KR102352758B1 - Novel redox polymer comprising transition metal complex and Electrochemical biosensor using the same - Google Patents

Abstract

Oxidation-reduction containing a transition metal complex, the immobilization rate of the transition metal complex is increased and the introduction of a functional group or linker is easy, because the present invention has a unique structure It relates to a polymer, a method for preparing the same, and an electrochemical biosensor comprising the oxidation-reduction polymer.

Description

Novel redox polymer comprising transition metal complex and electrochemical biosensor using the same

The present invention relates to an oxidation-reduction polymer comprising a transition metal complex and a method for preparing the same, which can be prepared in simple steps compared to the prior art, and the immobilization rate of the transition metal complex is increased and the introduction of a functional group or linker is easy will be.

Recently, interest in the development of biosensors for quantitative and qualitative analysis of target analytes is increasing day by day from the medical field to the environmental and food fields. In particular, the biosensor using an enzyme is a chemical sensor used to selectively detect and measure chemical substances contained in a sample by using the biological detection function in which an organism sensitively reacts with a specific substance, such as a functional substance of an organism or a microorganism. It was developed for medical measurement applications such as sensors, and research is being actively conducted in other applications in food engineering or environmental measurement fields.

Periodic measurement of blood sugar is very important in diabetes management, and various blood sugar meters are being manufactured so that blood sugar can be easily measured using a portable measuring instrument. The operating principle of such a biosensor is based on an optical method or an electrochemical method, and this electrochemical biosensor can reduce the effect of oxygen unlike the conventional optical method biosensor, It has the advantage that it can be used without a separate pretreatment. Accordingly, various types of electrochemical biosensors with accuracy and precision are widely used.

The currently commercialized electrochemical blood glucose sensor mainly uses an enzyme electrode, and more specifically, has a structure in which a glucose oxidase is fixed by a chemical or physical method on an electrode capable of converting an electrical signal. The electrochemical blood glucose sensor is based on the principle of measuring the concentration of glucose in the analyte by measuring the current generated by transferring electrons generated when glucose in an analyte such as blood is oxidized by an enzyme to an electrode. In the case of a biosensor using an enzyme electrode, since the distance from the active center of the enzyme is too far, it is not easy to directly transfer electrons generated by oxidation of the substrate to the electrode. Therefore, a redox mediator, that is, an electron transport mediator, is essential to facilitate this electron transport reaction. Therefore, the type of enzyme used and the characteristics of the electron transfer mediator are the most influential in determining the characteristics of the electrochemical biosensor for measuring blood sugar.

The development trend of blood glucose sensor is an enzymatic reaction instead of GOX in which oxygen participates in the enzymatic reaction with glucose in the blood in order to block the measurement value change due to the difference in _{oxygen partial pressure (pO 2} ) that varies depending on the blood (venous blood, capillary blood, etc.) In the case of electron transport, ferricyanide, which is sensitive to humidity, has been replaced with GDH, which excludes oxygen. They are being replaced by organometallic compounds such as ruthenium hexamine and osmium complexes.

The most commonly used electron transport medium is potassium ferricyanide [K ₃ Fe(CN) ₆ ], which is useful for all sensors using FAD-GOX, PQQ-GDH or FAD-GDH because of its low price and high responsiveness. do. However, the sensor using this electron transfer medium is manufactured and stored because measurement error occurs due to interfering substances such as uric acid or gentisic acid present in the blood and is easily deteriorated by temperature and humidity. Particular attention should be paid to this, and it is difficult to accurately detect low-concentration glucose due to changes in the background current after long-term storage.

Hexaamine ruthenium chloride [Ru(NH ₃ ) ₆ Cl ₃ ] has a higher redox stability than ferricyanide, so the biosensor using this electron transfer medium is easy to manufacture and store, and the change in the background current is small even after long-term storage, so it is stable Although this has a high advantage, there is a disadvantage that it is difficult to manufacture a commercially useful sensor because the reactivity does not match for use with FAD-GDH.

In addition, when using such a biosensor, it is a very important problem in terms of maximizing user convenience to obtain accurate and fast measurement values with a small amount of sample.

Therefore, the development of a new electron transport media that can achieve the shortcomings of the conventional electron transport media and the reduction of the measurement time is still in demand.

On the other hand, a continuous glucose monitoring (CGM) system is used to continuously monitor blood sugar to manage diseases such as diabetes. It cannot be used for these CGMs because it limits the frequency of measurement. In order to solve this problem, an improved version of an enzyme sensor that can be attached to the body and minimizes invasion has been developed. In the case of such a continuous blood glucose monitoring enzyme sensor, since a part of the sensor enters the human body, polyvinylpyrrolidone or polyvinyl By fixing it with a polymer backbone such as imidazole, it is intended to prevent problems due to the loss of electron transport mediators in the human body.

As such, in the case of oxidation-reduction polymers linked with electron transport mediators, in the prior art, N-hydroxysuccinimide (NHS), which is an active ester, is mainly used to efficiently fix the transition metal complex to the polymer main skeleton. ) was mainly used for the coupling reaction. However, according to this conventional synthesis method, it is necessary to go through very complicated steps until it is finally synthesized into an oxidation-reduction polymer, and the immobilization efficiency of the transition metal complex due to hydrolysis during the coupling reaction using NHS is actually reduced. It is not high, and there was a problem that it was difficult to introduce other types of linkers or functional groups into the main skeleton of the polymer, and there is an increasing demand for the development of oxidation-reduction polymers for biosensors that can solve these problems.

The present invention has been devised to solve the above problems, and an object of the present invention is that it can be prepared in a simple step compared to the conventional one, the immobilization rate of the transition metal complex is increased, and the introduction of a functional group or a linker is easy , to provide an oxidation-reduction polymer comprising a transition metal complex and a method for preparing the same.

Another object of the present invention is to provide an electrochemical biosensor comprising a redox polymer including a transition metal complex.

In order to achieve the above object, the present invention uses a click reaction such as a cycloaddition reaction of azide-alkyne using a copper catalyst and heat and a thiol-ene reaction using light, and is a simple and improved transition metal complex It provides an oxidation-reduction polymer including a transition metal complex and an electrochemical biosensor, such as a blood glucose sensor, which exhibits an immobilization rate of , and is easy to introduce additional functional groups or linkers.

The oxidation-reduction polymer comprising the transition metal complex according to the present invention includes a polymer backbone such as polyvinylpyridine (Poly(vinylpyridine): PVP) or polyvinylimidazole (Poly(vinylimidazole): PVI) and osmium; To include a transition metal complex including a transition metal such as ruthenium, iridium, rhodium, iron, cobalt, and the like and a ligand thereof, and a linker structure connecting the polymer backbone and the transition metal complex, specifically, Formulas 1 to 4 has the structure of:

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In the above formula,

M is one type of transition metal selected from the group consisting of Os, Rh, Ru, Ir, Fe and Co;

In the above formula, L1 and L2 are taken together to form a bidentate ligand selected from the following formulas 5 to 7;

L3 and L4 are each taken together to form a bidentate ligand selected from the following formulas 5 to 7;

L5 and L6 are each taken together to form a bidentate ligand selected from Formulas 5 to 7 below;

[Formula 5]

[Formula 6]

[Formula 7]

Wherein R1, R2 and R'1 are each independently a substituted or unsubstituted C1 to C10 alkyl group, a substituted or unsubstituted C1 to C20 alcohol group, or a substituted or unsubstituted C1 to C20 alkyl halogen group, a substituted or unsubstituted C1 to C20 thiol group, a substituted or unsubstituted C3 to C20 alkylazide group, a substituted or unsubstituted C7 to C30 arylazide group, a substituted or unsubstituted It is selected from the group consisting of an 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,

R3 to R20 are each independently a substituted or unsubstituted C1 to C10 alkyl group, a substituted or unsubstituted C3 to C40 cycloalkyl group, a substituted or unsubstituted C6 to C50 aryl group, a substituted or unsubstituted A heteroaryl group having 3 to 50 carbon atoms, a substituted or unsubstituted C 1 to C 20 alkoxy group, a substituted or unsubstituted C 1 to C 20 alcohol group, a substituted or unsubstituted C 1 to C 20 alkyl halogen group , a substituted or unsubstituted C1 to C20 thiol group, a substituted or unsubstituted C3 to C20 alkylazide group, a substituted or unsubstituted C7 to C30 arylazide group, a substituted or unsubstituted carbon number A 6 to 30 aryloxy group, a substituted or unsubstituted C 1 to C 20 alkylamino group, a substituted or unsubstituted C 6 to C 30 arylamino group, a substituted or unsubstituted C 1 to C 20 alkylsilyl group, substituted or An 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 alky having 2 to 40 carbon atoms selected from the group consisting of a nyl group, a cyano group, a halogen group, deuterium and hydrogen;

The Ad is a primary and secondary amine group, ammonium group, halogen group, epoxy group, azide group, acrylate group, alkenyl group, alkynyl group, thiol group, isocyanate, alcohol group, ?? selected from the group consisting of silane groups;

wherein X is a counter ion;

wherein a is an integer from 1 to 15;

b is an integer from 1 to 15;

wherein c is an integer from 1 to 15;

wherein m is an integer from 10 to 600;

wherein n is an integer from 10 to 600;

and o is an integer from 0 to 600.

The oxidation-reduction polymer comprising the transition metal complex provided in the present invention has a unique linker structure, so it can be prepared in a simple step by reducing the synthesis step compared to the conventional one, and the immobilization rate of the transition metal complex is increased and has the advantage that it is easy to introduce a functional group or a linker. In addition, the oxidation-reduction polymer comprising the transition metal complex provided in the present invention preferably has three kinds of bidentate ligands. Therefore, the electrochemical biosensor, preferably the continuous blood glucose monitoring sensor, containing such a redox polymer has advantages in that it is economical to manufacture, toxicity and side effects due to transition metals are significantly reduced, and a yield is high during manufacture.

An example of the present invention relates to an electrochemical biosensor manufactured by applying an enzyme capable of redoxing a liquid biological sample to the oxidation-reduction polymer having Formulas 1 to 4 on a substrate having at least two electrodes and drying it. it's about Examples of the electrode may be a working electrode and a counter electrode, and for example, an enzyme and a transition metal polymer may be applied to or located adjacent to the working electrode.

Although the embodiment of the present invention exemplifies a biosensor for measuring glucose as an applicable example of the electrochemical biosensor, cholesterol, lactate, creatinine, hydrogen peroxide, It can be applied to biosensors for quantification of various substances such as alcohol, amino acids, and glutamate.

Hereinafter, the present invention will be described in more detail.

Preferably, in the oxidation-reduction polymer of Formulas 1 to 4 according to the present invention, the counterion of X is an anion, for example, a halide, sulfate, which may be selected from the group consisting of F, Cl, Br and I; phosphate, hexafluorophosphate, tetrafluoroborate, or a cation (preferably a monovalent cation), for example, one selected from lithium, sodium, potassium, tetraalkylammonium and ammonium. More preferably, X may be chloride.

Preferably, a may be an integer of 2 to 10.

Preferably, b may be an integer of 2 to 10.

Preferably, c may be an integer of 2 to 10.

Preferably, m may be an integer of 15 to 550.

Preferably, n may be an integer of 15 to 550.

Preferably, o may be an integer of 0 to 300.

The redox polymer mediator according to the present invention may also have a structure represented by the following Chemical Formula 8 or 9, but is not limited thereto.

[Formula 8]

[Formula 9]

상기 화학식 8 또는 9에서,
상기 m은 10 내지 600 의 정수이고;
상기 n은 10 내지 600 의 정수이고;
상기 o는 0 내지 600 의 정수이다.

In Formula 8 or 9,
wherein m is an integer from 10 to 600;
wherein n is an integer from 10 to 600;
and o is an integer from 0 to 600.

The transition metal complex in the oxidation-reduction polymer having a structure selected from Chemical Formulas 1 to 4 and Chemical Formulas 8 and 9 according to the present invention may specifically include an osmium complex, for example, a trivalent osmium complex and a divalent osmium complex. and preferably a compound in an oxidation state (a trivalent Os compound). The oxidizing agent used in the oxidation treatment of the present invention is not particularly limited, and specific examples include NaOCl, H ₂ O ₂ , O ₂ , O ₃ , PbO ₂ , MnO ₂ , KMnO ₄ , ClO ₂ , F ₂ , Cl ₂ , H ₂ CrO ₄ , N ₂ O, Ag ₂ O, OsO ₄ , H ₂ S ₂ O ₈ , pyridinium chlorochromate, and 2,2'-dipyridyldisulfide (2,2'-Dipyridyldisulfide) It may be at least one selected from the group consisting of. In addition, in the case of a mixture containing a compound in an oxidation state and a reduced state as a transition metal complex, oxidation treatment may be performed to provide a transition metal complex in an oxidation state or a salt compound of a transition metal complex in an oxidation state.

The transition metal complex according to the present invention may be in the form of a salt, and since the salt compound has high solubility, it is more preferable. The salt compound may be a salt compound of at least one alkali metal selected from the group consisting of Li salt, Na salt, K salt, Rb salt, Cs salt and Fr salt, but is not limited thereto.

In a specific embodiment, the oxidation-reduction polymer according to the present invention can be synthesized by reacting the polymer backbone with the transition metal complex by a click reaction. Specifically, the compound according to Formula 1 among the oxidation-reduction polymers according to the present invention can be prepared by the following azide-alkyne Huisgen cycloaddition reaction, which is shown in Scheme 1, , but is not limited thereto.

[Scheme 1]

In addition, the compound of Formula 2 may be prepared by the following thiol-ene reaction, and may be represented according to Scheme 2, but is not limited thereto.

[Scheme 2]

Preferably, as a starting material for preparing the oxidation-reduction polymer according to Scheme 1 or 2, polyvinylpyridine or polyvinylimidazole may be functionalized to obtain a polyvinylpyridine or polyvinylimidazole precursor. The functionalized polyvinylpyridine or polyvinylimidazole precursor may have a structure represented by the following Chemical Formula 10 or Chemical Formula 11, respectively.

[Formula 10]

[Formula 11]

In the polymer precursor structure, R _a and R _b are each independently a substituted or unsubstituted C 1 to C 10 alkyl group, a substituted or unsubstituted C 1 to C 20 alcohol group, or substituted or unsubstituted C 1 to C 20 alkyl A halogen group, a substituted or unsubstituted C1 to C20 thiol group, a substituted or unsubstituted C3 to C20 alkylazide group, a substituted or unsubstituted C7 to C30 arylazide group, substituted or unsubstituted It is selected from the group consisting of a cyclic alkenyl group having 2 to 40 carbon atoms, and a substituted or unsubstituted alkynyl group having 2 to 40 carbon atoms.

The functionalized polyvinylpyridine or polyvinylimidazole precursor may be synthesized, for example, as shown in Scheme 3 below.

[Scheme 3]

Preferably, as a starting material for preparing an oxidation-reduction polymer according to Scheme 1 or 2, a transition metal complex may be functionalized. Such a functionalized transition metal complex can be synthesized, for example, as shown in Schemes 4 and 5 below.

[Scheme 4]

[Scheme 5]

Accordingly, as a further aspect, the present invention relates to a method for preparing an oxidation-reduction polymer of Formula 1 or 2, comprising the steps of:

(i) preparing a polyvinylpyridine precursor or a polyimidazole precursor by functionalizing polyvinylpyridine or polyimidazole;

(ii) functionalizing the transition metal complex; and

(iii) oxidation-reduction of Formula 1 or 2 by reacting the polyvinylpyridine precursor or polyimidazole precursor prepared in step (i) and the functionalized transition metal complex prepared in step (ii) by a click reaction preparing the polymer.

Specific aspects of each step are as described above.

The transition metal complex of the oxidation-reduction polymer according to the present invention not only enables accurate, reproducible, rapid and continuous analysis of a target material, but also can be easily and economically prepared in high yield, and transition metal leakage It has the advantage of remarkably low toxicity or side effects that may occur due to this.

The redox polymer according to the present invention transfers electrons between the working electrode and the analyte through an enzyme by being applied or laminated on the working electrode or positioned around the working electrode (eg, a structure surrounding the electrode in a solution). do. These redox polymers can form an impermeable coating on the working electrode in an electrochemical biosensor.

A further aspect of the present invention relates to an electrochemical biosensor composition comprising an enzyme capable of redoxing a liquid biological sample and the redox polymer.

The oxidoreductase is a generic term for enzymes that catalyze the redox reaction of a living body. In the present invention, a target material to be measured, for example, a biosensor, refers to an enzyme that is reduced by reacting with the target material to be measured. The reduced enzyme reacts with the electron transfer mediator, and the target substance is quantified by measuring a signal such as a change in current generated at this time. The oxidoreductase usable in the present invention may be at least one selected from the group consisting of various dehydrogenases, oxidases, esterases, etc., depending on the redox or detection target substance, From among the enzymes belonging to the enzyme group, an enzyme having the target material as a substrate may be selected and used.

More specifically, the oxidoreductase is glucose dehydrogenase, glutamate dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, rock. At least one selected from the group consisting of lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase, bilirubin oxidase, etc. can

On the other hand, the oxidoreductase may include a cofactor that serves to store the hydrogen taken by the oxidoreductase from the target substance to be measured (eg, the target substance), for example, flavin adenine dinu. It may be at least one selected from the group consisting of cleotatide (flavin adenine dinucleotide, FAD), nicotinamide adenine dinucleotide (NAD), and pyrroloquinoline quinone (PQQ).

For example, when measuring blood glucose concentration, glucose dehydrogenase (GDH) can be used as the oxidoreductase, and the glucose dehydrogenase is a flavin adenine dinucleotide containing FAD as a cofactor-glucose dehydrogenase. It may be an enzyme (flavin adenine dinucleotide-glucose dehydrogenase, FAD-GDH), and/or nicotinamide adenine dinucleotide-glucose dehydrogenase containing FAD-GDH as a cofactor.

In an embodiment, the usable oxidoreductase is FAD-GDH (eg, EC 1.1.99.10, etc.), NAD-GDH (eg, EC 1.1.1.47, etc.), PQQ-GDH (eg, EC1.1.5.2, etc.) , glutamic acid dehydrogenase (eg, EC 1.4.1.2, etc.), glucose oxidase (eg, EC 1.1.3.4, etc.), cholesterol oxidase (eg, EC 1.1.3.6, etc.), cholesterol esterase (eg, EC 3.1. 1.13, etc.), lactate oxidase (eg, EC 1.1.3.2, etc.), ascorbic acid oxidase (eg, EC 1.10.3.3, etc.), alcohol oxidase (eg, EC 1.1.3.13, etc.), alcohol dehydrogenase (eg , EC 1.1.1.1, etc.), bilirubin oxidase (eg, EC 1.3.3.5, etc.) may be at least one selected from the group consisting of.

The composition according to the present invention may contain 20 to 700 parts by weight of the redox polymer, such as 60 to 700 parts by weight or 30 to 340 parts by weight, based on 100 parts by weight of the oxidoreductase. The content of the oxidation-reduction polymer may be appropriately adjusted according to the activity of the oxidoreductase.

In addition, the composition according to the present invention may further include a crosslinking agent .

On the other hand, the composition according to the present invention contains one or more additives selected from the group consisting of surfactants, water-soluble polymers, quaternary ammonium salts, fatty acids, thickeners, etc. as a dispersant for dissolving reagents, adhesive for preparing reagents, stabilizers for long-term storage, etc. may be additionally included for the role of

The surfactant may serve to distribute the composition evenly over the electrode when dispensing the composition to be dispensed with a uniform thickness. 1 selected from the group consisting of Triton X-100, sodium dodecyl sulfate, perfluorooctane sulfonate, sodium stearate, etc. as the surfactant More than one species can be used. In the reagent composition according to the present invention, the surfactant is added to 100 parts by weight of the oxidoreductase in order to properly perform the role of dispensing the reagent evenly over the electrode and dispensing the reagent to a uniform thickness when dispensing the reagent. to 25 parts by weight, such as 10 to 25 parts by weight. For example, when using an oxidoreductase having an activity of 700 U/mg, 10 to 25 parts by weight of a surfactant may be contained based on 100 parts by weight of the oxidoreductase, and when the activity of the oxidoreductase is higher than this, the content of the surfactant can be adjusted lower than this.

The water-soluble polymer may serve to assist in stabilizing and dispersing the enzyme as a polymer support of the reagent composition. As the water-soluble polymer, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyfluorosulfonate (perfluoro sulfonate), hydroxyethyl cellulose (HEC), hydroxy At least one selected from the group consisting of propyl cellulose (hydroxypropyl cellulose; HPC), carboxy methyl cellulose (CMC), cellulose acetate, polyamide, and the like may be used. The reagent composition according to the present invention contains 10 to 70 parts by weight of the water-soluble polymer based on 100 parts by weight of the oxidoreductase, such It may be contained in an amount of 30 to 70 parts by weight. For example, when an oxidoreductase having an activity of 700 U/mg is used, it may contain 30 to 70 parts by weight of a water-soluble polymer based on 100 parts by weight of the oxidoreductase, and when the activity of the oxidoreductase is higher than this, the content of the water-soluble polymer can be adjusted lower than this.

The water-soluble polymer may have a weight average molecular weight of about 2,500 to 3,000,000, for example, about 5,000 to 1,000,000, in order to effectively perform a dynamic that helps stabilize and disperse the support and the enzyme.

The thickener serves to firmly attach the reagent to the electrode. As the thickener, at least one selected from the group consisting of natrozole, diethylaminoethyl-dextran hydrochloride (DEAE-Dextran hydrochloride), and the like may be used. In the electrochemical sensor according to the present invention, 10 to 90 parts by weight, such as 30 to 90 parts by weight, of the thickener based on 100 parts by weight of the oxidoreductase, in order for the redox polymer according to the present invention to be firmly attached to the electrode. It may contain in negative amounts. For example, when using an oxidoreductase having an activity of 700 U/mg, it may contain 30 to 90 parts by weight of a thickener based on 100 parts by weight of the oxidoreductase, and when the activity of the oxidoreductase is higher than this, the content of the thickener can be adjusted lower than this.

In a further aspect, the present invention provides an electrochemical biosensor comprising the redox polymer.

Specifically, the type of the electrochemical biosensor is not limited, but may preferably be a continuous blood glucose monitoring sensor.

With the configuration of such a continuous blood glucose monitoring sensor, the present invention provides, for example, an electrode, an insulator, a substrate, a sensing layer including the redox polymer and a redox enzyme, and a diffusion layer ), a protection layer, and the like. In the case of the electrode, two types of electrodes such as a working electrode and a counter electrode may be included, and three types of electrodes such as a working electrode, a counter electrode and a reference electrode may be included. In one embodiment, the biosensor according to the present invention is capable of redoxing the redox polymer having Formula 1 or 2 and a liquid biological sample on a substrate having at least two, preferably two or three electrodes. It may be an electrochemical biosensor manufactured by applying a reagent composition containing an enzyme present and drying it. For example, in the electrochemical biosensor, a working electrode and a counter electrode are provided on opposite surfaces of a substrate, and a sensing film containing the redox polymer according to the present invention is laminated on the working electrode, the working electrode and the counter electrode There is provided a planar electrochemical biosensor characterized in that an insulator, a diffusion film and a protective film are sequentially stacked on both surfaces of the provided substrate.

In a specific embodiment, the substrate may be made of one or more materials selected from the group consisting of polyethylene terephthalate (PET), polycarbonate (PC), and polyimide (PI).

In addition, the working electrode may use a carbon, gold, platinum, silver or silver/silver chloride electrode.

In addition, in the case of an electrochemical biosensor having two electrodes, since the counter electrode also serves as a reference electrode, a gold, platinum, silver, or silver/silver chloride electrode can be used as the counter electrode. In the case of the electrochemical biosensor, a gold, platinum, silver, or silver/silver chloride electrode may be used as a reference electrode, and a carbon electrode may be used as a counter electrode.

Nafion, cellulose acetate, silicone rubber may be used as the diffusion film, and silicone rubber, polyurethane, polyurethane-based copolymer, etc. may be used as the protective film, but is not limited thereto.

As a non-limiting example, in the case of two electrodes, silver chloride or silver chloride may be used because the counter electrode serves as a reference electrode, and in the case of three electrodes, silver chloride or silver is used as the reference electrode, and a carbon electrode may be used as the counter electrode. have.

The oxidation-reduction polymer according to the present invention is economical due to the small number of process steps during production, the immobilization rate of the transition metal complex is increased, and the introduction of functional groups or linkers is easy. There is an advantage that

1 is a view showing the structure of a biosensor according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Preparation Example 1: Synthesis of oxidation-reduction polymer compound represented by [Formula 8]

1-1. 2,2'-biimidazole synthesis

Put 79 mL (0.69 mol) of a 40% aqueous solution of glyoxal in a 500 mL three-necked round-bottom flask, and ^{after cooling to 0 o} C, 370 mL (2.76 mol) of ammonium acetate was added to the temperature through a dropping funnel for 3 hours. It was slowly added dropwise, paying attention to the rise ( ^{below 30 o C).} After completion of the dropwise addition ^{, the mixture was stirred at 45-50 o} C overnight and then cooled to room temperature. After filtration, the resulting solid was dissolved in ethyle glycol and purified by a hot-filter to finally obtain 2,2'-biimidazole. (10.1 g , yield: 33%)

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

Put 2 g (15 mmol) of 2,2'-biimidazole in a 250 mL 3-neck round-bottom flask, dissolve in 60 mL of anhydrous dimethylformamide, and then cool to ^{0 o C.} Sodium hydride 0.6 g (15 mmol) is added little by little while paying attention to the temperature rise. After stirring the mixture at 0 ^o C for 1 hour, 1 mL (15 mmol) of iodomethane is 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 is added to the final reaction solution, and the resulting sodium iodide is removed by filtration. The filtrate is concentrated under reduced pressure to remove the solvent, and the remaining solid is 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

Put 5 g (37 mmol) of 2,2'-biimidazole in a 500 mL three-necked round-bottom flask, dissolve in 60 mL of anhydrous dimethylformamide, and then cool to ^{0 o C.} 3 g (40 mmol) of sodium hydride is added little by little while paying attention to the temperature rise. After stirring this mixture at 0 ^o C for 1 hour, 2.5 mL (40 mmol) of iodomethane is slowly added dropwise through a syringe pump. After completion of the dropwise addition, the mixture was stirred at room temperature for 24 hours. Water is added to the final reaction solution, and the mixture is extracted with ethyl acetate (200 mL X 3), and the organic layers are 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 , 84%)

1-4. N -Butynyl- N' -Methyl-2,2'-biimidazole synthesis

1.5 g (10 mmol) of N -methyl-2,2'-biimidazole was placed in a 100 mL three-necked round-bottom flask, dissolved in 30 mL of anhydrous dimethylformamide under nitrogen, and 0.5 g (13 mmol) of sodium hydride was added. do. After stirring the mixture at room temperature for one hour, 1.7 g (13 mmol) of 4-bromo-1-butyne and 1.5 g (10 mmol) of sodium iodide are added. The reaction solution was ^{heated to 80 o} C under nitrogen and stirred for 24 hours. The final reaction solution is cooled to room temperature, extracted with water (100 mL) and ethyl acetate (200 mL X 3), and the organic layers are 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 , 74%)

1-5. [Osmium (III) ( N, N' -dimethyl-2,2'-biimidazole) ₂ ( N -Butynyl- N' -Methyl-2,2'-biimidazole)] (hexafluorophosphine) ₃ synthesis

Equipped with a reflux condenser, gas inlet and thermometer in a 100 mL 3-neck round bottom flask, 2 g (13 mmol) of N,N' -dimethyl-2,2'-biimidazole, ammonium hexachloro osmate (IV) 3 g (6.5 mmol) 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 is dissolved in 10 mL of ethylene glycol, and then put into the reaction mixture using a syringe. The mixture was again ^{stirred at 140 °} C under nitrogen for 24 hours. After the reaction, the reaction mixture is cooled to room temperature, and the red residue produced is removed by filtration. After diluting the filtrate with 300 mL of water, add AG1X4 chloride resin and stir for 24 hours to sufficiently oxidize in air. The solution is added dropwise to an aqueous ammonium hexafluorophosphine solution to obtain a precipitate of an ion-exchanged metal complex. The resulting solid is filtered and washed several times with water, and then dried in a vacuum oven to obtain the final compound osmium (III) complex. (5 g , 67%)

1-6. Poly(4-(2-azidoethyl)pyridinium)- co -(4-(2-aminoethyl)pyridinium)- co -4-vinylpyridine) synthesis

Equipped with a reflux condenser, gas inlet and thermometer in a 250 mL three-necked round-bottom flask, add 20 g of poly(4-vinylpyridine): number average molecular weight: ~160,000 g/mol) to 150 mL of dimethylformamide melt in To this solution, 4.5 g (30 mmol) of 1-azido-2-bromoethane and 6.0 g (30 mmol) of 2-bromoethylamine are added. ^{The solution is stirred at 90 o} C for 24 hours using a mechanical stirrer. After the reaction, the reaction mixture is cooled to room temperature and poured into an ethyl acetate solution to form a precipitate. The solvent is discarded, and the resulting solid is dissolved again in 300 mL of methanol and concentrated under reduced pressure (150 mL) to form a precipitate again in diethyl ether. The resulting solid is dried in a vacuum oven to obtain a polyvinylpyridine precursor. (27 g , 90%)

1-7. Oxidation-reduction polymer 1 synthesis [Formula 8]

In culture tubes with 50 mL of poly (4- (2-O map ethyl) pyridinium) - co - (4- (2-aminoethyl) pyridinium) - -4- co-vinylpyridine) with distilled water of 10 g to 0.5 mL [Osmium (III) ( N,N' -dimethyl-2,2'-biimidazole) ₂ ( N -butynyl- N' -methyl-2,2'- Biimidazole)] (hexafluorophosphine) ₃ 0.8 g is added. 25 mg of copper(I) catalyst (CuTc: Copper(I)thiophene carboxylate) was added to the reaction mixture and stirred at room temperature for 12 hours. After the reaction, the reaction mixture is poured into an ethyl acetate solution to form a precipitate. Discard the solvent, re-dissolve the resulting solid in 50 mL of acetonitrile, add AG1X4 chloride resin and water (150 mL), and stir for 24 hours. After the polymer solution is concentrated under reduced pressure (50 mL), dialysis is performed to remove substances of low molecular weight (10,000 g/mol or less). The dialyzed polymer solution is freeze-dried to obtain the final oxidation-reduction polymer 1. (0.7 g)

Claims (12)

An oxidation-reduction polymer for an electron transport mediator of an electrochemical biosensor, having the structure of any one of the following formulas 1, 2 and 4:
[Formula 1]

[Formula 2]

[Formula 4]

In the above formula,
M is one type of transition metal selected from the group consisting of Os, Rh, Ru, Ir, Fe and Co;
In the above formula, L1 and L2 are taken together to form a bidentate ligand selected from the following formulas 5 to 7;
L3 and L4 are each taken together to form a bidentate ligand selected from the following formulas 5 to 7;
L5 and L6 are each taken together to form a bidentate ligand selected from Formulas 5 to 7 below;

[Formula 5]

[Formula 6]

[Formula 7]

Wherein R1, R2 and R'1 are each independently a substituted or unsubstituted C1 to C10 alkyl group, a substituted or unsubstituted C1 to C20 alcohol group, or a substituted or unsubstituted C1 to C20 alkyl halogen group, a substituted or unsubstituted C1 to C20 thiol group, a substituted or unsubstituted C3 to C20 alkylazide group, a substituted or unsubstituted C7 to C30 arylazide group, a substituted or unsubstituted It is selected from the group consisting of an 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,
R3 to R20 are each independently a substituted or unsubstituted C1 to C10 alkyl group, a substituted or unsubstituted C3 to C40 cycloalkyl group, a substituted or unsubstituted C6 to C50 aryl group, a substituted or unsubstituted A heteroaryl group having 3 to 50 carbon atoms, a substituted or unsubstituted C 1 to C 20 alkoxy group, a substituted or unsubstituted C 1 to C 20 alcohol group, a substituted or unsubstituted C 1 to C 20 alkyl halogen group , a substituted or unsubstituted C 1 to C 20 thiol group, a substituted or unsubstituted C 3 to C 20 alkylazide group, a substituted or unsubstituted C 7 to C 30 arylazide group, a substituted or unsubstituted C A 6 to 30 aryloxy group, a substituted or unsubstituted C 1 to C 20 alkylamino group, a substituted or unsubstituted C 6 to C 30 arylamino group, a substituted or unsubstituted C 1 to C 20 alkylsilyl group, substituted or An unsubstituted C6-C30 arylsilyl group, a substituted or unsubstituted C1-C50 arylalkylamino group, a substituted or unsubstituted C2-C40 alkenyl group, a substituted or unsubstituted C2-C40 alky selected from the group consisting of a nyl group, a cyano group, a halogen group, deuterium and hydrogen;
wherein Ad is selected from the group consisting of primary and secondary amine groups, ammonium groups, halogen groups, epoxy groups, azide groups, acrylate groups, alkenyl groups, alkynyl groups, thiol groups, isocyanates, alcohol groups, and silane groups;
wherein X is a counter ion;
wherein a is an integer from 1 to 15;
b is an integer from 1 to 15;
wherein c is an integer from 1 to 15;
wherein m is an integer from 10 to 600;
wherein n is an integer from 10 to 600;
and o is an integer from 0 to 600.
An oxidation-reduction polymer for an electron transport mediator, having the structure of the following formula (8) or formula (9):

[Formula 8]

[Formula 9]

In Formula 8 or 9,
wherein m is an integer from 10 to 600;
wherein n is an integer from 10 to 600;
and o is an integer from 0 to 600. (i) preparing a polyvinylpyridine precursor or a polyvinylimidazole precursor by functionalizing polyvinylpyridine or polyimidazole;
(ii) functionalizing the transition metal complex; and
(iii) reacting the polyvinylpyridine precursor or polyvinylimidazole precursor prepared in step (i), and the functionalized transition metal complex prepared in step (ii) by a click reaction to claim 1 or 2 A method for preparing a redox polymer according to claim 1 or 2, comprising the step of preparing the redox polymer according to claim 1 : The method according to claim 3, wherein the click reaction in step (iii) is an azide-alkyne Huisgen cycloaddition reaction (Azide-alkyne Huisgen cycloaddition) or a thiol-ene reaction (Thiol-ene reaction). . an enzyme capable of redoxing a liquid biological sample; and
A composition for an electrochemical biosensor comprising the oxidation-reduction polymer according to claim 1 or 2. 6. The method of claim 5, wherein the enzyme is
at least one oxidoreductase selected from the group consisting of dehydrogenase, oxidase, and esterase; or
One or more oxidoreductases selected from the group consisting of dehydrogenases, oxidases, and esterases, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), And pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ) will include one or more cofactors selected from the group consisting of, the composition. According to claim 5, wherein the enzyme is flavin adenine dinucleotide-glucose dehydrogenase (flavin adenine dinucleotide-glucose dehydrogenase, FAD-GDH), and nicotinamide adenine dinucleotide-glucose dehydrogenase (nicotinamide adenine dinucleotide-glucose dehydrogenase) One or more selected from the group consisting of, the composition. The composition of claim 5, further comprising at least one additive selected from the group consisting of a surfactant, a water-soluble polymer, and a thickener, and a crosslinking agent. An electrochemical biosensor comprising the redox polymer according to claim 1 or 2. The method according to claim 9, wherein a sensing layer, a diffusion layer, a protection layer, two or more kinds of electrodes, an insulator, and a substrate comprising the redox polymer and an oxidoreductase Which further comprises an electrochemical biosensor. The electrochemical biosensor according to claim 10, wherein the electrode is a two-electrode consisting of a working electrode and a counter electrode or a three-electrode consisting of a working electrode, a counter electrode and a reference electrode. The electrochemical biosensor of claim 9, wherein the electrochemical biosensor is a blood glucose sensor.

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