, YRTSOSH, maleimide, NHS ester, YF^ L, COONa, SO₃Na, COOK, SO₃K, YF^COONa, YR1 SO₃Na, YRTCOOK or YF^SC^K;

Y = C, O, S or N;

Ri = alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, and Ri can be the same or different; and

L = carbohydrates, proteins, peptides, DNA, RNA, antibodies, antigens, enzymes, bacteria, redox molecules, host molecules, guest molecules, haptens, lipids, microbes, sugars and aptamers, maleimide or NHS ester. [0054] TABLE 2

X = a single bond, C1 -C20 alkyl, aryl, substituted aryl, heteroaryl or a substituted heteroaryl; n = 0, 1 or an integer greater than 1 ;

M = substituted ferrocene or ferrocene derivatives;

R = OH, Br, I, CI, SH, COOH, SO₃H, NH₂, Ν⁺(R₁)₃, YRiCOOH, YRTSOSH, maleimide, NHS ester, YR₁L, COONa, SO₃Na, COOK, SO₃K, YF^COONa, YR1 SO₃Na, YRTCOOK or YR^O₃K;

Y = C, O, S or N;

R₁ = alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, and R-i can be the same or different; and L = carbohydrates, proteins, peptides, DNA, RNA, antibodies, antigens, enzymes, bacteria, redox molecules, host molecules, guest molecules, haptens, lipids, microbes, sugars and aptamers, maleimide or NHS ester.

[0055] TABLE 3

X = a single bond, C1 -C20 alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl;

M =

substituted ferrocene or ferrocene derivatives; R = OH, Br, I, CI, SH, COOH, SO₃H, NH₂, Ν⁺(R₁)₃, YR1COOH, YRTSOSH, maleimide, NHS ester, YR^, COONa, SO₃Na, COOK, SO₃K, YR^OONa,

YR1 SO₃Na, YRTCOOK or YR^OsK;

R-i = an alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, and R-i can be the same or different; and

L = carbohydrates, proteins, peptides, DNA, RNA, antibodies, antigens, enzymes, bacteria, redox molecules, host molecules, guest molecules, haptens, lipids, microbes, sugars and aptamers, maleimide or NHS ester.

[0056] According to yet another alternative aspect, the present invention provides a polymer comprising: a backbone having a conjugated polymer; a first side chain attached to the backbone, the first side chain having a ferrocene group or a derivative thereof; a second side chain attached to the backbone, the second side chain having an organic acid, a salt of an organic acid, or quaternary ammonium; and at least one of the first and second side chains having at least one of a caron atom, a nitrogen atom, an oxygen atom, and a sulfur atom. The conjugated polymer may comprise at least one of: a polythiophene, a polyaniline, a polyacetylene, a poly(p-phenylene), a polypyrrole, or derivatives thereof. The first chain may have 5 to 40 atoms between the ferrocene group and the conjugated polymer backbone. At least one of the first and second side chains can optionally comprise an ethylene oxide group. The second side chain may optionally comprise a carboxylic acid group, a carboxylate group, a sulfonic acid group or a sulfonate group. [0057] The versatile ferrocene-functionalized water-soluble polythiophenes can be obtained after post-functionalizing polymer precursors with functional molecules.

[0058] The polymers are soluble in water and various buffers and show good redox activity in various detection buffers. The solubility of the polymers in water may be more than 0.8 g/100 g at 25°C, preferably more than 1 .0 g/100 g at 25°C, more preferably more than 2.0 g/100 g at 25°C, and still more preferably more than 3.0 g/100 g at 25°C, where the concentration is a measure of the amount of polymer with respect to the amount of water. The solubility of the polymer in water may be less than 100 g /100 g at 25°C. Some sensors fabricated using these polymers show good sensing performance even at low concentrations (0-2 mM) of glucose.

Samples of polymer syntheses

[0059] Synthesis Example 1 : Synthesis of Polymer 1 , which is represented by the following structural formula, is shown in Schemes 1 to 3:

[0060] Scheme 1 below illustrates the synthesis of monomer 1 : 2,5-dibromo (2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethoxy)thiophene. Scheme 1

[0061] To a 500 ml_ three-necked round-bottomed flask, t-BuOK (26 g, 232 mmol), Cul (6.0 g, 31 .6 mmol), pyridine (30 ml_) and 2,2'-((oxybis(ethane-2, 1 - diyl))bis(oxy))diethanol (149 g, 767 mmol) were added. The mixture was stirred at room temperature for 30 minutes under a nitrogen atmosphere and then 3-bromothiophene (25.0 g, 153.4 mmol) was added. The mixture was then heated to 100 C for about 24 hrs until disappearance of the 3-bromothiophene, as monitored by TLC. The reaction mixture was cooled to room temperature, poured into 10% HCI solution, extracted with ethyl acetate ("EtOAc"), washed with 10% NH₄CI solution and/or NaCI solution, and dried over anhydrous MgS0 . After removal of the solvent, the crude mixture was purified by chromatography to give compound 1 as an oil. ¹ H NMR (500 MHz, CDCI3): δ 7.19 (m, 1 H), 5 6.81 (m, 1 H), 6.30 (m, 1 H), 4.15 (t, J=6.2 MHz, 2H), 3.87(t, J=6.2 MHz, 2H), 3.62-3.75 (m, 12H) ppm, 2.81 (m, 1 H) ppm. [0062] PPh₃ (9.5 g, 36.3 mmol) was suspended in 30 ml_ of CH₃CN under a nitrogen atmosphere at 0 C and Br₂ (2.9 g, 18.12 mmol) was slowly added. Then, compound 1 (5 g, 18.12 mmol) in 10 ml_ CH₃CN was added dropwise and the mixture was stirred from 0 C to room temperature for about 48 hrs. Any remaining solid was filtered and the filtrate was purified by chromatography to provide compound 2 as an oil. ¹ H N MR (500 MHz, CDCI3): 5 7.22 (m, 1 H), 5 6.83 (m, 1 H), 6.31 (m, 1 H), 4.17 (t, J=6.2 MHz, 2H), 3.71 -3.90 (m, 12H), 3.51 (t, J=6.2 MHz, 2H) ppm.

[0063] Compound 2 (4.15 g, 12.24 mmol) was dissolved in a mixture of 8 mL THF and 8 mL AcOH. /V-Bromosuccinimide (4.58 g, 25.73 mmol) was added and the mixture was stirred at room temperature for about 3 hrs. The reaction mixture was then poured into NaCI solution and extracted with EtOAc. Combined EtOAc was washed with NaCI solution and dried over anhydrous MgS0_4. After removal of the solvent, the crude mixture was purified by chromatography to give monomer 1 . ¹H NMR (500 MHz, CDCI3): 5 6.87 (s, 1 H), 4.22 (t, J=6.2 MHz, 2H), 3.71 -3.87 (m, 12H) ppm, 3.52 (t, J=6.2 MHz, 2H) ppm.

[0064] Scheme 2 below illustrates the synthesis of monomer 2.

Scheme 2

[0065] Ferrocenemethanol (4.8 g, 22.2mmol) was dissolved in dry THF and NaH (0.8 g, 33.3mmol) was added. The mixture was stirred at room temperature for about 20 minutes and then monomer 1 ( 10 g, 20.1 mmol) was added. The resulting mixture was stirred at room temperature for about 20 hrs until the disappearance of monomer 1 , as monitored by TLC. The reaction mixture was then poured into NaCI solution and extracted with EtOAc. Combined EtOAc was washed with NaCI solution and dried over anhydrous MgS0_4. After removal of the solvent, the crude mixture was purified by chromatography to give monomer 2. ¹ H NMR (500 MHz, CDCI3): δ 6.86 (s, 1 H), 4.36 (s, 2H), 4.28 (s, 2H), 4.18-4.20 (m, 7H), 3.83 (t, J=6.2 MHz, 2H), 3.61 -3.84 (m, 14H) ppm.

[0066] Scheme 3 below shows the co-polymerization of monomer 1 , thiophene- 2,5-diboronic acid and monomer 2 to produce polymer 1 precursor and polymer 1.

Scheme 3

[0067] 0.5 mol of monomer 1 , 0.5 mol of monomer 2, 1 .0 mol of 2,5-thiophene- diboronic acid, Pd(PPh₃) (5% of monomer 1 ), and K2CO3 were placed in a two-necked flask under a nitrogen atmosphere. 20 ml of THF and 6 ml of water were added, and the reaction mixture was heated to 70^°C for about 20 h. The reaction was cooled to room temperature and poured into CH₃OH, which resulted in the formation of a precipitate. The collected precipitate was washed with CH₃OH several times and dried by vacuum to give polymer 1 precursor as a dark sticky oil. The polymer 1 precursor was then dissolved in anhydrous DMF, and 2 equivalents of K2CO3 and 2 equivalents of sodium 2-mercaptoethanesulfonate were added. The mixture was stirred at room temperature for about 16 hrs, and transferred into a dialysis tube (CO 12,000) for dialysis against water. After dialysis, the solution in the dialysis tube was filtered to remove insoluble impurities and then freeze-dried to give polymer 1 .

[0068] Polymer 1 is highly soluble in water, and also soluble in various buffers. Solubility in water of Polymer 1 is more than 3 g/100g. [0069] To check the redox activity of Polymer 1 , cyclic voltammetry (CV) was carried out in a solution of 2.5 mg of Polymer 1 dissolved in 1 mL 0.1 M PBS buffer using a glassy carbon electrode. Fig. 1 A is a first CV scan for Polymer 1 , which shows the redox peaks of the mediator ferrocenes. After the CV scans, a Polymer 1 film was formed on the electrode surface. Fig. 1 B are the CV curves of the Polymer 1 film before (solid line) and after 10 minutes of sonication in PBS buffer (broken line), which shows the Polymer 1 film is quite stable and also has good redox activity.

[0070] Synthesis Example 2: Synthesis of Polymer 2, which is represented by the following structural formula, is shown in Scheme 4:

Scheme 4

[0071] The polymer 1 precursor was dissolved in anhydrous DMF, and then 2 equivalents of K2CO3 and 2 equivalents of sodium 2-mercaptoacetate were added. The mixture was stirred at room temperature for about 16 hrs, and then transferred into a dialysis tube (CO 12,000) for dialysis against water. After dialysis, the solution in the dialysis tube was filtered to remove insoluble impurities and then freeze-dried to give polymer 2.

[0072] Polymer 2 is highly soluble in water, and various buffers. The solubility of Polymer 2 in water is more than 3 g/100 g. The cyclic voltammetry curve of Polymer 2 is similar to that of polymer 1 in a 0.1 M PBS buffer, as shown in Fig. 2. Polymer 2 also has reactive sites (-COOH), which can be used to further couple with other substrates like enzymes.

[0073] Synthesis Example 3: Synthesis of Polymer 3, represented by the following structural formula, is shown in Schemes 5-7:

[0074] Scheme 5 below illustrates the synthesis of monomer 3:

Scheme 5

[0075] To a 500 mL three-necked round-bottomed flask, t-BuOK (34 g, 0.3 mol), Cul (8.0 g, 40 mmol), pyridine (50ml_) and 2,2'-((oxybis(ethane-2, 1 - diyl))bis(oxy))diethanol (200 g, 1 .03 mol) were added. The mixture was stirred at room temperature for about 30 minutes under a nitrogen atmosphere. Then, 3,4- dibromothiophene (25.0 g, O. l mmol) was added, and the mixture was heated to 100 C for about 24 hrs until the disappearance of 3,4-dibromothiophene, as monitored by TLC. The reaction mixture was cooled to room temperature, poured into 10% HCI solution, and extracted with ethyl acetate ("EtOAc"). The combined EtOAc solution was washed with 10% saturated NH CI solution and/or NaCI solution and dried over anhydrous MgSO₄. After removal of the solvent, the crude mixture was purified by chromatography to give compound 3 as an oil. ¹ H NMR (500 MHz, CDCI3): δ 6.27 (s, 2H), 4.17 (t, J=6.2 MHz, 4H), 3.87(t, J=6.2 MHz, 4H), 3.62-3.75 (m, 24H) ppm, 2.84 (m, 2H) ppm.

[0076] PPh₃ (18.8 g, 71 .76 mmol) was suspended in 40 ml_ of CH₃CN under a nitrogen atmosphere at 0 C and Br₂ (5.75 g, 35.94 mmol) was slowly added. After all of the Br₂ was added, compound 3 (8.4 g, 17.95 mmol) in 15 mL CH₃CN was added dropwise and the mixture was stirred from 0 C to room temperature for about 48 hrs. After completion of the reaction, the solid in the mixture was filtered out and the filtrate was collected and purified by chromatography to provide compound 4 as an oil. ¹ H NMR (500 MHz, CDCI3): δ 6.28 (s, 2H), δ 4.19 (t, J=6.2 MHz, 4H), 3.71 -3.90 (m, 24H), 3.51 (t, J=6.2 MHz, 4H) ppm.

[0077] Compound 4 (7.6 g, 12.79 mmol) was dissolved in a mixture of 10 mL THF and 10 mL AcOH. /V-Bromosuccinimide (4.78 g, 26.85 mmol) was added to the mixture, and the mixture was stirred at room temperature for about 4 hrs. The reaction mixture was then poured into NaCI solution and extracted with EtOAc. Combined EtOAc was washed with NaCI solution and dried over anhydrous MgS0_4. After removal of the solvent, the crude mixture was purified by chromatography to give monomer 3. ¹ H NMR (500 MHz, CDCI3): δ 4.29 (t, J=6.2 MHz, 4H), 3.71 -3.86 (m, 24H) ppm, 3.52 (t, J=6.2 MHz, 4H) ppm.

[0078] Scheme 6 below illustrates the synthesis of monomer 4.

Scheme 6

[0079] Ferrocenemethanol (2.3 g, 10.65mmol) was dissolved in dry THF and NaH (0.25 g, 10.41 mmol) was added. The mixture was stirred at room temperature for about 20 minutes and then monomer 3 (3.0 g, 3.99mmol) was added. The mixture was then stirred at room temperature for about 20 hrs until the disappearance of monomer 3, as monitored by TLC. The reaction mixture was then poured into NaCI solution and extracted with EtOAc. Combined EtOAc was washed with NaCI solution and dried over anhydrous MgS0_4. After removal of the solvent, the crude mixture was purified by chromatography to give monomer 4. ¹ H NMR (500 MHz, CDCI3): δ 4.37 (s, 4H), 4.28 (s, 4H), 4.17-4.22 (m, 14H), 3.79 (t, J=6.2 MHz, 4H), 3.62-3.79 (m, 28H) ppm,

[0080] Scheme 7 below shows the co-polymerization of monomer 3, thiophene- 2,5-diboronic acid and monomer 4 to produce polymer 3 precursor and polymer 3. Scheme 7

[0081] 0.5 mol of monomer 3, 0.5 mol of monomer 4, 1 .0 mol of 2,5-thiophene- diboronic acid, Pd(PPh₃)₄ (10% of monomer 3), and K₂C0₃ were placed in a two- necked flask under a nitrogen atmosphere. 20 ml of THF and 6 ml of water were added, and the reaction mixture was heated to 70^°C for about 20 hrs. The reaction mixture was then cooled to room temperature and poured into CH₃OH, which resulted in the formation of a precipitate. The collected precipitate was washed with CH₃OH several times and dried by vacuum to give polymer 3 precursor as a dark sticky oil. The polymer 3 precursor was then dissolved in anhydrous DMF, and 2 equivalents of K₂C0₃ and 2 equivalents of sodium 2-mercaptoethanesulfonate were added. The mixture was stirred at room temperature for about 16 hrs, and then transferred into a dialysis tube (CO 12,000) for dialysis against water. After dialysis, the solution in the dialysis tube was filtered to remove insoluble impurities and then freeze-dried to give polymer 3.

[0082] Polymer 3 is highly soluble in water, and also soluble in various buffers. Solubility of Polymer 3 in water is more than 3 g/100 g.

[0083] To check the redox activity of Polymer 3, cyclic voltammetry (CV) was carried out in a solution of 2.5 mg of Polymer 3 in 1 ml_ of 0.1 M PBS buffer using a glassy carbon electrode. Fig. 3A is the initial CV curve for Polymer 3, which shows the redox peaks of the mediator ferrocenes. After CV scans, a Polymer 3 film was formed on an electrode surface. Fig. 3B shows the CV curves of the Polymer 3 film in PBS buffer, which shows Polymer 3 film has good redox activity.

[0084] Synthesis Example 4: Synthesis of Polymer 4, which is represented by the following structural formula, using the polymer 3 precursor is shown in Scheme 8:

Scheme 8

[0085] The polymer 3 precursor was dissolved in anhydrous DMF, and 2 equivalents of K₂CO₃ and 2 equivalents of sodium 2-mercaptoacetate were added. The mixture was stirred at room temperature for about 16 hrs, and then transferred into a dialysis tube (CO 12,000) for dialysis against water. After dialysis, the solution in the dialysis tube was filtered to remove insoluble impurities and then freeze-dried to give Polymer 4.

[0086] Polymer 4 is highly soluble in water and various buffers, and its cyclic voltammetry curve is similar to that of polymers 1 , 2 and 3 in buffer, as shown in Fig. 4. Polymer 4 also has reactive sites (-COOH), which can be used to couple with other substrates, e.g., enzymes.

[0087] Synthesis Example 5: Synthesis of Polymer 5

[0088] Scheme 9 below illustrates the synthesis of compounds 4 - 6.

Scheme 9

[0089] Synthesis of compound 4: A suspension of AiCb (6.5g, 49.1 mmol) in 20mL CH2CI2 was formed and 6-bromohexanoyl chloride (10.5g, 49.1 mmol) was added at 0°C and N₂ atmosphere. Then a solution of 1 , 1 '-Dimethylferrocene (10g, 46.73mmol) in 10ml_ CH2CI2 was also added. The whole mixture was reacted at 0°C first and then gradually to room temperature for 12 hours. The reaction mixture was poured into 40ml of saturated NaCI solution and extracted with CH2CI2, and combined CH2CI2 solution was washed with saturated NaCI solution and dried over anhydrous MgS0₄. After removal of solvent, the crude mixture was purified by chromatography to give

compound 4 as orange oil. ¹ H NMR (500 MHz, CDCI3): δ 4.66 (s, 1 H), δ 4.65 (s 1 H), 4.06 (s, 1 H), 3.48-3.51 (m, 4H), 3.49(t, J=6.2 MHz, 2H), 2.73 (t, J=6.2 MHz, 2H), 2.01 (s, 3H), 1 .96 (m, 2H), 1 .95 (s, 3H), 1 .77 (m, 2H), 1 .60 (m, 2H) ppm.

[0090] Synthesis of compound 5: To a solution of compound 4 (4.8g, 12.28mmol) in 1 QmL CH₂CI₂ under N₂ atmosphere was added to a solution of BH₃ SMe₂ (0.94g, 12.37mmol). The reaction mixture was stirred at room temperature for 12 hrs, after which time the mixture was quenched with saturated aqueous NH₄CI (30mL) and extracted with CH₂Ci2. Combined CH₂CI₂ solution was washed with saturated NaCI solution and dried over anhydrous MgS0 , After removal of solvent, the crude mixture was purified by chromatography to give compound 5 as orange oil. ¹ H NMR (500 MHz, CDCI3): δ 3.90-3.97 (m, 7H), 3.47 (t, J=6.2 MHz, 2H), 2.32 (t, J=6.2 MHz, 2H), 2.01 (s, 3H), 1 .99 (s, 3H), 1 .92 (m, 2H), 1.49-1 .54 (m, 4H), 1 .40-1 .42 (m, 2H) ppm.

[0091] Synthesis of compound 6: To a 20 ml_ ethanol solution containing compound 5 (2.93g, 7.77mmol) was added thiourea (0.65g, 8.55mmol) under N₂ atmosphere and the mixture was refluxed for 18 hrs. After being cooled to room temperature, the reaction mixture was concentrated in vacuo, followed by adding NaOH solution (0.37g, 9.32mmol in 35ml degassed water). The reaction mixture was again refluxed for 1 hr under N₂ atmosphere. The solution was then cooled to room

temperature and extracted with CH₂CI₂. The combined CH₂CI₂ was dried over anhydrous MgSO . After removal of solvent, the crude product was purified by chromatography to give compound 6. ¹H NMR (500 MHz, CDCI3): δ 3.93 (m, 7H), 2.61 (m, 2H), 2.31 (m,2H), 2.01 (s, 3H), 1.98 (s, 3H), 1 .68 (m, 2H), 1 .54 (m, 2H), 1.45 (m, 2H), 1 .37-1 .41 (m, 3H) ppm.

[0092] Scheme 10 below shows the synthesis of monomer 5.

Scheme 10

[0093] A solution of compound 6 (0.5g, 1 .52mmol) in anhydrous DMF (15ml) was added to a mixture of K₂C0₃ (1 .8g, 13.6mmol) and compound 3 (5.6g, 7.5mmol) under a N₂ atmosphere. The mixture was stirred at room temperature for about 24 hrs. The reaction mixture was then poured into 40ml of saturated NaCI solution and extracted with ethyl acetate and the combined solution was washed with saturated NaCI solution and dried over anhydrous MgS0₄. After removal of solvent, the crude mixture was separated by chromatography to obtain monomer 5. ¹H NMR (500 MHz, CDCI3): δ 4.17-4.19 (m, 1 1 H), 3.82-3.94 (m, 10H), 3.51 -3.76 (m, 14H), 3.52 (t, J=6.2 MHz, 2H), 2.76 (t, J=6.2 MHz, 2H), 2.59 (t, J=6.2 MHz, 2H), 1 .96 (m, 3H), 1 .95 (s, 3H), 1 .63 (m, 4H), 1 .41 (m, 6H) ppm.

[0094] Scheme 1 1 below shows the synthesis of polymer 5 precursor. Scheme 11

[0095] A mixture of Pd(PPh₃)₄ (0.14g, 0.12mmol), K₂C0₃ (0.8g, 6.0mmol) and 2,5-bis(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2yl)thiophene (0.42g, 1 .2mmol) was formed. Under N₂ atmosphere, a solution of monomer 5 (1.2g, 1 .2mmol) in 15ml degassed THF and 3ml degassed H₂0 was added to the mixture to form a reaction mixture. The reaction mixture was then heated to 70°C to react for 24 hrs. Then, the reaction mixture was cooled to room temperature and poured into a saturated NaCI solution and extracted with EtOAc. The combined EtOAc was dried over anhydrous MgSO_4. After removing solvent, the residue was dried by vacuum to give crude polymer 5 precursor, which was taken to next step reaction to post-functionaiize without further purification.

[0096] Scheme 12 below shows the synthesis of polymer 5. Scheme 12

[0097] To a mixture of K₂CO₃ (0.8g, 5.8mmol) and sodium 2

mercaptoethanesulfonate (1 .Og, 6.1 mmol) was added a solution of polymer 5 precursor (1.5g) in anhydrous DMF (10ml) under N₂ atmosphere and the mixture was stirred at room temperature for about 24 hrs. Then, a small amount of water (5ml) was added into the mixture and the whole mixture was transferred into dialysis tube (CO 12,000) to perform dialysis in deionized ("Dl") water. After dialysis, the clear water solution (about 1 .5g polymer 5 in 20ml Dl H₂O) in the dialysis tube went through a filter (0.1 pm) and the filtered solution was then freeze-dried to give polymer 5.

[0098] Polymer 5 is highly soluble in water and various buffers, for example 50mg polymer 5 can easily dissolve in 1 ml Dl water or 0.1 M PBS buffer to give a clear yellow solution. [0099] Fig.5 is a cyclic voltammetry (CV) curve of Polymer 5 in 0.1 M PBS buffer (~2mg/1 ml_) using glassy carbon electrode, which shows its redox activity in a low potential range (0-0.2V).

[00100] Because of their redox activity, water solubility and ease of further coupling with versatile functional species, the polymers discussed in this application can be used as sensor interface materials for various biosensor developments. Also, due to their easy processibility in water and buffers, the sensors can be fabricated using already established drop casting or printing technology, and thus, these sensors can be mass produced easily. The redox mediator macromolecular materials of this application can also be used to functionalize other electro-conductive materials to create new materials with functional surfaces for other applications like implantable materials.

[00101] Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification be considered exemplary only, with the scope and spirit of the invention being indicated by the claims.

[00102] As various changes could be made in the above methods and

compositions without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

[00103] None of the features recited herein should be interpreted as invoking 35 U.S.C. § 1 12, unless the term "means" is explicitly used.

Claims

WE CLAIM:

1 . A polymer comprising at least one repeating unit (A), and at least one of

repeating units (B) or (C), wherein repeating unit (A) is one or more monomers represented by following formulae (1 -1 ) to (1 -10); repeating unit (B) is one or more monomers represented by following formulae (2-1 ) to (2-10); and repeating unit (C) is one or more monomers represented by following formulae (3-1 ) to (3-9):

and wherein: a single bond, C1 -C20 alkyl, aryl, substituted aryl, heteroaryl or substituted

heteroaryl;

0, 1 or an integer greater than 1 ;

substituted ferrocene or ferrocene derivatives;

R = OH, Br, I, CI, SH, COOH, SO₃H, NH₂, Ν⁺(R₁)₃, YRiCOOH, YRTSOSH, maleimide, NHS ester, YR^, COONa, SO₃Na, COOK, SO₃K, YR^OONa, YR1 SO₃Na, YR^OOK or YR^OsK;

Y = C, O, S or N;

R₁ = alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, and R-i can be the same or different;

2. The polymer of claim 1 , wherein the polymer comprises at least one repeating unit (A) and at least one repeating unit (B).

3. The polymer of claim 1 , wherein the polymer comprises at least one repeating unit (A) and at least one repeating unit (C).

4. The polymer of claim 1 , wherein the polymer comprises at least one repeating unit (A), at least one repeating unit (B), and at least one repeating unit (C).

5. The polymer of claim 1 , wherein the monomers are conjugated by a single bond, a double bond or a triple bond.

6. The polymer of claim 1 , wherein the group M is covalently bonded to one or more of side-chains of the polymer.

7. The polymer of claim 1 , wherein one or more side-chains further comprises a functional group.

8. The polymer of claim 7, wherein the group M is linked to the functional group.

9. The polymer of claim 1 , wherein the polymer is water-soluble.

10. A homopolymer comprising a repeating unit (A),

and wherein: X = a single bond, C1 -C20 alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl; n = 0, 1 or an integer greater than 1 ;

substituted ferrocene or ferrocene derivatives;

Y = C, O, S or N;

R₂ and R₃ = H or and

1 1 . The polymer of claim 10, wherein the monomers are conjugated by a single bond, a double bond or a triple bond.

12. The polymer of claim 10, wherein the group M is covalently bonded to one or more of side-chains of the polymer.

13. The polymer of claim 10, wherein one or more side-chains further comprises a functional group.

14. The polymer of claim 13, wherein the group M is linked to the functional group.

15. The polymer of claim 10, wherein the polymer is water-soluble.

16. The polymer of claim 15, wherein the solubility of the polymer in water is more than 0.8 g/100 g.

17. A polymer comprising:

a structural unit (A), a structural unit (Β'), and a structural unit (C);

wherein the polymer comprises 20-40 mol% of structural unit (Α'), 20-40 mol% of structural unit (Β'), and 20-40 mol% of structural unit (C); and

wherein structural unit (A), structural unit (Β'), and structural unit (C) are represented by the following formulas, respectively:

wherein X = S or N, Y = ferrocene or its derivatives,

Z = a carboxylic acid group, a carboxylate group, a sulfonic group or a sulfonate group,

R² = a divalent organic group,

R⁴ = a divalent organic group, and

m = 1 or 2, n = 1 or 2, p = 2 to 20 and q = 2 to 20.

18. A polymer comprising:

a backbone comprising a conjugated polymer;

a first side chain attached to the backbone, the first side chain comprising a ferrocene group or derivative thereof;

a second side chain attached to the backbone, the second side chain comprising an organic acid, a salt of an organic acid or quaternary ammonium; and

at least one of the first and second side chains comprising at least one of a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom.

19. The polymer of claim 18, wherein the conjugated polymer comprises at least one of: a polythiophene, a polyaniline, a polyacetylene, a poly(p-phenylene), a polypyrrole or derivatives thereof.

20. The polymer of claim 18, wherein the first side chain comprises 5 to 40 atoms between the ferrocene group or derivative thereof and the conjugated polymer backbone.

21. The polymer of claim 18, wherein the first side chain comprises 5 to 40 atoms between the organic acid, salt of an organic acid or quaternary ammonium and the conjugated polymer backbone.

22. The polymer of claim 18, wherein at least one of the first and second side chains comprise an ethylene oxide group.

23. The polymer of claim 18, wherein the second side chain comprises a carboxylic acid group, a carboxylate group, a sulfonic acid group or a sulfonate group.