OXIDATION-REDUCTION POLYMER INCLUDING TRANSITION METAL COMPLEX, AND ELECTROCHEMICAL BIOSENSOR USING SAME

Abstract

The present disclosure relates to an oxidation-reduction polymer including a transition metal complex, which has a unique structure, and so can be prepared in a simpler step compared to a conventional method, and can increase the immobilization rate of the transition metal complex and facilitates the introduction of a functional group or a linker, a method for preparing the same and an electrochemical biosensor comprising the oxidation-reduction polymer.

Claims

1. An oxidation-reduction polymer for an electron transport medium of an electrochemical biosensor, having any one structure of the following Chemical Formulas 1 to 4: ##STR00020## ##STR00021## wherein, M is a transition metal selected from the group consisting of Os, Rh, Ru, Ir, Fe and Co; L.sub.1 and L.sub.2 are combined with each other to form a bidentate ligand selected from the following Chemical Formulas 5 to 7; L.sub.3 and L.sub.4 are combined with each other to form a bidentate ligand selected from the following Chemical Formulas 5 to 7; L.sub.5 and L.sub.6 are each combined with each other to form a bidentate ligand selected from the following Chemical Formulas 5 to 7; ##STR00022## the R.sub.1, R.sub.2 and R′.sub.1 are each independently selected from the group consisting of a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, a substituted or unsubstituted alcohol group having 1 to 20 carbon atoms, a substituted or unsubstituted alkylhalogen group having 1 to 20 carbon atoms, a substituted or unsubstituted thiol group having 1 to 20 carbon atoms, a substituted or unsubstituted alkyl azide group having 3 to 20 carbon atoms, a substituted or unsubstituted aryl azide group having 7 to 30 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 40 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 40 carbon atoms, a cyano group, a halogen group, deuterium and hydrogen, the R.sub.3 to R.sub.20 are each independently selected from the group consisting of a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 40 carbon atoms, a substituted or unsubstituted aryl group having 6 to 50 carbon atoms, a substituted or unsubstituted heteroaryl group having 3 to 50 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 20 carbon atoms, a substituted or unsubstituted alcohol group having 1 to 20 carbon atoms, a substituted or unsubstituted alkylhalogen group having 1 to 20 carbon atoms, a substituted or unsubstituted thiol group having 1 to 20 carbon atoms, a substituted or unsubstituted alkyl azide group having 3 to 20 carbon atoms, a substituted or unsubstituted aryl azide group having 7 to 30 carbon atoms, a substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms, a substituted or unsubstituted alkylamino group having 1 to 20 carbon atoms, a substituted or unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted alkylsilyl group having 1 to 20 carbon atoms, a substituted or unsubstituted arylsilyl group having 6 to 30 carbon atoms, a substituted or unsubstituted arylalkylamino group having 1 to 50 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 40 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 40 carbon atoms, a cyano group, a halogen group, deuterium and hydrogen; the Ad is selected from the group consisting of a primary and secondary amine group, an ammonium group, a halogen group, an epoxy group, an azide group, an acrylate group, an alkenyl group, an alkynyl group, a thiol group, isocyanate, an alcohol group, and a silane group; the X is a counter ion; the a is an integer of 1 to 15; the b is an integer of 1 to 15; the c is an integer of 1 to 15; the m is an integer of 10 to 600; the n is an integer of 10 to 600; and the o is an integer of 0 to 600.

2. The oxidation-reduction polymer for an electron transport medium according to claim 1, wherein the compound of Chemical Formula 1 has a structure represented by the following Chemical Formula 8 or 9. ##STR00023##

3. A method for preparing an oxidation-reduction polymer as set forth in claim 1, the method comprising the steps of: (i) functionalizing polyvinylpyridine or polyimidazole to produce a polyvinylpyridine precursor or a polyimidazole precursor; (ii) functionalizing the transition metal complex; and (iii) reacting the polyvinylpyridine precursor or polyimidazole precursor produced in step (i) and the functionalized transition metal complex produced in step (ii) by a click reaction to prepare the oxidation-reduction polymer as set forth in claim 1.

4. The preparation method according to claim 3, wherein the click reaction of step (iii) is azide-alkyne Huisgen cycloaddition or thiol-ene reaction.

5. A composition for an electrochemical biosensor comprising: an enzyme capable of subjecting a liquid biological sample to an oxidation-reduction reaction; and the oxidation-reduction polymer as set forth in claim 1.

6. The composition according to claim 5, wherein the enzyme comprises: at least one oxidoreductase selected from the group consisting of dehydrogenase, oxidase, and esterase; or at least one oxidoreductase selected from the group consisting of dehydrogenase, oxidase, and esterase, and at least one cofactor selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and pyrroloquinoline quinone (PQQ).

7. The composition according to claim 5, wherein the enzyme is at least one selected from the group consisting of flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) and nicotinamide adenine dinucleotide-glucose dehydrogenase.

8. The composition according to claim 5, further comprising one or more additives selected from the group consisting of surfactants, water-soluble polymers, and thickeners, and a crosslinking agent.

9. An electrochemical biosensor comprising the oxidation-reduction polymer according to claim 1.

10. The electrochemical biosensor according to claim 9, further comprising a sensing layer, a diffusion layer, a protection layer, two or more electrodes, an insulator and a substrate, comprising the oxidation-reduction polymer as set forth in claim 1.

11. The electrochemical biosensor according to claim 10, wherein the electrode is a 2-electrode consisting of an working electrode and a counter electrode, or a 3-electrode consisting of an working electrode, a counter electrode and a reference electrode.

12. The electrochemical biosensor according to claim 9, wherein the biological sample is blood.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 is a diagram showing the structure of a biosensor according to an embodiment of the present disclosure.

[0082] FIG. 2 is a graph showing the results of measuring a cyclic voltammetry curve using the compound of Chemical Formula 8 which is the oxidation-reducing polymer for the electron transport medium according to the present disclosure, and a single Os complex.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0083] Hereinafter, the present disclosure will be described in more detail.

[0084] The present disclosure will be described in more detail by way of examples. However, the present disclosure is for illustrative purposes only and the scope of the present disclosure is not limited thereto.

Preparation Example 1: Synthesis of an Oxidation-Reduction Polymer Compound Represented by [Chemical Formula 8]

1-1. Synthesis of 2,2′-biimidazole

[0085] ##STR00012##

[0086] 79 mL (0.69 mol) of 40% glyoxal aqueous solution was added to a 500 mL three-neck round-bottom flask, cooled to 0° C., and then 370 mL (2.76 mol) of ammonium acetate was slowly added dropwise through a dropping funnel for 3 hours, while paying attention to temperature rise (less than 30° C.). After completion of the dropwise addition, the mixture was stirred overnight at 45˜50° C., and then cooled to room temperature. The resulting solid was filtered, then dissolved in ethyl glycol, and purified by a hot-filter. Finally, 2,2′-biimidazole was obtained. (10.1 g, yield: 33%)

1-2. Synthesis of N-methyl-2,2′-biimidazole

[0087] ##STR00013##

[0088] 2 g (15 mmol) of 2,2′-biimidazole was added to a 250 mL three-neck round-bottom flask, dissolved in 60 mL of anhydrous dimethylformamide, and then cooled to 0° C. 0.6 g (15 mmol) of sodium hydride was added little by little, while paying attention to temperature rise. The mixture was stirred at 0° C. for 1 hour, and then 1 mL (15 mmol) of iodomethane was slowly added dropwise through a syringe pump. After completion of the dropwise addition, the mixture was stirred at room temperature for 12 hours. 100 mL of ethyl acetate was added to the final reaction solution, and the resulting sodium iodide was removed by filtration. The filtrate was concentrated under reduced pressure to remove the solvent, and then the remaining solid was purified by column chromatography using ethyl acetate and hexane as developing solvents. Finally, N-methyl-2,2′-biimidazole was obtained. (0.8 g, yield: 37%)

1-3. Synthesis of N,N′-dimethyl-2,2′-biimidazole

[0089] ##STR00014##

[0090] 5 g (37 mmol) of 2,2′-biimidazole was added to a 500 mL three-neck round-bottom flask, dissolved in 60 mL of anhydrous dimethylformamide, and then cooled to 0° C. 3 g (40 mmol) of sodium hydride was added little by little, while paying attention to temperature rise. The mixture was stirred at 0° C. for 1 hour, and then 2.5 mL (40 mmol) of iodomethane was slowly added dropwise through a syringe pump. After completion of the dropwise addition, the mixture was stirred at room temperature for 24 hours. Water was added to the final reaction solution, extracted with ethyl acetate (200 mL×3), and then the organic layer was collected and dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to remove the solvent, and then purified by column chromatography using ethyl acetate and hexane as developing solvents. Finally, N,N′-dimethyl-2,2′-biimidazole was obtained. (5.1 g, yield: 84%)

1-4. Synthesis of N-butynyl-N′-methyl-2,2′-biimidazole

[0091] ##STR00015##

[0092] 1.5 g (10 mmol) of N-methyl-2,2′-biimidazole was added to a 100 mL three-neck round-bottom flask, dissolved in 30 mL of anhydrous dimethylformamide under nitrogen, and then sodium hydride 0.5 g (13 mmol) was added thereto. The mixture was stirred at room temperature for 1 hour, and then 1.7 g (13 mmol) of 4-bromo-1-butyne and 1.5 g (10 mmol) of sodium iodide were added thereto. The reaction solution was heated to 80° C. under nitrogen and stirred for 24 hours. The final reaction solution was cooled to room temperature, extracted with water (100 mL) and ethyl acetate (200 mL×3), and then the organic layer was collected and dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to remove the solvent, and purified by column chromatography using ethyl acetate and hexane as developing solvents. Finally, N-butynyl-N′-methyl-2,2′-biimidazole was obtained. (1.5 g, yield: 74%)

1-5. Synthesis of [Osmium (III) (N,N′-dimethyl-2,2′-biimidazole).SUB.2 .(N-butynyl-N′-methyl-2,2′-biimidazole)](hexafluorophosphine).SUB.3

[0093] ##STR00016##

[0094] A 100 mL three-neck round-bottom flask was equipped with a reflux condenser, a gas inlet and a thermometer, and 2 g (13 mmol) of N,N′-dimethyl-2,2′-biimidazole, 3 g (6.5 mmol) of ammonium hexachloro osmate (IV) and 50 mL of ethylene glycol were stirred under nitrogen at 140° C. for 24 hours. 1.3 g (6.5 mmol) of N-butynyl-N′-methyl-2,2′-biimidazole was dissolved in 10 mL of ethylene glycol, and then added to the reaction mixture using a syringe. The mixture was again stirred at 140° C. under nitrogen for 24 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, and the resulting red residue was removed by filtration. The filtrate was diluted with 300 mL of water, and then AG1X4 chloride resin was added and stirred for 24 hours to sufficiently oxidize in air. The solution is added dropwise to an aqueous ammonium hexafluorophosphine solution to obtain a precipitate of the ion-exchanged metal complex. The resulting solid was filtered, washed several times with water, and then dried in a vacuum oven to obtain the final compound osmium (III) complex. (5 g, yield: 67%)

1-6. Synthesis of poly(4-(2-azidoethyl)pyridinium)-co-(4-(2-aminoethyl)pyridinium)-co-4-vinylpyridine)

[0095] ##STR00017##

[0096] A 250 mL three-neck round-bottom flask was equipped with a reflux condenser, a gas inlet and a thermometer, and 20 g of poly(4-vinylpyridine): number average molecular weight: ˜160,000 g/mol) was dissolved in 150 mL of dimethylformamide. 4.5 g (30 mmol) of 1-azido-2-bromoethane and 6.0 g (30 mmol) of 2-bromoethylamine were added to this solution. The solution was stirred at 90° C. for 24 hours using a mechanical stirrer. After completion of the reaction, the reaction mixture was cooled to room temperature and poured into ethyl acetate solution to form a precipitate. The solvent was drained off, and the resulting solid was dissolved again in 300 mL of methanol, concentrated under reduced pressure (150 mL), and a precipitate was again formed in diethyl ether. The resulting solid was dried in a vacuum oven to obtain a polyvinylpyridine precursor. (27 g, yield: 90%)

1-7. Synthesis of Oxidation-Reduction Polymer 1 [Chemical Formula 8]

[0097] ##STR00018##

[0098] 0.5 g of poly(4-(2-azidoethyl)pyridinium)-co-(4-(2-aminoethyl) pyridinium)-co-4-vinylpyridine) was added to a 50 mL culture tube and dissolved in 10 mL of distilled water. Then, 0.8 g of [Osmium(III)(N,N′-dimethyl-2,2′-biimidazole).sub.2(N-butynyl-N′-methyl-2,2′-biimidazole)](hexafluorophosphine).sub.3 dissolved in 5 mL of dimethylformamide was added thereto. 25 mg of a copper (I) catalyst (CuTc: Copper(I)thiophene carboxylate) was added to the reaction mixture and stirred at room temperature for 12 hours. After completion of the reaction, the reaction mixture was poured into ethyl acetate solution to form a precipitate. The solvent was drained off and the resulting solid was dissolved again in 50 mL of acetonitrile, and AG1X4 chloride resin and water (150 mL) were added and stirred for 24 hours. The polymer solution is concentrated under reduced pressure (50 mL), and then dialyzed to remove substances of low molecular weight (10,000 g/mol or less). The dialyzed polymer solution was lyophilized to obtain a final oxidation-reduction polymer 1. (0.7 g)

Experimental Example: Confirmation of the Electrochemical Properties of the Oxidation-Reduction Polymer [Formula 8] and Os Complex for an Electron Transfer Medium According to the Present Disclosure Using Cyclic Voltammetry

[0099] In order to confirm the performance of the oxidation-reduction polymer including the Os complex according to the present disclosure as an electron transfer medium, electrochemical properties were measured using the cyclic voltammetry method according to the following experimental method.

Experimental Method

[0100] {circle around (1)} Two kinds of osmium complexes of the following Chemical Formulas 12 and 13 (Os(mbim).sub.3, Os(mbim).sub.3-A), and 20 mg of each of the osmium-polymers of Chemical Formula 8 according to the present disclosure were dissolved in deionized water and 5 mL of 0.1M sodium chloride solution. [0101] {circle around (2)} Degassed with argon for 10 minutes to remove oxygen in the solution. [0102] {circle around (3)} The working electrode, the reference electrode, and the counter electrode were connected to the oxygen-degassed solution, and changes in the electrical signal due to changes in the voltage were measured under argon. [0103] {circle around (4)} The results of the experiment are shown in FIG. 2 and Table 1, respectively.

##STR00019##

Experimental Materials/Conditions

[0104] Working electrode: glass carbon electrode (dia: 3.0 mm)

[0105] Reference electrode: Ag/AgCl electrode

[0106] Counter electrode: platinum rod

[0107] Test parameters [0108] Equipment: EmStat (PalmSens Co.) [0109] Technique: cyclic voltammetry [0110] Potential range: −1.0˜1.0 V [0111] Scan rate: 10 mV/s

TABLE-US-00001 TABLE 1 Compound E.sub.pc (V) E.sub.pa (V) Os(mbim).sub.3-A −0.150 −0.263 Os(mbim).sub.3 −0.165 −0.284 Os-polymer −0.160 −0.290

[0112] As shown in FIG. 2 and Table 1, as a result of measuring the cyclic voltammetry curve of an oxidation-reduction polymer [Chemical Formula 8] together with osmium tris(III) (N,N′-dimethyl-2,2′-biimidazole) and osmium(III) (N,N′-dimethyl-2,2′-biimidazole).sub.2(N-butynyl-N′-methyl-2,2′-biimidazole), all three compounds showed an oxidation-reduction potential at approximately the same position. Therefore, it was indirectly confirmed that the performance of the oxidation-reduction polymer according to the present disclosure as an electron transport medium is the same as that of a single osmium complex.