WO2022051889A1 - 提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器 - Google Patents

提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器 Download PDF

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WO2022051889A1
WO2022051889A1 PCT/CN2020/113935 CN2020113935W WO2022051889A1 WO 2022051889 A1 WO2022051889 A1 WO 2022051889A1 CN 2020113935 W CN2020113935 W CN 2020113935W WO 2022051889 A1 WO2022051889 A1 WO 2022051889A1
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oxidoreductase
glucose oxidase
solution
glucose
purified
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高志强
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三诺生物传感股份有限公司
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    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

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  • the present invention relates to the field of electrochemistry, in particular to an oxidoreductase with improved electrochemical activity and a biosensor containing the oxidoreductase.
  • biosensors have been widely used in environmental detection, food industry, clinical medicine and other fields.
  • various glucose sensors developed based on biosensing technology have benefited millions of diabetic patients.
  • the implantable continuous glucose monitoring system which has developed rapidly in recent years, is favored by more and more diabetic patients, especially type I diabetic patients, due to its convenient use and real-time monitoring.
  • the performance of the glucose biosensor directly determines the performance and service life of the implantable continuous glucose monitoring system.
  • the glucose biosensors used in the existing implantable continuous glucose monitoring systems are developed based on the first and second generation biosensing technologies.
  • the first generation of biosensing technology is to monitor glucose indirectly by electrochemically detecting the hydrogen peroxide generated or the oxygen consumed during glucose oxidation.
  • Medtronic's Guardian and iPro2 and Dexcom's Dexcom G5 and G6 are developed based on the first-generation biosensing technology. They detect the hydrogen peroxide generated during the catalytic oxidation of glucose by electrochemical methods. Glucose is monitored. Since the electrochemical detection of hydrogen peroxide requires very strict electrodes, only a few materials such as platinum and platinum alloys can be used for the fabrication of such glucose biosensors, which greatly increases the number of implantable continuous glucose monitoring systems. the cost of the sensor. In addition, the electrochemical detection of hydrogen peroxide requires a high detection potential, thus greatly reducing the anti-interference ability of the implantable continuous glucose monitoring system, especially the anti-interference ability of commonly used antipyretics such as acetaminophen.
  • the second-generation biosensing technology realizes direct electrochemical detection of glucose by introducing redox mediators in glucose biosensors.
  • glucose oxidase has a large molecular weight (160KDa), and its molecular structure, especially the three-dimensional structure of the catalytic active center, is very complex, and it is located inside the glucose oxidase and is deeply wrapped by various peptide chains. write. Therefore, glucose oxidase cannot directly exchange electrons with electrodes.
  • redox mediators redox small molecules such as ferricyanide or redox macromolecules
  • glucose biosensors Oxidase can exchange electrons with electrodes through these mediators.
  • the second generation of biosensing technology has been widely used in biosensors, especially glucose biosensors, including various disposable blood glucose detection test strips and implantable continuous glucose monitoring systems, such as Abbott Diabetes Care FreeStyle Libre.
  • glucose detection can be achieved at lower potentials, thereby greatly improving the anti-interference ability of implantable continuous glucose monitoring systems, especially for commonly used antipyretics such as acetyl Anti-interference ability of aminophenols. Since this type of glucose monitoring system performs direct electrochemical detection of glucose through redox mediators, its sensitivity is also significantly improved.
  • the redox mediator is a small molecule or a polymer material, it is difficult to accurately control the preparation, and there is also the possibility of the redox mediator leaking out of the implantable glucose biosensor, which is very important for the implantable continuous glucose biosensor. The performance of monitoring systems introduces considerable uncertainty.
  • the present invention provides an oxidoreductase with improved electrochemical activity and a biosensor containing the oxidoreductase.
  • the catalytic oxidation efficiency of glucose by this glucose oxidase is two orders of magnitude higher than that of native glucose oxidase by oxygen.
  • the present invention provides formulations for preparing biosensors, comprising one or more of the following compositions:
  • Composition 1 including enzyme denaturant, redox small molecules with superior electrochemical performance, cross-linking agent, cross-linking agent auxiliary and buffer solution;
  • the enzyme denaturing agent includes urea or guanidine hydrochloride; the redox small molecule with excellent electrochemical performance includes ruthenium or osmium complexes with free amino groups; the cross-linking agent includes carbodiimide or suberic acid Disuccinimide; the cross-linking agent auxiliary includes N-hydroxysuccinimide or N-hydroxysuccinimide; the buffer solution includes PBS buffer solution;
  • Composition 2 including an aldehyde-based reagent, a redox small molecule with superior electrochemical performance, and a reducing agent;
  • the aldehyde-ylation reagent includes sodium periodate;
  • the redox small molecule with excellent electrochemical performance includes ruthenium or osmium complexes with free amino groups;
  • the reducing agent includes sodium borohydride;
  • Composition 3 including a bifunctional chemical cross-linking agent and a buffer solution;
  • the bifunctional chemical crosslinking agent includes glutaraldehyde, epichlorohydrin, N,N-methylenebisacrylamide, acetic anhydride, diglycidyl ether, methyl suberimidate, polyethylene glycol
  • the buffer solution includes PBS buffer solution;
  • the preparation comprises at least one composition.
  • composition one comprises the following components:
  • the concentration °C of the enzyme denaturant in the buffer solution of the enzyme denaturant is 1-8 mol/L.
  • the present invention also provides the application of the preparation in improving the electrochemical activity of oxidoreductase.
  • the present invention also provides a method for improving the electrochemical activity of oxidoreductase, based on the preparation of the present invention, comprising the following steps:
  • Step 1 The oxidoreductase is mixed with the buffer solution of the enzyme denaturant and then cultivated to obtain the developing solution of the oxidoreductase;
  • Step 2 mixing the redox small molecules with superior electrochemical performance with the developing solution of the redox enzyme prepared in step 1 to prepare a mixed solution;
  • Step 3 take the mixed solution obtained in step 2 and mix it with the cross-linking agent and the auxiliary of the cross-linking agent in turn, and then react to obtain a modification solution;
  • Step 4 Take the modification solution obtained in Step 3, separate and purify.
  • step 1 is specifically: mixing 1-10 mg/mL glucose oxidase (preferred value: 2) with a PBS buffer solution containing 1-8 mol/L urea (preferred value: 3) in Incubate at 4°C for 12-24h (preferred value: 12).
  • concentration of glucose oxidase is 2 mg/mL.
  • concentration of urea is 3 mol/L.
  • the incubation time is 12h.
  • step 2 is specifically: combining 1-10 mg/mL (preferred value: 2) of redox small molecules with superior electrochemical performance with 1-10 mg/mL (preferred value: 2) of redox small molecules
  • the oxidoreductase developing solutions are mixed.
  • the concentration of redox small molecules with excellent electrochemical performance is 2 mg/mL.
  • the concentration of the developing solution of the oxidoreductase is 1-10 mg/mL.
  • step 3 is specifically as follows: taking the mixed solution prepared in step 2 and adding 1-10 mmol/L (preferred value: 2) of crosslinking agent and 0.1-1 mmol/L (preferably Value: 0.5) after mixing the auxiliary crosslinking agent, react at 4° C. for 12-24 h (preferred value: 12).
  • the concentration of the crosslinking agent is 2 mmol/L.
  • the concentration of the crosslinking agent auxiliary is 0.5 mmol/L.
  • the incubation time is 12h.
  • step 4 is specifically: taking the modification solution obtained in step 3 and dialysis with ultrafiltration bag (cutting molecular weight: 1000-30000) (preferred value: 10000) to improve the electrochemical activity oxidoreductase. Separate and purify.
  • the cut molecular weight of the ultrafiltration bag is 10,000.
  • Step 5 mixing, separating and purifying the oxidoreductase purified in step 4 and the aldolization reagent to obtain a purified oxidoreductase solution;
  • Step 6 mixing the purified oxidoreductase solution obtained in step 5 with the redox small molecules with excellent electrochemical performance to prepare a mixed solution;
  • Step 7 The mixed solution obtained in Step 6 is mixed with the reducing agent, and the electrochemically activated glucose oxidase is separated and purified.
  • step 5 is specifically: mixing the oxidoreductase purified in step 4 with 0.1-1 g/mL (preferred value: 0.3) of the aldehyde-ylation reagent at 20-30 (preferred value) : 25) °C for 1-5 (preferred value: 2) h, and the glucose oxidase with aldolized sugar molecules was separated by ultrafiltration bag dialysis (molecular weight cutting: 1000-30000) (preferred value: 10000). Purified to obtain a purified oxidoreductase solution.
  • the concentration of the aldolization reagent is 3 g/mL.
  • the culture temperature is 25°C and the time is 2h.
  • the cut molecular weight of the ultrafiltration bag is 10,000.
  • step 6 is specifically: taking the purified oxidoreductase solution and mixing 1-10 mg/mL (preferred value: 2) of the redox small molecule with superior electrochemical performance Then, react at 4° C. for 2-24 h (preferred value: 8) to obtain a mixed solution.
  • concentration of the redox small molecule with excellent electrochemical performance is 2 mg/mL.
  • the reaction time is 8h.
  • step 7 is specifically as follows: take the mixed solution and 2-20 mg/mL (preferred value: 5) of the reducing agent, mix and react at 4°C for 1-4 h (preferred value: 5). : 2), after the reaction, the electrochemically activated glucose oxidase is separated and purified by ultrafiltration bag dialysis (molecular weight cut: 1000-30000) (preferred value: 10000).
  • the concentration of the reducing agent is 2-20 mg/mL.
  • the reaction time is 2h.
  • the cut molecular weight of the ultrafiltration bag is 10,000.
  • the present invention also provides the oxidoreductase with improved electrochemical activity prepared by the method.
  • the invention also provides the application of the oxidoreductase with improved electrochemical activity in the preparation of oxidoreductase sensor, oxidoreductase detection system or food industry.
  • the present invention also provides an oxidoreductase sensor, including the oxidoreductase enzyme with improved electrochemical activity.
  • the present invention also provides a method for preparing the oxidoreductase sensor.
  • the oxidoreductase improving electrochemical activity is mixed with a bifunctional chemical crosslinking agent, and the chemical crosslinking method is prepared by a drop coating method or a dip-pulling method.
  • the oxidoreductase enzyme is then used to make an oxidoreductase sensor on the surface of the electrode.
  • 5-200 mg/mL (preferred value: 150) electrochemically activated glucose oxidase is prepared in PBS buffer solution and 0.1-5% (preferred value: 1) glutaraldehyde solution After mixing, after 30-180 min (preferred value: 60), the chemically cross-linked glucose oxidase is prepared on the electrode surface by drop coating method or dip-pulling method to make an oxidoreductase sensor.
  • the concentration of the electrochemically activated glucose oxidase is 150 mg/mL.
  • the concentration of the glutaraldehyde solution is 0.1 to 5%.
  • the mixing time is 60 min.
  • the present invention also provides an oxidoreductase detection system, including the oxidoreductase enzyme with improved electrochemical activity or the oxidoreductase enzyme sensor.
  • the oxidoreductase includes glucose oxidase.
  • the present invention chemically modifies the free carboxyl groups on the surface and inside of the glucose oxidase molecule—covalently bonds redox small molecules with superior electrochemical properties, such as ruthenium or osmium complexes, to the glucose oxidase, and from the inside Once an electron channel is established outside, the catalytic active center of glucose oxidase can directly exchange electrons with the electrode very quickly.
  • the electrochemically activated glucose oxidase not only maintained its catalytic oxidation performance of glucose, but its catalytic oxidation efficiency of glucose by direct electrochemical oxidation was two orders of magnitude higher than that of natural glucose oxidase by oxygen.
  • the present invention aldolizes these sugar molecules, and then reacts with ruthenium or osmium complexes with free amino groups, thereby introducing a large number of electron exchange nodes on the surface of glucose oxidase molecules to form an efficient electron transfer network. In this way, the electrons transferred from the catalytic active center of glucose oxidase can quickly reach the electrode surface through this electron transfer network. After the above two chemical treatments, the glucose oxidase has been completely electrochemically activated from the inside out.
  • Figure 1 shows a cyclic voltammogram; in which curve a shows glucose oxidase treated in the absence of the carbodiimide/N-hydroxysuccinimide cross-linker and curve b is shown in the carbodiimide/N-hydroxysuccinimide Cyclic voltammogram of glucose oxidase cyclic voltammogram treated in the presence of imine crosslinker;
  • Fig. 2 shows cyclic voltammogram; wherein curve a shows the cyclic voltammogram containing electrochemically activated glucose oxidase in PBS buffer solution; curve b shows the cyclic voltammogram after adding 10 mmol/L of glucose;
  • Figure 3 shows the cyclic voltammogram
  • the curve a shows the cyclic voltammogram of the glucose biosensor containing electrochemically activated glucose oxidase in PBS buffer solution
  • the curve b shows the first circle after adding 5.0 mmol/L glucose
  • curve c shows the cyclic voltammogram of the 100th cycle; where, show curve a; show curve b; shows curve c.
  • the invention discloses an oxidoreductase with improved electrochemical activity and a biosensor containing the oxidoreductase.
  • the third-generation biosensing technology is a biosensing technology developed by direct electrochemistry of oxidoreductases (Measuring Technology, 2006, (Volume 26, Supplement) 92-96, Chem. Rev. 2008, 108, 814-825) .
  • oxidoreductases Measurement Technology, 2006, (Volume 26, Supplement) 92-96, Chem. Rev. 2008, 108, 814-825) .
  • improve the sensitivity, accuracy, stability, specificity and anti-interference ability of glucose dynamic detection and prolong the service life of the implantable continuous glucose monitoring system, at the same time
  • we have successfully developed an ultra-long-life glucose biosensor based on the third-generation biosensor technology we have successfully developed an ultra-long-life glucose biosensor based on the third-generation biosensor technology.
  • the glucose biosensor can not only be used to manufacture the high-performance glucose biosensor urgently needed for implantable continuous glucose monitoring systems, but also can be applied to other fields such as the food industry.
  • other various oxidoreductase-containing biosensors can also be fabricated based on this technology.
  • glucose oxidase is also composed of twenty kinds of natural amino acids.
  • chemically active groups in and on the surface of glucose oxidase such as arginine, lysine, glutamine and asparagine free amino groups, aspartic acid And the free carboxyl group of glutamic acid and the sulfhydryl group of cysteine, etc.
  • these chemically active groups can be used as electrons for electron transfer from the catalytic active center of glucose oxidase to the electrode surface after chemical modification.
  • the relay node establishes a high-speed electron channel for the electron exchange between the catalytic active center of glucose oxidase and the electrode, so as to realize the direct electrochemistry of glucose oxidase.
  • the huge molecular weight and complex three-dimensional structure of glucose oxidase pose great challenges to realize its direct electrochemistry.
  • the present invention firstly denatures the glucose oxidase in high concentration of urea (3.0mol/L) to open the glucose oxidase (Biochemistry 2002, 41, 3819-3827), and then By chemically modifying the free carboxyl groups on the surface and interior of glucose oxidase molecules—covalently bonding redox small molecules with superior electrochemical properties, such as ruthenium or osmium complexes, to glucose oxidase, from the inside to the outside Once an electron channel is established, the catalytically active center of glucose oxidase can directly exchange electrons with the electrode very rapidly.
  • the specific method is: firstly incubate 1-10 mg/mL glucose oxidase (preferred value: 2) in PBS buffer solution containing 1-8 mol/L urea (preferred value: 3) at 4°C for 12-24 h (preferred value: 12), fully expand the glucose oxidase.
  • glucose oxidase To achieve direct electron exchange with the electrode, glucose oxidase must be optimally positioned on the electrode surface—the exit of its electron channel must be in close contact with the electrode surface, since the contact of glucose oxidase in solution with the electrode surface is random and dynamic Yes, we can simply increase the concentration of glucose oxidase to increase the probability that the outlet of the electron channel is in close contact with the electrode surface, thereby improving the efficiency of glucose oxidase catalyzing the oxidation of glucose, but this approach is only applicable in the solution state applications such as disposable blood glucose test strips.
  • the surface of the glucose oxidase molecule is also wrapped by a large number of sugar molecules, such as glucose extracted from Aspergillus niger
  • sugar molecules such as glucose extracted from Aspergillus niger
  • the surface of oxidase has about 16% neutral sugars and 2% amino sugars, and we can aldolylate these sugar molecules first, and then react with ruthenium or osmium complexes with free amino groups to oxidize them in glucose.
  • a large number of electron exchange nodes are introduced on the surface of the enzyme molecule to form an efficient electron transfer network. In this way, the electrons transferred from the catalytic active center of glucose oxidase can quickly reach the electrode surface through this electron transfer network.
  • the specific method is: incubate the purified glucose oxidase in a sodium periodate solution containing 0.1-1 g/mL (preferred value: 0.3) at 20-30 °C (preferred value: 25) for 1-5 h (preferred value: 2 ), use ultrafiltration bag dialysis (cut molecular weight: 1000-30000) (preferred value: 10000) to separate and purify the glucose oxidase with aldolized sugar molecules.
  • the electrochemically activated glucose oxidase prepared in Examples 4-6 is prepared with a bifunctional chemical cross-linking agent such as glutaraldehyde, epichlorohydrin, N,N-methylenebisacrylamide, acetic anhydride, Glyceryl ether, methyl suberimidate, polyethylene glycol diglycidyl ether, etc. can be chemically cross-linked under mild conditions (aqueous solution, normal temperature, normal pressure), and a stable glucose biosensing membrane can be prepared.
  • a bifunctional chemical cross-linking agent such as glutaraldehyde, epichlorohydrin, N,N-methylenebisacrylamide, acetic anhydride, Glyceryl ether, methyl suberimidate, polyethylene glycol diglycidyl ether, etc.
  • a bifunctional chemical cross-linking agent such as glutaraldehyde, epichlorohydrin, N,N-methylenebisacrylamide, acetic anhydride, Glyceryl
  • the specific method is: thoroughly mix 5-200mg/mL (preferred value: 150) electrochemically activated glucose oxidase in PBS buffer solution and 0.1-5% (preferred value: 1) glutaraldehyde solution, 30- After 180min (preferred value: 60), the chemically cross-linked glucose oxidase was formed on the electrode surface to form a glucose biofilm by drop coating method or dip-pulling method.
  • the chemically cross-linked glucose oxidase still maintained their direct electrochemical properties, such as the glutaraldehyde chemically cross-linked glucose biosensor containing the modified glucose oxidase exhibited good electrochemical performance on the electrode, and is a typical surface electrochemical phenomenon (peak potential difference is much less than 59 mV) (Fig.
  • the oxidoreductase with improved electrochemical activity provided by the present invention and the raw materials and reagents used in the biosensor containing the oxidoreductase can be purchased from the market.
  • glucose oxidase was incubated in a PBS buffer solution containing 3 mol/L urea (preferred value: 3) at 4°C for 12 h to fully expand the glucose oxidase. Then 2 mg/mL of ruthenium or osmium complex with free amino group was thoroughly mixed with 2 mg/mL of glucose oxidase, and then 2 mmol/L of carbodiimide and 0.5 mmol/L of N-hydroxysuccinyl were added successively. imine, mixed thoroughly, and reacted at 4°C for 12h. Then, the modified perglucose oxidase was separated and purified by ultrafiltration bag dialysis (molecular weight cut: 10000).
  • glucose oxidase was incubated in a PBS buffer solution containing 8 mol/L urea at 4°C for 24 h to fully expand the glucose oxidase. Then 1 mg/mL of ruthenium or osmium complex with free amino groups was thoroughly mixed with 10 mg/mL of glucose oxidase, and then 1 mmol/L of carbodiimide and 1 mmol/L of N-hydroxysuccinyl were added successively. imine, mixed thoroughly, and reacted at 4°C for 24h. Then, the modified perglucose oxidase was separated and purified by ultrafiltration bag dialysis (cut molecular weight: 30000).
  • glucose oxidase was incubated in a PBS buffer solution containing 1 mol/L urea at 4°C for 18 h to fully expand the glucose oxidase. Then 10 mg/mL of ruthenium or osmium complex with free amino group was thoroughly mixed with 1 mg/mL of glucose oxidase, and then 10 mmol/L of carbodiimide and 0.1 mmol/L of N-hydroxysuccinic acid were added successively. imide, mixed thoroughly, and reacted at 4°C for 18h. Then, the modified glucose oxidase was isolated and purified by ultrafiltration bag dialysis (molecular weight cut: 1000).
  • glucose oxidase has been modified (electrochemically activated) to successfully bond redox small molecules with superior electrochemical properties to glucose oxidase, it is still necessary to confirm that this treatment does not affect the catalytically active center of glucose oxidase. Therefore, the catalytic activity of the electrochemically activated glucose oxidase was also evaluated.
  • Glucose was added to the PBS buffer solution containing electrochemically activated glucose oxidase (produced in Example 1), and the electrochemically activated glucose oxidase was characterized by cyclic voltammetry again (Fig. 2). As shown in Figure 2, the cyclic voltammogram of the electrochemically activated glucose oxidase after adding glucose clearly demonstrated a typical electrochemical catalytic process ( Figure 2(b)). Further experiments showed that the electrochemically activated glucose oxidase not only maintained its catalytic oxidation performance to glucose, but its catalytic oxidation efficiency of glucose by direct electrochemical oxidation was two times higher than that of natural glucose oxidase by oxygen. order of magnitude.
  • the purified glucose oxidase prepared in Example 1 was incubated in a sodium periodate solution containing 0.3 g/mL at 25 °C for 2 h, and then dialyzed with an ultrafiltration bag (molecular weight cut: 10000) for aldolized sugars.
  • Molecular glucose oxidase was isolated and purified.
  • 2 mg/mL of ruthenium or osmium complex with free amino groups was added to the purified glucose oxidase solution, mixed thoroughly and reacted at 4°C for 8 h, and then 5 mg/mL of sodium borohydride was added to the solution, and mixed thoroughly
  • the modified glucose oxidase was separated and purified by ultrafiltration bag dialysis (molecular weight cut: 10000).
  • the purified glucose oxidase prepared in Example 2 was incubated in a sodium periodate solution containing 0.1 g/mL at 30° C. for 1 h, and then dialyzed with an ultrafiltration bag (cutting molecular weight: 30,000) for aldolized sugars.
  • Molecular glucose oxidase was isolated and purified.
  • 1 mg/mL of ruthenium or osmium complex with free amino groups was added to the purified glucose oxidase solution, mixed thoroughly and reacted at 4°C for 2 h, and then 2 mg/mL of sodium borohydride was added to the solution, and mixed thoroughly Then, the reaction was carried out at 4°C for 4 h.
  • the modified glucose oxidase was separated and purified by ultrafiltration bag dialysis (molecular weight cut: 1000).
  • the purified glucose oxidase prepared in Example 3 was incubated in a sodium periodate solution containing 1 g/mL at 20 °C for 5 h, and then dialyzed with an ultrafiltration bag (cutting molecular weight: 1000) to aldolized sugar molecules.
  • the glucose oxidase was isolated and purified.
  • electrochemically activated glucose oxidase prepared in Example 4
  • PBS buffer solution 150 mg/mL was thoroughly mixed in PBS buffer solution and 1% glutaraldehyde solution, and after 60 min, drip coating method or dip-pulling method was used to remove the enzyme.
  • the chemically cross-linked glucose oxidase forms a glucose biofilm on the electrode surface.
  • the 5-mg/mL electrochemically activated glucose oxidase (prepared in Example 5) was thoroughly mixed in PBS buffer solution and 0.1% glutaraldehyde solution, and after 30 minutes, it was applied by drop coating method or dipping and pulling.
  • the chemically cross-linked glucose oxidase is formed on the electrode surface to form a glucose biofilm.

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Abstract

电化学领域的、特别涉及提高电化学活性的氧化还原酶及含有氧化还原酶的生物传感器。经过化学交联的葡萄糖氧化酶仍然保持其直接电化学活性，例如戊二醛化学交联的含有经过修饰的葡萄糖氧化酶的葡萄糖生物传感器在电极上呈现出良好的电化学性能，且是一个典型的表面电化学现象。化学交联没有对电化学活化后的葡萄糖氧化酶产生显著的影响，为电化学活化后的葡萄糖氧化酶在植入式持续葡萄糖监测系统中的应用铺平了道路。

Description

提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器 技术领域
本发明涉及电化学领域，特别涉及提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器。
背景技术
自1962年Clark和Lyon成功地研制出第一个生物传感器以来，经过50多年的发展，生物传感器已经在环境检测，食品工业，临床医学等领域得到了非常广的应用。例如基于生物传感技术发展起来的各种葡萄糖传感器已经造福了千百万的糖尿病患者。其中，近几年迅速发展起来的植入式持续葡萄糖监测系统以其使用方便和实时监测等特点，受到越来越多的糖尿病患者的青睐，特别是I型糖尿病患者。作为植入式持续葡萄糖监测系统的核心部件，葡萄糖生物传感器的性能直接决定了植入式持续葡萄糖监测系统的性能和使用寿命。现有的植入式持续葡萄糖监测系统所使用的葡萄糖生物传感器都是基于第一和第二代生物传感技术发展起来的。第一代生物传感技术是通过电化学方法检测葡萄糖氧化过程中生成的过氧化氢或消耗的氧气来间接地对葡萄糖进行监测。例如美敦力的Guardian和iPro2和德康的Dexcom G5和G6都是基于第一代生物传感技术开发出来的，它们通过电化学方法检测葡萄糖在葡萄糖氧化酶催化氧化过程中生成的过氧化氢来对葡萄糖进行监测。由于电化学方法检测过氧化氢对电极的要求非常苛刻，只有铂和铂合金等极少数几种材料能用于这类葡萄糖生物传感器的制作，这就大大地增加了植入式持续葡萄糖监测系统的传感器的成本。另外，过氧化氢的电化学检测要求较高的检测电位，因而大大地降低了植入式持续葡萄糖监测系统的抗干扰能力，特别是对常用的退烧药如乙酰氨基酚的抗干扰能力。
第二代生物传感技术是通过在葡萄糖生物传感器中引入氧化还原媒介体来实现对葡萄糖进行直接的电化学检测。与普通的蛋白质分子不同，葡萄糖氧化酶的分子量很大(160KDa)，其分子结构特别是催化活性中心 的立体结构非常复杂，而且位于葡萄糖氧化酶的内部，并被各种肽链深深地包裹着。因此，葡萄糖氧化酶不能直接与电极进行电子交换。Heller等人(Acc.Chem.Res.23(1990)128-134)发现在葡萄糖生物传感器中引入氧化还原物质—氧化还原媒介体(氧化还原小分子如铁氰化物或氧化还原高分子),葡萄糖氧化酶可以通过这些媒介体实现与电极进行电子交换。基于此原理发展起来第二代生物传感技术目前已被广泛应用于生物传感器，特别是葡萄糖生物传感器，包括各种一次性血糖检测试纸条和植入式持续葡萄糖监测系统，例如雅培糖尿病护理的FreeStyle Libre。通过对氧化还原媒介体的分子设计和优化,葡萄糖的检测可以在较低的电位下实现,从而大大地提高了植入式持续葡萄糖监测系统的抗干扰能力，特别是对常用的退烧药如乙酰氨基酚的抗干扰能力。由于这类葡萄糖监测系统是通过氧化还原媒介体对葡萄糖进行直接的电化学检测，其灵敏度也得到了显著的改善。但由于氧化还原媒介体为小分子或高分子材料，使其制备难于得到精确的控制，同时也存在氧化还原媒介体从植入式葡萄糖生物传感器中渗出的可能性，给植入式持续葡萄糖监测系统的性能带来相当多的不确定性。
发明内容
有鉴于此，本发明提供了提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器。该葡萄糖氧化酶对葡萄糖的催化氧化效率比天然葡萄糖氧化酶通过氧气对葡萄糖的催化氧化效率提高了两个数量级。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了用于制备生物传感器的制剂，包括如下组合物中的一个或多个：
组合物一：包括酶变性剂、电化学性能优越的氧化还原小分子、交联剂、交联剂辅助物和缓冲溶液；
所述酶变性剂包括尿素或盐酸胍；所述电化学性能优越的氧化还原小分子包括带有游离氨基的钌或锇的络合物；所述交联剂包括碳化二亚胺或辛二酸二琥珀酰亚胺；所述交联剂辅助物包括N-羟基琥珀酰亚胺或N-羟基硫代琥珀酰亚胺；所述缓冲溶液包括PBS缓冲溶液；
和/或
组合物二：包括醛基化试剂、电化学性能优越的氧化还原小分子和还原剂；
所述醛基化试剂包括高碘酸钠；所述电化学性能优越的氧化还原小分子包括带有游离氨基的钌或锇的络合物；所述还原剂包括硼氢化钠；
和/或
组合物三：包括双功能化学交联剂和缓冲溶液；
所述双功能化学交联剂包括戊二醛、环氧氯丙烷、N,N-亚甲基双丙烯酰胺、乙酸酐、二缩水甘油基乙醚、辛二亚氨酸甲酯、聚乙二醇二缩水甘油醚中的一个或两者以上的组合物；所述缓冲溶液包括PBS缓冲溶液；
其中，所述制剂至少包含组合物一。
在本发明的一些具体实施方案中，以重量份计，所述组合物一包括如下组分：
Figure PCTCN2020113935-appb-000001
所述酶变性剂的缓冲溶液中所述酶变性剂的浓℃为1～8mol/L。
在上述研究的基础上，本发明还提供了所述的制剂在提高氧化还原酶电化学活性中的应用。
本发明还提供了提高氧化还原酶电化学活性的方法，基于本发明所述的制剂，包括如下步骤：
步骤1：将氧化还原酶与酶变性剂的缓冲溶液混合后培养，制得氧化还原酶的展开溶液；
步骤2：将电化学性能优越的氧化还原小分子与步骤1制得的所述氧化还原酶的展开溶液混合，制得混合溶液；
步骤3：取步骤2制得的所述混合溶液依次与交联剂、交联剂辅助物混合后反应，获得修饰液；
步骤4：取步骤3制得的所述修饰液，分离，纯化。
在本发明的一些具体实施方案中，步骤1具体为：将1～10mg/mL的葡萄糖氧化酶(优选值:2)与含有1～8mol/L尿素(优选值:3)的PBS缓冲溶液在4℃培养12～24h(优选值:12)。作为优选，葡萄糖氧化酶的浓度为2mg/mL。作为优选，尿素的浓度为3mol/L。作为优选，培养时间为12h。
在本发明的一些具体实施方案中，步骤2具体为：将1～10mg/mL(优选值:2)的电化学性能优越的氧化还原小分子与1～10mg/mL(优选值:2)的所述氧化还原酶的展开溶液混合。作为优选，电化学性能优越的氧化还原小分子的浓度为2mg/mL。作为优选，所述氧化还原酶的展开溶液的浓度为1～10mg/mL。
在本发明的一些具体实施方案中，步骤3具体为：取步骤2制得的所述混合溶液依次与1～10mmol/L(优选值:2)的交联剂和0.1～1mmol/L(优选值:0.5)的交联剂辅助物混合后，在4℃反应12～24h(优选值:12)。作为优选，交联剂的浓度为2mmol/L。作为优选，交联剂辅助物的浓度为0.5mmol/L。作为优选，培养时间为12h。
在本发明的一些具体实施方案中，步骤4具体为：取步骤3制得的所述修饰液经超滤袋透析(切割分子量:1000～30000)(优选值:10000)对提高电化学活性的氧化还原酶。进行分离、提纯。作为优选，超滤袋的切割分子量为10000。
在本发明的一些具体实施方案中，还包括如下步骤：
步骤5：将步骤4纯化后的氧化还原酶与所述醛基化试剂混合，分离，纯化，制得纯化后的氧化还原酶溶液；
步骤6：取步骤5制得的所述纯化后的氧化还原酶溶液与所述电化学性能优越的氧化还原小分子混合，制得混合溶液；
步骤7：取步骤6制得的混合溶液与所述还原剂混合，对电化学活化后的葡萄糖氧化酶进行分离和纯化。
在本发明的一些具体实施方案中，步骤5具体为：将步骤4纯化后的氧化还原酶与0.1～1g/mL(优选值:0.3)的所述醛基化试剂于20～30(优选值:25)℃培养1～5(优选值:2)h，经超滤袋透析(切割分子量:1000～30000)(优选值:10000)对带有醛基化糖分子的葡萄糖氧化酶进行分离，纯化，制得 纯化后的氧化还原酶溶液。作为优选，所述醛基化试剂的浓度为3g/mL。作为优选，培养温度为25℃，时间为2h。作为优选，超滤袋的切割分子量为10000。
在本发明的一些具体实施方案中，步骤6具体为：取所述纯化后的氧化还原酶溶液与1～10mg/mL(优选值:2)的所述电化学性能优越的氧化还原小分子混合后，在4℃反应2～24h(优选值:8)，制得混合溶液。所述电化学性能优越的氧化还原小分子的浓度为2mg/mL。作为优选，反应时间为8h。
在本发明的一些具体实施方案中，步骤7具体为：取所述混合溶液与2～20mg/mL(优选值:5)的所述还原剂，混合后于4℃反应1～4h(优选值:2)，反应结束后，经超滤袋透析(切割分子量:1000～30000)(优选值:10000)对电化学活化后的葡萄糖氧化酶进行分离和纯化。作为优选，所述还原剂的浓度为2～20mg/mL。作为优选，所述反应时间为2h。作为优选，超滤袋的切割分子量为10000。
在上述研究的基础上，本发明还提供了所述的方法制得的提高电化学活性的氧化还原酶。
本发明还提供了所述提高电化学活性的氧化还原酶在制备氧化还原酶传感器、氧化还原酶检测系统或食品工业中的应用。
更重要的是，本发明还提供了氧化还原酶传感器，包括所述的提高电化学活性的氧化还原酶。
本发明还提供了所述氧化还原酶传感器的制备方法，将所述提高电化学活性的氧化还原酶与双功能化学交联剂混合，用滴落涂布法或浸渍提拉法制得化学交联后的氧化还原酶在电极表面制成氧化还原酶传感器。
在本发明的一些具体实施方案中，将5～200mg/mL(优选值:150)电化学活化后的葡萄糖氧化酶在PBS缓冲溶液与0.1～5％(优选值:1)的戊二醛溶液进行混合，30～180min(优选值:60)后，用滴落涂布法或浸渍提拉法将化学交联后的葡萄糖氧化酶在电极表面制成氧化还原酶传感器。作为优选，所述电化学活化后的葡萄糖氧化酶的浓度为150mg/mL。作为优选，戊二醛溶液的浓度为0.1～5％。作为优选，混合的时间为60min。
本发明还提供了氧化还原酶检测系统，包括所述的提高电化学活性的氧化还原酶或所述的氧化还原酶传感器。
在本发明中，所述氧化还原酶包括葡萄糖氧化酶。
本发明通过对葡萄糖氧化酶分子表面和内部的游离羧基进行化学修饰—将电化学性能优越的氧化还原小分子,例如钌或锇的络合物等共价键合到葡萄糖氧化酶上,从内到外建立起一条电子通道,葡萄糖氧化酶催化活性中心就可以直接与电极进行非常快速的电子交换。电化学活化后的葡萄糖氧化酶不仅保持了其对葡萄糖的催化氧化性能,其通过直接电化学对葡萄糖的催化氧化效率比天然葡萄糖氧化酶通过氧气对葡萄糖的催化氧化效率提高了两个数量级。
此外，本发明将这些糖分子醛基化，然后与带有游离氨基的钌或锇的络合物反应，从而在葡萄糖氧化酶分子表面引入大量的电子交换节点，形成一个高效的电子传递网络。这样一来，葡萄糖氧化酶催化活性中心传递出来的电子可以通过这个电子传递网络快速地到达电极表面。经过以上两次化学处理以后，葡萄糖氧化酶从内到外已经被彻底电化学活化。
申请人发现经过化学交联的葡萄糖氧化酶仍然保持着它们的直接电化学活性，例如戊二醛化学交联的含有经过修饰的葡萄糖氧化酶的葡萄糖生物传感器在电极上呈现出良好的电化学性能，而且是一个典型的表面电化学现象。以上实验结果证明化学交联没有对电化学活化后的葡萄糖氧化酶产生显著的影响，这就为电化学活化后的葡萄糖氧化酶在植入式持续葡萄糖监测系统中的应用铺平了道路。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示循环伏安图；其中曲线a示没有碳化二亚胺/N-羟基琥珀酰亚胺交联剂存在时处理的葡萄糖氧化酶和曲线b示在碳化二亚胺/N-羟基琥珀酰亚胺交联剂存在时处理的葡萄糖氧化酶循环伏安图的循环伏安图；
图2示循环伏安图；其中曲线a示含有电化学活化的葡萄糖氧化酶在 PBS缓冲溶液中的循环伏安图；曲线b示加入10毫摩尔/升的葡萄糖后的循环伏安图；
图3示循环伏安图；其中曲线a示含有电化学活化后的葡萄糖氧化酶葡萄糖生物传感器在PBS缓冲溶液中的循环伏安图，曲线b示加入5.0毫摩尔/升葡萄糖后的第一圈和曲线c示第100圈的循环伏安图；其中，
Figure PCTCN2020113935-appb-000002
示曲线a；
Figure PCTCN2020113935-appb-000003
示曲线b；
Figure PCTCN2020113935-appb-000004
示曲线c。
具体实施方式
本发明公开了提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
第三代生物传感技术是利用氧化还原酶的直接电化学发展起来的生物传感技术(计测技术,2006,(26卷,增刊)92-96,Chem.Rev.2008,108,814-825)。为了克服第一和第二代生物传感技术的缺点,提高葡萄糖动态检测的灵敏度、准确性、稳定性、专一性和抗干扰能力,并延长植入式持续葡萄糖监测系统的使用寿命,同时大大地降低葡萄糖生物传感器的成本,我们成功地研制出了基于第三代生物传感技术的超长寿命的葡萄糖生物传感器。该葡萄糖生物传感器不仅可以用于制造植入式持续葡萄糖监测系统所急需的高性能葡萄糖生物传感器,而且还可以应用于食品工业等其它领域。另外，基于此技术也可以制造其它各种含有氧化还原酶的生物传感器。
与其它蛋白质分子一样,作为一类特殊的蛋白质分子,葡萄糖氧化酶也是由二十种天然氨基酸构成的。除了形成肽链所必须的氨基和羧基外,葡萄糖氧化酶内部和表面还有大量的化学活性基团例如精氨酸、赖氨酸、谷氨酰胺和天冬酰胺的游离氨基，天冬氨酸和谷氨酸的游离羧基和半胱氨酸的巯基等。只要不显著地破坏葡萄糖氧化酶的三维结构，不严重地影响其 催化活性,这些化学活性基团在经过化学修饰以后都可以用做从葡萄糖氧化酶的催化活性中心向电极表面进行电子传递的电子接力节点，为葡萄糖氧化酶的催化活性中心与电极之间的电子交换建立一条高速电子通道，从而实现葡萄糖氧化酶的直接电化学。然而，葡萄糖氧化酶的巨大分子量和复杂的三维结构对实现其直接电化学带来了极大的挑战。对葡萄糖氧化酶的晶体结构研究发现其可利用的游离氨基(主要来自赖氨酸)主要分布在葡萄糖氧化酶的表面,距其催化活性中心有相当大的距离
Figure PCTCN2020113935-appb-000005
(J.Mol.Biol.229(1993)153-172)。只对它们进行化学修饰将大大降低葡萄糖氧化酶的催化氧化效率(J.Am.Chem.Soc.115(1993)7053-7060)。另一方面，虽然其催化活性中心附近有几个可以利用的游离羧基
Figure PCTCN2020113935-appb-000006
对这为数不多的游离羧基进行化学修饰也难得到催化氧化效率很高的葡萄糖氧化酶。
为了得到催化氧化效率很高的葡萄糖氧化酶，本发明首先将葡萄糖氧化酶在高浓度的尿素(3.0mol/L)中变性─将葡萄糖氧化酶打开(Biochemistry 2002,41,3819-3827)，然后通过对葡萄糖氧化酶分子表面和内部的游离羧基进行化学修饰─将电化学性能优越的氧化还原小分子,例如钌或锇的络合物等共价键合到葡萄糖氧化酶上，从内到外建立起一条电子通道，葡萄糖氧化酶催化活性中心就可以直接与电极进行非常快速的电子交换。
具体做法是：先将1-10mg/mL的葡萄糖氧化酶(优选值:2)在含有1～8mol/L尿素(优选值:3)的PBS缓冲溶液在4℃培养12～24h(优选值:12)，将葡萄糖氧化酶充分展开。然后将1～10mg/mL(优选值:2)的带有游离氨基的钌或锇的络合物与1～10mg/mL(优选值:2)的葡萄糖氧化酶充分混合，然后依次加入1～10mmol/L(优选值:2)的碳化二亚胺和0.1-1mmol/L(优选值:0.5)的N-羟基琥珀酰亚胺，充分混合后，在4℃反应12～24h(优选值:12)。然后利用超滤袋透析(切割分子量:1000～30000)(优选值:10000)对修饰过葡萄糖氧化酶进行分离和提纯。
要实现直接与电极进行电子交换，葡萄糖氧化酶必须在电极表面有个最佳定位—它的电子通道的出口必须与电极表面密切接触，由于葡萄糖氧化酶在溶液与电极表面的接触是随机和动态的，我们可以简单地通过提高 葡萄糖氧化酶的浓度来增加电子通道的出口与电极表面密切接触的几率，进而提高葡萄糖氧化酶催化氧化葡萄糖的效率，但是，这种做法只适用于在溶液状态下的应用，如一次性血糖检测试纸条。要实现在植入式持续葡萄糖监测系统中的应用，即制备出具有高效催化性能的固态生物传感膜，我们必须保证在生物传感膜里的葡萄糖氧化酶的一致性，也就是说所有的葡萄糖氧化酶分子都可以参与到催化氧化葡萄糖的过程中，与它们在电极上/生物传感膜里的定位没有任何关系。要实现这一目的，我们不但需要从内(催化活性中心)到外(葡萄糖氧化酶分子表面)建立起一条电子通道，还必须在葡萄糖氧化酶分子表面建立起电子传递网络，使得从一个葡萄糖氧化酶分子的催化活性中心传递出来的电子可通过这个电子传递网络与电极或与其它葡萄糖氧化酶分子进行快速的电子交换。我们知道，葡萄糖氧化酶属于一类糖蛋白，除了由各种氨基酸组成的肽链的蛋白质的基本结构以外，葡萄糖氧化酶分子表面还被大量的糖分子包裹着，例如从黑曲霉中提取的葡萄糖氧化酶的表面有约16％的中性糖和2％的氨基糖，我们可以先将这些糖分子醛基化，然后与带有游离氨基的钌或锇的络合物反应，从而在葡萄糖氧化酶分子表面引入大量的电子交换节点，形成一个高效的电子传递网络。这样一来，葡萄糖氧化酶催化活性中心传递出来的电子可以通过这个电子传递网络快速地到达电极表面。
具体做法是：将提纯后的葡萄糖氧化酶在含有0.1～1g/mL(优选值:0.3)的高碘酸钠溶液在20～30℃(优选值:25)培养1～5h(优选值:2)后，利用超滤袋透析(切割分子量:1000～30000)(优选值:10000)对带有醛基化糖分子的葡萄糖氧化酶进行分离和提纯。然后在提纯葡萄糖氧化酶溶液中加入1～10mg/mL(优选值:2)的带有游离氨基的钌或锇的络合物,充分混合后在4℃反应2～24h(优选值:8),然后在溶液中加入2～20mg/mL(优选值:5)的硼氢化钠，充分混合后在4℃反应1～4h(优选值:2),反应结束后，再次利用超滤袋透析(切割分子量:1000～30000)(优选值:10000)对修饰过葡萄糖氧化酶进行分离和提纯。经过以上两次化学处理以后，葡萄糖氧化酶从内到外已经被彻底电化学活化。
将实施例4～6制得的电化学活化后的葡萄糖氧化酶用双功能化学交 联剂例如戊二醛、环氧氯丙烷、N,N-亚甲基双丙烯酰胺、乙酸酐、二缩水甘油基乙醚、辛二亚氨酸甲酯、聚乙二醇二缩水甘油醚等在温和条件下(水溶液、常温、常压)进行化学交联，就可以制备出稳定的葡萄糖生物传感膜。具体做法是：将5-200mg/mL(优选值:150)电化学活化后的葡萄糖氧化酶在PBS缓冲溶液与0.1-5％(优选值:1)的戊二醛溶液进行充分混合，30-180min(优选值:60)后,用滴落涂布法或浸渍提拉法将化学交联后的葡萄糖氧化酶在电极表面制成葡萄糖生物传膜。我们发现经过化学交联的葡萄糖氧化酶仍然保持着它们的直接电化学，例如戊二醛化学交联的含有经过修饰的葡萄糖氧化酶的葡萄糖生物传感器在电极上呈现出良好的电化学性能，而且是一个典型的表面电化学现象(峰电位差远远小于59毫伏)(图3(a))。当在PBS缓冲溶液中加入5.0mM的葡萄糖后，与电化学活化后的葡萄糖氧化酶在溶液中相似，葡萄糖生物传感膜的循环伏安图清晰地展示了一个典型的电化学催化过程(图3(b))。以上实验结果证明化学交联没有对电化学活化后的葡萄糖氧化酶产生显著的影响，这就为电化学活化后的葡萄糖氧化酶在植入式持续葡萄糖监测系统中的应用铺平了道路。
本发明提供的提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
先将2mg/mL的葡萄糖氧化酶在含有3mol/L尿素(优选值:3)的PBS缓冲溶液在4℃培养12h，将葡萄糖氧化酶充分展开。然后将2mg/mL的带有游离氨基的钌或锇的络合物与2mg/mL的葡萄糖氧化酶充分混合，然后依次加入2mmol/的碳化二亚胺和0.5mmol/L的N-羟基琥珀酰亚胺，充分混合后，在4℃反应12h。然后利用超滤袋透析(切割分子量:10000)对修饰过葡萄糖氧化酶进行分离和提纯。
实施例2
先将1mg/mL的葡萄糖氧化酶在含有8mol/L尿素的PBS缓冲溶液在 4℃培养24h，将葡萄糖氧化酶充分展开。然后将1mg/mL的带有游离氨基的钌或锇的络合物与10mg/mL的葡萄糖氧化酶充分混合，然后依次加入1mmol/L的碳化二亚胺和1mmol/L的N-羟基琥珀酰亚胺，充分混合后，在4℃反应24h。然后利用超滤袋透析(切割分子量:30000)对修饰过葡萄糖氧化酶进行分离和提纯。
实施例3
先将10mg/mL的葡萄糖氧化酶在含有1mol/L尿素的PBS缓冲溶液在4℃培养18h，将葡萄糖氧化酶充分展开。然后将10mg/mL的带有游离氨基的钌或锇的络合物与1mg/mL的葡萄糖氧化酶充分混合，然后依次加入10mmol/L的碳化二亚胺和0.1mmol/L的N-羟基琥珀酰亚胺，充分混合后，在4℃反应18h。然后利用超滤袋透析(切割分子量:1000)对修饰过葡萄糖氧化酶进行分离和提纯。
效果例1
为了确保对葡萄糖氧化酶的化学修饰(电化学活化)的成功，我们首先利用循环伏安法对实施例1制得的修饰过葡萄糖氧化酶进行表征(图1)。图1曲线b清楚地表明经过化学交联剂处理后，电化学性能优越的氧化还原小分子已经成功地键合到葡萄糖氧化酶上。与之相反，在没有碳化二亚胺/N-羟基琥珀酰亚胺化学交联剂存在时，经过上述处理的葡萄糖氧化酶没有任何的电化学活性(图1曲线a)。
效果例2
虽然葡萄糖氧化酶经过修饰(电化学活化)后，成功地将电化学性能优越的氧化还原小分子键合到葡萄糖氧化酶上，但是还需要确认这种处理不会对葡萄糖氧化酶的催化活性中心产生明显的影响，因此还要对电化学活化后的葡萄糖氧化酶的催化活性进行评估。
在含有电化学活化过的葡萄糖氧化酶(实施例1制得)的PBS缓冲溶液中加入葡萄糖，再次用循环伏安法对电化学活化后的葡萄糖氧化酶进 行表征(图2)。如图2所示，在加入葡萄糖后，电化学活化后的葡萄糖氧化酶的循环伏安图清晰地展示了一个典型的电化学催化过程(图2(b))。进一步的实验表明电化学活化后的葡萄糖氧化酶不仅保持了其对葡萄糖的催化氧化性能,其通过直接电化学对葡萄糖的催化氧化效率比天然葡萄糖氧化酶通过氧气对葡萄糖的催化氧化效率提高了两个数量级。
以上实验结果表明，经过以上处理，已经在葡萄糖氧化酶中从内到外—从葡萄糖氧化酶催化活性中心到其表面，建立起了一条电子通道，葡萄糖氧化酶催化活性中心现在可以直接与电极进行非常快速的电子交换，成功地实现了葡萄糖氧化酶的直接电化学。
实施例4
将实施例1制得的提纯后的葡萄糖氧化酶在含有0.3g/mL的高碘酸钠溶液在25℃培养2h后，利用超滤袋透析(切割分子量:10000)对带有醛基化糖分子的葡萄糖氧化酶进行分离和提纯。然后在提纯葡萄糖氧化酶溶液中加入2mg/mL的带有游离氨基的钌或锇的络合物，充分混合后在4℃反应8h，然后在溶液中加入5mg/mL的硼氢化钠，充分混合后在4℃反应2h，反应结束后，再次利用超滤袋透析(切割分子量:10000)对修饰过葡萄糖氧化酶进行分离和提纯。
实施例5
将实施例2制得的提纯后的葡萄糖氧化酶在含有0.1g/mL的高碘酸钠溶液在30℃培养1h后，利用超滤袋透析(切割分子量:30000)对带有醛基化糖分子的葡萄糖氧化酶进行分离和提纯。然后在提纯葡萄糖氧化酶溶液中加入1mg/mL的带有游离氨基的钌或锇的络合物，充分混合后在4℃反应2h，然后在溶液中加入2mg/mL的硼氢化钠，充分混合后在4℃反应4h，反应结束后，再次利用超滤袋透析(切割分子量:1000)对修饰过葡萄糖氧化酶进行分离和提纯。
实施例6
将实施例3制得的提纯后的葡萄糖氧化酶在含有1g/mL的高碘酸钠溶液在20℃培养5h后，利用超滤袋透析(切割分子量:1000)对带有醛基化糖分子的葡萄糖氧化酶进行分离和提纯。然后在提纯葡萄糖氧化酶溶液中加入10mg/mL的带有游离氨基的钌或锇的络合物，充分混合后在4℃反应24h，然后在溶液中加入20mg/mL的硼氢化钠，充分混合后在4℃反应1h，反应结束后，再次利用超滤袋透析(切割分子量:30000)对修饰过葡萄糖氧化酶进行分离和提纯。
实施例7
将150mg/mL电化学活化后的葡萄糖氧化酶(实施例4制得)在PBS缓冲溶液与1％的戊二醛溶液进行充分混合，60min后，用滴落涂布法或浸渍提拉法将化学交联后的葡萄糖氧化酶在电极表面制成葡萄糖生物传膜。
实施例8
将5-mg/mL电化学活化后的葡萄糖氧化酶(实施例5制得)在PBS缓冲溶液与0.1％的戊二醛溶液进行充分混合，30min后，用滴落涂布法或浸渍提拉法将化学交联后的葡萄糖氧化酶在电极表面制成葡萄糖生物传膜。
实施例9
将200mg/mL电化学活化后的葡萄糖氧化酶(实施例6制得)在PBS缓冲溶液与5％的戊二醛溶液进行充分混合，180min后，用滴落涂布法或浸渍提拉法将化学交联后的葡萄糖氧化酶在电极表面制成葡萄糖生物传膜。
效果例3
申请人发现经过化学交联的葡萄糖氧化酶(实施例7制得)仍然保持着它们的直接电化学，例如戊二醛化学交联的含有经过修饰的葡萄糖氧化 酶的葡萄糖生物传感器在电极上呈现出良好的电化学性能，而且是一个典型的表面电化学现象(峰电位差远远小于59毫伏)(图3(a))。当在PBS缓冲溶液中加入5.0mM的葡萄糖后，与电化学活化后的葡萄糖氧化酶在溶液中相似，葡萄糖生物传感膜的循环伏安图清晰地展示了一个典型的电化学催化过程(图3(b))。以上实验结果证明化学交联没有对电化学活化后的葡萄糖氧化酶产生显著的影响，这就为电化学活化后的葡萄糖氧化酶在植入式持续葡萄糖监测系统中的应用铺平了道路。
以上对本发明所提供的提高电化学活性的氧化还原酶及含有该氧化还原酶的生物传感器进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (19)

  1. 用于制备生物传感器的制剂，其特征在于，包括如下组合物中的一个或多个：
    组合物一：包括酶变性剂、电化学性能优越的氧化还原小分子、交联剂、交联剂辅助物和缓冲溶液；
    所述酶变性剂包括尿素或盐酸胍；所述电化学性能优越的氧化还原小分子包括带有游离氨基的钌或锇的络合物；所述交联剂包括碳化二亚胺或辛二酸二琥珀酰亚胺；所述交联剂辅助物包括N-羟基琥珀酰亚胺或N-羟基硫代琥珀酰亚胺；所述缓冲溶液包括PBS缓冲溶液；
    和/或
    组合物二：包括醛基化试剂、电化学性能优越的氧化还原小分子和还原剂；
    所述醛基化试剂包括高碘酸钠；所述电化学性能优越的氧化还原小分子包括带有游离氨基的钌或锇的络合物；所述还原剂包括硼氢化钠；
    和/或
    组合物三：包括双功能化学交联剂和缓冲溶液；
    所述双功能化学交联剂包括戊二醛、环氧氯丙烷、N,N-亚甲基双丙烯酰胺、乙酸酐、二缩水甘油基乙醚、辛二亚氨酸甲酯、聚乙二醇二缩水甘油醚中的一个或两者以上的组合物；所述缓冲溶液包括PBS缓冲溶液；
    其中，所述制剂至少包含组合物一。
  2. 如权利要求1所述的制剂，其特征在于，以重量份计，所述组合物一包括如下组分：
    Figure PCTCN2020113935-appb-100001
    所述酶变性剂的缓冲溶液中所述酶变性剂的浓度为1～8mol/L。
  3. 如权利要求1或2所述的制剂在提高氧化还原酶电化学活性中的 应用。
  4. 提高氧化还原酶电化学活性的方法，其特征在于，基于如权利要求1或2所述的制剂，包括如下步骤：
    步骤1：将氧化还原酶与酶变性剂的缓冲溶液混合后培养，制得氧化还原酶的展开溶液；
    步骤2：将电化学性能优越的氧化还原小分子与步骤1制得的所述氧化还原酶的展开溶液混合，制得混合溶液；
    步骤3：取步骤2制得的所述混合溶液依次与交联剂、交联剂辅助物混合后反应，获得修饰液；
    步骤4：取步骤3制得的所述修饰液，分离，纯化。
  5. 如权利要求4所述的方法，其特征在于，步骤1具体为：将1～10mg/mL的葡萄糖氧化酶与含有1～8mol/L尿素的PBS缓冲溶液在4℃培养12～24h。
  6. 如权利要求4或5所述的方法，其特征在于，步骤2具体为：将1～10mg/mL的电化学性能优越的氧化还原小分子与1～10mg/mL的所述氧化还原酶的展开溶液混合。
  7. 如权利要求4至6任一项所述的方法，其特征在于，步骤3具体为：取步骤2制得的所述混合溶液依次与1～10mmol/L的交联剂和0.1～1mmol/L的交联剂辅助物混合后，在4℃反应12～24h。
  8. 如权利要求4至7任一项所述的方法，其特征在于，步骤4具体为：取步骤3制得的所述修饰液经切割分子量为1000～30000的超滤袋透析对提高电化学活性的氧化还原酶；进行分离、提纯。
  9. 如权利要求4至8任一项所述的方法，其特征在于，还包括如下步骤：
    步骤5：将步骤4纯化后的氧化还原酶与所述醛基化试剂混合，分离，纯化，制得纯化后的氧化还原酶溶液；
    步骤6：取步骤5制得的所述纯化后的氧化还原酶溶液与所述电化学性能优越的氧化还原小分子混合，制得混合溶液；
    步骤7：取步骤6制得的混合溶液与所述还原剂混合，对电化学活化 后的葡萄糖氧化酶进行分离和纯化。
  10. 如权利要求9所述的方法，其特征在于，步骤5具体为：将步骤4纯化后的氧化还原酶与0.1～1g/mL的所述醛基化试剂于20～30℃培养1～5h，经切割分子量为1000～30000的超滤袋透析对带有醛基化糖分子的葡萄糖氧化酶进行分离，纯化，制得纯化后的氧化还原酶溶液。
  11. 如权利要求9或10所述的方法，其特征在于，步骤6具体为：取所述纯化后的氧化还原酶溶液与1～10mg/mL的所述电化学性能优越的氧化还原小分子混合后，在4℃反应2～24h，制得混合溶液。
  12. 如权利要求9～11中任一项所述的方法，其特征在于，步骤7具体为：取所述混合溶液与2～20mg/mL的所述还原剂，混合后于4℃反应1～4h，反应结束后，经切割分子量为1000～30000的超滤袋透析对电化学活化后的葡萄糖氧化酶进行分离和纯化。
  13. 如权利要求4至12任一项所述的方法制得的提高电化学活性的氧化还原酶。
  14. 如权利要求13所述提高电化学活性的氧化还原酶在制备氧化还原酶传感器、氧化还原酶检测系统或食品工业中的应用。
  15. 氧化还原酶传感器，其特征在于，包括如权利要求13所述的提高电化学活性的氧化还原酶。
  16. 如权利要求15所述氧化还原酶传感器的制备方法，其特征在于，将所述提高电化学活性的氧化还原酶与双功能化学交联剂混合，用滴落涂布法或浸渍提拉法制得化学交联后的氧化还原酶在电极表面制成氧化还原酶传感器。
  17. 如权利要求16所述的制备方法，其特征在于，将5～200mg/mL电化学活化后的葡萄糖氧化酶在PBS缓冲溶液与0.1～5％的戊二醛溶液进行混合，30～180min后，用滴落涂布法或浸渍提拉法将化学交联后的葡萄糖氧化酶在电极表面制成氧化还原酶传感器。
  18. 氧化还原酶检测系统，其特征在于，包括如权利要求13所述的提高电化学活性的氧化还原酶或如权利要求15所述的氧化还原酶传感器。
  19. 如权利要求1或2所述的制剂、如权利要求3所述的应用、如权利要求4至12任一项所述的方法、如权利要求13所述的提高电化学活性的氧化还原酶、如权利要求14所述的应用、如权利要求15所述的氧化还原酶传感器、如权利要求16或17所述的制备方法、如权利要求18所述的氧化还原酶检测系统，其特征在于，所述氧化还原酶包括葡萄糖氧化酶。
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