CN114609213A

CN114609213A - Glucose sensor and preparation method and application thereof

Info

Publication number: CN114609213A
Application number: CN202210262656.5A
Authority: CN
Inventors: 高志强
Original assignee: Suzhou Zhongxing Medical Technology Co ltd
Current assignee: Suzhou Zhongxing Medical Technology Co ltd
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2022-06-10

Abstract

The invention discloses a glucose biosensor and a preparation method and application thereof, wherein the preparation method of the glucose biosensor comprises the following steps: A) and (3) preparing a triblock polymer: B) and coating the triblock polymer. The invention uses the triblock polymer containing the biocompatible group to prepare the permselectivity membrane, has very small temperature coefficient, can effectively and accurately regulate and control glucose when being applied to the dynamic glucometer, and utilizes the impedance of the glucose sensor to carry out in-situ temperature correction.

Description

Glucose sensor and preparation method and application thereof

Technical Field

The invention relates to a glucose sensor and a preparation method and application thereof.

Background

With the development of economy and improvement of life, diabetes has become worldwide and cardiovascular and cerebrovascular diseases and cancer become three major diseases affecting human health. Diabetes mellitus is a metabolic disease with hyperglycemia as a typical clinical symptom, and various complications can be caused by long-term hyperglycemia, and even the life of a patient is threatened. Although there is no medical treatment for radically treating diabetes, many years of clinical studies have demonstrated that if diabetic patients can control blood sugar as normal as possible by regulating diet and living habits and assisting drug therapy, the occurrence of complications can be greatly reduced or the risk of complications can be reduced. As one of the main tools for regulating and controlling blood sugar, the blood sugar meter becomes the standard of the diabetics. Particularly, the dynamic blood glucose meter which is rapidly developed in recent years provides a powerful tool for regulating and controlling blood glucose for diabetics, and the real-time and uninterrupted performance of the dynamic blood glucose meter enables the diabetics to regulate and control blood glucose more timely, scientifically, conveniently and effectively.

As the only interface of the dynamic blood glucose meter in direct contact with the human body, the performance of the biocompatible film directly determines the accuracy and the service life of the dynamic blood glucose meter. High hydrophilicity is a basic property of such biocompatible films, so that hydrogels or polymers with hydrogels as the main component are not the choice for such biocompatible films (e.g. US9777307, US9668685, US9014774, US 9042954). Although the glucose can be effectively regulated and controlled, the permeability of the glucose can be greatly influenced by temperature, and the signal of the dynamic blood glucose meter can be changed by 8-10% when the temperature is changed by one degree centigrade, so that the accuracy of blood glucose monitoring is directly influenced, and people have to introduce a temperature correction mechanism. Temperature measurements are not performed in-vivo with glucose sensors, however, and temperature correction based on in-vitro temperature measurements can only improve the accuracy of blood glucose monitoring to a limited extent. In addition, in the existing formulation of hydrogel-based biocompatible membrane, there is a chemical crosslinking reaction, which greatly shortens the service life of the biocompatible membrane solution, and virtually increases the production cost of the dynamic blood glucose meter (for example, US patent No. 9777307, US 9042954), and more seriously, as the service time of the dynamic blood glucose meter increases, the chemical crosslinking reaction increases, and the viscosity of the biocompatible membrane solution also increases, thus seriously affecting the consistency of the product.

Disclosure of Invention

Aiming at the defects in the prior art, the invention uses the triblock polymer containing the biocompatible groups to prepare the permselectivity membrane, and the permselectivity membrane is applied to the dynamic glucometer, so that the glucose can be effectively and accurately regulated.

In order to achieve the purpose, the invention provides the following technical scheme: the preparation method of the glucose sensor comprises the following steps:

A) preparation of triblock polymer:

A1) adding 10-50 parts by weight of hydrophilic group compound and 2-15 parts by weight of biocompatible group compound into an alcohol solvent, and carrying out deoxidization treatment to obtain a reaction solution I;

A2) adding 0.005-0.015 part by weight of cuprous bromide and 0.01-0.04 part by weight of 2, 2' -bipyridyl into the reaction liquid I obtained in the step A1), and reacting under an oxygen-free condition to obtain a reaction liquid II;

A3) adding 50-120 parts by weight of hydrophobic framework compound into the reaction liquid II obtained in the step A2), and continuously reacting under an oxygen-free condition to obtain a triblock polymer;

A4) adding an acetone solvent to precipitate the generated triblock polymer, and separating and drying;

A5) adding an alcohol solvent into the dried triblock polymer for dissolving, simultaneously adding acetone for precipitating again, then separating and drying, and repeating the step for multiple times;

B) coating of triblock polymer:

B1) mixing 0.05-0.4 part by weight of nanogold-treated glucose oxidase and 0.00005-0.001 part by weight of glutaraldehyde, and coating the mixed solution on the surface of a printing electrode containing aminated graphite for culture to obtain a glucose biosensor membrane;

B2) coating the triblock polymer solution prepared in the step A5) on the glucose biosensing membrane to form a biocompatible outer membrane.

Preferably, the hydrophilic group compound comprises brominated polypropylene oxide or brominated polyethylene glycol; the biocompatible group compound includes styrylalanine, amino acids with a vinyl or acetyl group, 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, betaine or vinylpyrrolidone with a vinyl or acetyl group; the hydrophobic framework compound comprises styrene, vinylpyridine, vinyl acetate, acrylate or crotonate.

Preferably, in the step B1), the glucose oxidase is 0.1-0.3 part by weight, and the glutaraldehyde is 0.0001-0.0004 part by weight.

Preferably, in the step B2), the concentration of the triblock polymer is 50-400 mg/ml.

Preferably, the concentration of the triblock polymer is 180-300 mg/ml.

Preferably, in the step B2), the triblock polymer solution further contains 1 to 30% of one or more solutions of polyethylene oxide, a copolymer containing polyethylene oxide, polypropylene oxide, a copolymer containing polypropylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polylactic acid, hyaluronic acid and derivatives thereof, chitosan and derivatives thereof, cellulose and derivatives thereof, and alginic acid and derivatives thereof.

Preferably, in the step B2), one or more than two solutions of polyvinyl pyridine, styrene and vinyl pyridine copolymer, styrene and vinyl pyrrole copolymer and styrene and acrylamide copolymer are also added into the triblock polymer solution.

Preferably, in step B2), the coating method is a drop coating method, a spin coating method, a spray coating method, or a dip-draw method.

The glucose sensor prepared by the preparation method of the glucose sensor is provided.

The glucose sensor is applied to in-situ temperature correction, and the temperature correction is carried out in situ by using the impedance of the glucose sensor.

In conclusion, the invention has the following beneficial effects:

1. the invention uses the triblock polymer containing the biocompatible group to prepare the permselectivity membrane, and applies the permselectivity membrane to the dynamic blood glucose meter, so that the glucose can be effectively and accurately regulated, and meanwhile, the influence of the temperature on the permeability of the glucose is reduced to 2-3%;

2. the invention avoids using chemical cross-linking agent, improves the consistency of the dynamic glucometer, thereby preparing the dynamic glucometer with high accuracy and long service life;

3. the invention also develops an in-situ temperature correction mechanism, and the in-situ correction is carried out on the 2-3% temperature influence, so that the temperature influence is basically eliminated within the range of 35-40 ℃.

Drawings

FIG. 1 is a gel permeation chromatogram of a triblock polymer (1, before formation, 2, 6 hours after addition of styrylalanine, 3, 24 hours after addition of styrene);

FIG. 2A is a graph of the current-glucose concentration response of a glucose sensor in PBS solution;

FIG. 2B is a graph showing the operation of a glucose sensor in PBS;

FIG. 3 is a graph showing the effect of temperature on the response of a glucose sensor in PBS buffer containing 10mmol/L glucose;

FIG. 4 is a graph illustrating a test of the effect of temperature on the impedance of a glucose sensor;

FIG. 5 is a graph of the current-glucose concentration response of a temperature corrected glucose sensor (concentration from 0 to 30mmol/L with a concentration gradient of 5 mmol/L).

Detailed Description

The invention is further described with reference to the accompanying drawings.

In order to improve the accuracy and the service life of dynamic detection of glucose and greatly reduce the cost of a glucose biosensor, we have recently successfully implemented direct electrochemistry of glucose oxidase, i.e., a third-generation biosensing technology (see chinese patent CN 113325058A). Due to the high sensitivity of direct electrochemical glucose detection, a glucose sensor of a dynamic blood glucose meter must be provided with a highly biocompatible permselective membrane, so that glucose can be effectively regulated. More importantly, to ensure the accuracy of the dynamic detection of glucose, the permselective membrane must be temperature-independent or temperature-independent as little as possible while ensuring a high degree of biocompatibility, otherwise the accuracy of the dynamic blood glucose meter is severely limited.

Through detailed research and experiments, the inventors found that when a high molecular material developed based on a triblock polymer containing a biocompatible group is applied to a dynamic blood glucose meter as a permselectivity membrane, glucose can be regulated and controlled very effectively and accurately; meanwhile, the influence of the temperature on the permeability of the composite material can be reduced to 2-3%. In order to further eliminate the influence of temperature, an in-situ temperature correction mechanism is developed, and the influence of temperature is basically eliminated within the range of 35-40 ℃ by carrying out in-situ correction on the influence of the temperature of 2-3%. In addition, the application also avoids using a chemical cross-linking agent, greatly improves the consistency of the dynamic blood glucose meter, and can prepare the dynamic blood glucose meter with high accuracy and long service life. The specific preparation method of the glucose sensor is as follows:

the invention provides a preparation method of a glucose sensor, which comprises the following steps:

A) preparation of triblock polymer:

A1) adding 10-50 parts by weight of hydrophilic group compound and 2-15 parts by weight of biocompatible group compound into a methanol solvent, and carrying out argon deoxidization for 20-60 minutes to obtain a reaction solution I; wherein the hydrophilic group compound comprises brominated polypropylene oxide or brominated polyethylene glycol, and the biocompatible group compound comprises styryl alanine, amino acid with vinyl or acetyl, 3- [ [2- (methacryloyloxy) ethyl ] dimethyl ammonium ] propionate, betaine or vinyl pyrrolidone with vinyl or acetyl;

A2) adding 0.005-0.015 part by weight of cuprous bromide and 0.01-0.04 part by weight of 2, 2' -bipyridyl into the reaction liquid I obtained in the step A1), and reacting at room temperature for 5-10 hours under the protection of argon to obtain a reaction liquid II;

A3) adding 50-120 parts by weight of hydrophobic skeleton compound into the reaction liquid II obtained in the step A2), and reacting at room temperature for 12-36 hours under the protection of argon gas to obtain a triblock polymer; wherein the hydrophobic skeleton compound comprises styrene, vinylpyridine, vinyl acetate, acrylate or crotonate;

A4) adding an acetone solvent to precipitate the generated triblock polymer, and performing centrifugal separation and vacuum drying at the drying temperature of 50-120 ℃ for 12-36 hours;

A5) adding ethanol into the dried triblock polymer for dissolving, simultaneously adding acetone for secondary precipitation, performing centrifugal separation and vacuum drying at the drying temperature of 50-120 ℃ for 12-36 hours, and repeating the steps for multiple times;

A6) finally, drying the triblock polymer obtained in the step A5) for at least 12-36 hours at the temperature of 50-120 ℃ in vacuum;

B) coating of triblock polymer:

B1) adding 0.05-0.4 part by weight of nanogold-treated glucose oxidase and 0.00005-0.001 part by weight of glutaraldehyde into a PBS buffer solution for mixing, coating the mixed solution on the surface of a printing electrode containing aminated graphite by using a dripping coating method after 20-50 minutes, and culturing for 8-16 hours at the temperature of 20-30 ℃ to obtain a biosensing membrane;

B2) coating the ethanol solution of the triblock polymer prepared in the step A6) on the biosensing membrane by a dripping coating method, a spin coating method, a spraying method or a dip-coating method, wherein the concentration of the triblock polymer is 50-400 mg/ml; then, drying at room temperature to form a biocompatible outer membrane, and repeating the steps for multiple times; finally, vacuum drying is carried out for 12 hours at the temperature of 37 ℃, and the glucose sensor is prepared; preferably, the concentration of the triblock polymer is 180-300 mg/ml;

synthesis of the above brominated polypropylene oxide:

dissolving 50-180 g of polypropylene oxide in 100-1000 ml of dichloromethane and 20-80 g of triethylamine, and cooling to 0 ℃; then, dropwise adding 3-15 g of dibromo isobutyryl bromide, and reacting for 12-48 hours at 15-40 ℃; pouring the reaction solution into 1000-5000 ml of ethyl acetate, and fully washing the precipitate with 100-1000 ml of ethyl acetate for three times; meanwhile, vacuum drying is carried out for 12-48 hours at the temperature of 40-80 ℃, so that brominated polypropylene oxide is obtained, and the yield is 85-90%.

In a preferred embodiment, in the step B), the glucose oxidase is 0.1-0.3 part by weight, and the glutaraldehyde is 0.0001-0.0004 part by weight.

Preferably, the biocompatibility function can be further optimized and adjusted by directly adding 1-30% of high molecular weight polymer solution with good hydrophilicity and high biocompatibility, such as polyethylene oxide, copolymer containing polyethylene oxide, polypropylene oxide, copolymer containing polypropylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polylactic acid, hyaluronic acid and its derivatives, chitosan and its derivatives, cellulose and its derivatives, alginic acid and its derivatives, and the like, into the triblock polymer solution.

Preferably, in addition to adjusting the content of the acrylate in the copolymer, 1-25% of polymer solution with excellent mechanical properties, such as polyvinylpyridine, styrene-vinylpyridine copolymer, styrene-vinylpyrrole copolymer, styrene-acrylamide copolymer, etc., can be further added into the triblock polymer solution.

All of the above formulations of biocompatible membranes are based on synthetic and purified polymers, provided that they are dissolved in a suitable solvent, such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, water, N-dimethylacrylamide, dimethylsulfoxide, sulfolane, tetrahydrofuran, dioxane, etc., and the prepared solution can be used indefinitely.

The invention also provides a glucose sensor prepared by the preparation method of the glucose sensor, and the glucose sensor is a temperature-insensitive glucose sensor.

The specific embodiment is as follows:

example S1:

(1) and synthesizing brominated polypropylene oxide:

100g of polypropylene oxide are dissolved in 500ml of dichloromethane and 50g of triethylamine and cooled to 0 ℃; then, 10g of dibromo-isobutyryl bromide is dropwise added to react for 24 hours at the temperature of 25 ℃; the reaction solution was then poured into 2000ml of ethyl glacial ether, and the precipitate was washed thoroughly three times with 1000ml of ethyl glacial ether and simultaneously vacuum-dried at 60 ℃ for 24 hours to give brominated polypropylene oxide in 89% yield.

(2) Synthesis of triblock polymer:

adding 30g of brominated polypropylene oxide and 10g of styrylalanine into 1000ml of methanol, and deoxidizing for 30 minutes by argon; then, 10mg of cuprous bromide and 25mg of 2, 2' -bipyridyl are added, and the mixture reacts for 6 hours at room temperature under the protection of argon; then, 100g of styrene is added, and the mixture reacts for 24 hours at room temperature under the protection of argon; then, 5 liters of acetone is added to precipitate the generated block polymer, and centrifugal separation and vacuum drying are carried out, wherein the drying temperature is 80 ℃, and the drying time is 24 hours; dissolving the dried block polymer in ethanol, adding 5L of acetone for precipitation, performing centrifugal separation again, and performing vacuum drying for 6 times at 80 ℃ for 24 hours; finally, the block polymer was dried under vacuum at 80 ℃ for 24 hours.

Before the addition of styrylalanine, the polymer reaction solution had only brominated polypropylene oxide, which had a gel permeation chromatogram with only one polymer peak, as shown in FIG. 1, curve 1, and which completely disappeared after 6 hours of reaction with styrylalanine, instead of a high molecular weight polymer peak, as shown in FIG. 1, curve 2. Finally, after addition of styrene monomer and reaction at room temperature for 24 hours, this high molecular weight polymer peak also completely disappeared, and was replaced by a higher molecular weight polymer peak, as shown in FIG. 1, curve 3. The above experimental results clearly demonstrate the formation of the triblock polymer.

(3) Coating of triblock polymer:

adding 200mg of nanogold-treated glucose oxidase and 0.2mg of glutaraldehyde into PBS buffer solution for fully mixing, coating the mixed solution on the surface of a printing electrode containing aminated graphite by using a dripping coating method after 30 minutes, and culturing for 12 hours at the temperature of 25 ℃ to obtain a glucose biosensor membrane; then, uniformly coating the synthesized ethanol solution of the triblock polymer with the concentration of 250mg/ml on a glucose biosensing membrane by a dripping coating method, a rotary coating method, a spraying method or a dip-coating method, and drying at room temperature to form a biocompatible outer membrane, wherein the steps are repeated for 4 times; finally, drying in a vacuum drying oven for 12 hours at 37 ℃ to obtain the glucose sensor.

As shown in FIG. 2A and FIG. 2B, the glucose concentration and the current signal of the sensor show a good linear relationship in the concentration range of 0-30 mmol/L, the linear regression coefficient is greater than 0.99, and the clinical requirement on glucose monitoring is completely met.

As shown in FIG. 3, the sensitivity of the glucose sensor increases with increasing temperature, and the temperature coefficient is around (2-3%)/deg.C, which is much smaller than the temperature coefficient (8-10%/deg.C) of other glucose sensors. When the body temperature changes within +/-2 ℃, namely when the body temperature is 34.5-38.5 ℃ (36.5 +/-2 ℃), the maximum error of the glucose sensor caused by the body temperature changes is about 6%, the error is within 15% of the maximum error of clinical requirements, and no obvious influence can be brought to blood sugar detection.

Through further research, the impedance of the glucose sensor, more precisely, the impedance of the triblock polymer of the glucose sensor as a biocompatible outer membrane, is also closely related to the temperature in order to detect the blood glucose more accurately. As shown in FIG. 4, the impedance of the glucose sensor rapidly decreases with increasing temperature, and is in a linear relationship between 30 ℃ and 45 ℃. This linear temperature coefficient can be used to correct the temperature coefficient of sensitivity of the glucose sensor in situ. The specific correction method comprises the following steps: when detecting the current signal of the glucose sensor, the impedance of the glucose sensor is monitored, and the obtained impedance is used for correcting the influence of temperature on blood glucose detection. As shown in FIG. 5, the sensitivity of the glucose sensor remains substantially unchanged between 35 and 40 ℃ after temperature correction.

Examples S2-S5 and comparative example S6:

the process parameters are the same as those of S1, except that the hydrophilic group compound and its amount, the biocompatible group compound and its amount, and the hydrophobic skeleton compound and its amount are adjusted as shown in table 1 below:

TABLE 1

In conclusion, the glucose sensor taking the triblock polymer with the extremely low temperature coefficient as the biocompatible outer membrane assists in-situ temperature correction based on impedance, can effectively eliminate the influence of temperature on the glucose sensor, and realizes accurate measurement of blood sugar, thereby paving a way for manufacturing a user calibration-free dynamic blood glucose meter. In addition, this triblock polymer can also be applied to other biosensors such as uric acid, blood ketone and lactic acid biosensors.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

1. The preparation method of the glucose sensor is characterized by comprising the following steps:

A) preparation of triblock polymer:

B) coating of triblock polymer:

2. The method of claim 1, wherein the hydrophilic group compound comprises brominated polypropylene oxide or brominated polyethylene glycol; the biocompatible group compound includes styrylalanine, amino acids with a vinyl or acetyl group, 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate, betaine or vinylpyrrolidone with a vinyl or acetyl group; the hydrophobic framework compound comprises styrene, vinylpyridine, vinyl acetate, acrylate or crotonate.

3. The method for producing the glucose sensor according to claim 1, wherein in step B1), the glucose oxidase is 0.1 to 0.3 parts and the glutaraldehyde is 0.0001 to 0.0004 parts by weight.

4. The method for producing a glucose sensor according to claim 3, wherein the concentration of the triblock polymer in step B2) is 50 to 400 mg/ml.

5. The method for producing a glucose sensor according to claim 4, wherein the concentration of the triblock polymer is 180 to 300 mg/ml.

6. The method for preparing a glucose sensor according to claim 1, wherein in step B2), the triblock polymer solution further contains 1 to 30% of one or more solutions selected from the group consisting of polyethylene oxide, a copolymer containing polyethylene oxide, polypropylene oxide, a copolymer containing polypropylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polylactic acid, hyaluronic acid and derivatives thereof, chitosan and derivatives thereof, cellulose and derivatives thereof, and alginic acid and derivatives thereof.

7. The method of claim 6, wherein in step B2), the triblock polymer solution further comprises one or more than two of 1-25% of polyvinyl pyridine, styrene-vinyl pyridine copolymer, styrene-vinyl pyrrole copolymer, and styrene-acrylamide copolymer.

8. The method of claim 7, wherein the coating of step B2) comprises drop coating, spin coating, spray coating, or dip-coating.

9. A glucose sensor produced by the method for producing a glucose sensor according to any one of claims 1 to 8.

10. Use of the glucose sensor of claim 9 for in situ temperature correction, using its impedance to perform temperature correction in situ.