CN116381025A - Dynamic glucose sensor enzyme membrane and preparation method thereof - Google Patents
Dynamic glucose sensor enzyme membrane and preparation method thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention relates to the field of electrochemical sensors, in particular to a dynamic glucose sensor enzyme membrane, which comprises the following components: an enzyme, a protective agent, a film forming agent, a first crosslinking agent and a second crosslinking agent; the enzyme is mixed with a protective agent to form a protective solution; the film forming agent reacts with the first cross-linking agent to form a hydrogel solution; the protective solution is mixed with the hydrogel solution to form an enzyme intermediate solution; the enzyme intermediate solution reacts with a second cross-linking agent to form an enzyme solution; the enzyme solution is processed by a coating and drying process to obtain an enzyme film; the enzyme is used for catalyzing glucose, the protective agent is used for protecting the activity of the enzyme, and the film forming agent provides a framework support network of an enzyme film; also relates to a preparation method, which comprises the following steps: s1: preparing phosphate buffer solution, namely PB solution; s2: preparing a glucose oxidase and bovine serum albumin mixed solution, and respectively marking the mixed solution as GOD and BSA; s3: crosslinking polyvinyl alcohol with citric acid to generate a second solution; solves the problems of low strength and poor toughness of the enzyme membrane of single hydrogel.
Description
Technical Field
The invention relates to the field of electrochemical sensors, in particular to a dynamic glucose sensor enzyme membrane and a preparation method thereof.
Background
Diabetes is currently one of the most common chronic diseases in the world with the greatest number of patients, and blood glucose monitoring is the most effective means of diabetes management. Unlike in vitro single-time glucose test strips, the implantable dynamic glucose sensor can be implanted in human tissue fluid to continuously monitor glucose concentration, and more accurately provide glucose control guidance for patients.
The enzyme is the most critical substance of the dynamic glucose sensor, and how to expose the active site to the greatest extent and keep the activity from being damaged is the guarantee that the sensor obtains excellent sensitivity and long-term service. The addition of e.g. bovine serum albumin protectant is a common method for blending with enzymes, but such physical blending in free form has no effective effect on the exposure of the active site, and the free, easily local agglomeration and easily leakage properties result in very weak protection of the enzyme activity. Therefore, scholars have inspired from schiff base crosslinking reaction, and considering that the enzyme is a macromolecule with a plurality of amino groups, carboxyl groups and other functional groups, they immobilize the free enzyme through the crosslinking reaction by adding a crosslinking agent such as glutaraldehyde, and develop the active site of the enzyme as much as possible. However, solutions composed of only the enzyme and the first crosslinking agent are poor in film forming property, and the film is easily damaged to cause a decrease or even a loss of the enzyme activity. At present, besides the protective agent cross-linking agent, hydrogel with excellent hydrophilicity is added as a supporting framework to optimize the film forming performance. However, many researches simply mix the hydrogel into the enzyme solution, and few consideration is given to the fact that the mechanical properties of a single hydrogel skeleton are not enough to maintain the stability and the overall uniformity of the enzyme film, such as the most commonly used single polyvinyl alcohol has strong degradability, and the single polyvinyl alcohol is extremely easy to become fragile and weak in a complex environment of human tissue fluid, so that the enzyme activity is reduced or even fails.
One of the common components of the existing dynamic glucose sensor enzyme membrane comprises enzyme (comprising glucose oxidase or glucose dehydrogenase) and protein protecting agent (such as bovine serum albumin), and the enzyme membrane is formed on a substrate electrode by means of dripping, spraying, spin coating, dipping, lifting and the like after the enzyme membrane and the protein protecting agent are uniformly mixed. This technique has the following disadvantages: 1. because of the lack of polymer network support, the formed enzyme film is uneven, brittle and weak, is extremely easy to crack and break to cause enzyme loss, activity is reduced, and sensor performance is unstable. 2. The enzyme in the system is in a free state, and besides easy loss and leakage, active sites are difficult to be stably exposed and contacted with glucose, so that the response sensitivity of the sensor is low, and the time required for response balance is long.
The second common composition of the dynamic glucose sensor enzyme film comprises enzyme (comprising glucose oxidase or glucose dehydrogenase), protein protective agent (such as bovine serum albumin), enzyme first cross-linking agent and hydrogel film-forming auxiliary agent (such as polyvinyl alcohol), and the enzyme film is formed on the substrate electrode by means of dripping, spraying, dipping, pulling and the like after the enzyme first cross-linking agent and the hydrogel film-forming auxiliary agent are uniformly mixed. This technique has the following disadvantages: 1. although single-use hydrogels have the theoretical feasibility of crosslinking with certain enzyme primary crosslinking agents (such as glutaraldehyde), the reaction is difficult to carry out under the low-temperature and neutral pH conditions which can be borne by enzymes, so that the current enzyme membrane technology is in fact lack of hydrogel crosslinking treatment, and the commonly formed membranes have low strength, poor toughness and easy degradation. On one hand, the possibility that the dynamic glucose sensor damages an enzyme membrane by external force in the process of implanting tissue fluid is high, and the sensitivity and the precision of the sensor are reduced. On the other hand, the dynamic glucose sensor is very easy to become fragile and weak in a complex body fluid environment of human tissue fluid, so that the enzyme activity is reduced or even is invalid, the stability of the sensor is poor, and the service life is shortened. 2. The enzyme is uniformly embedded in the membrane holes to be in an optimal dispersion state, which is most favorable for exposing and maintaining enzyme active sites, while the size and uniformity of hydrogel mesh holes are difficult to control by adopting simple blending operation in the prior art, the enzyme is easy to be mismatched with the enzyme, the risk of enzyme loss is high, and the effective utilization rate is low.
Aiming at the problems of low strength and poor toughness of enzyme membranes containing single hydrogel in the prior art, the invention discloses an enzyme membrane with exposed active sites, long and uniform stability time, high strength and high toughness, which can benefit considerably when being used in a dynamic glucose sensor.
Disclosure of Invention
The invention provides a dynamic glucose sensor enzyme membrane and a preparation method thereof, which solve the problems of low strength and poor toughness of the enzyme membrane of a single hydrogel.
In order to solve the technical problems, the application provides the following technical scheme:
a dynamic glucose sensor enzyme membrane comprising: an enzyme, a protective agent, a film forming agent, a first crosslinking agent and a second crosslinking agent; the enzyme is mixed with a protective agent to form a protective solution; the film forming agent reacts with the first cross-linking agent to form a hydrogel solution; the protective solution is mixed with the hydrogel solution to form an enzyme intermediate solution; the enzyme intermediate solution reacts with a second cross-linking agent to form an enzyme solution; the enzyme solution is processed by a coating and drying process to obtain an enzyme film;
the enzyme is used for catalyzing glucose, the protective agent is used for protecting the activity of the enzyme, and the film forming agent provides a framework support network of an enzyme film; the first crosslinking agent comprises two or more compounds with different molecular chain lengths and flexibilities, and reacts with the film forming agent to improve the mechanical property, pore-forming size and uniformity of the film; the second crosslinking agent serves to immobilize the enzyme and expose the active site of the enzyme.
The basic principle and the beneficial effects of the scheme are that: the dynamic glucose sensor enzyme membrane provided by the invention consists of enzyme, a protective agent, a film forming agent, a first crosslinking agent and a second crosslinking agent. Enzyme 1 is a glucose catalyst and is the most central component of the sensor. The protective agent is a protein molecule, which is used for protecting the activity of the enzyme. The film former is a hydrogel providing a skeletal support network for the enzyme film. The first crosslinking agent comprises two or more compounds with different molecular chain lengths and flexibilities, and reacts with the film forming agent to improve the mechanical properties, pore-forming size and uniformity of the film. The second crosslinking agent reacts with enzyme with amino, carboxyl and hydroxyl, protein protecting agent and film forming agent mainly by Schiff base principle, and further fixes the enzyme and well exposes the active site of the enzyme. Specifically, the preparation of the enzyme solution is performed in four steps: 1. mixing the enzyme 1 with a protective agent 2 to form a protective solution; 2. the film forming agent 3 reacts with the first crosslinking agent 4 to form a hydrogel solution; 3. mixing the protective solution with the hydrogel solution to form an enzyme intermediate solution; 4. the enzyme intermediate solution reacts with the second cross-linking agent to form an enzyme solution. The enzyme solution is further processed to obtain the enzyme membrane.
According to the invention, the strength and toughness of the dynamic glucose enzyme membrane are improved through hydrogel mixing and crosslinking, and the pore structure and size of the membrane are regulated to adapt to the enzyme, so that the enzyme is uniformly and stably fixed in a hydrogel network. The method can not only improve the capability of resisting the external destructive power, but also well expose the active site of the enzyme and keep the active site for a long time, thereby improving the sensitivity, the precision and the service life of the sensor.
Aiming at the problems of low strength and poor toughness of an enzyme membrane containing single hydrogel in the prior art, the enzyme solution disclosed by the invention is added with the hydrogel, and the hydrogel is crosslinked, so that the acting force between molecular chains is enhanced, and the strength and toughness of the membrane are improved;
aiming at the problem that the pore-forming size, structure and uniformity of the enzyme membrane are not easy to control in the prior art, the invention innovatively adopts a mixed crosslinking mode, and flexibly designs the pore size, structure and uniformity of the membrane by means of the difference of the length and flexibility of molecular chains of different first crosslinking agents.
The scheme adopts a mixed crosslinking mode to control the acting force among molecular chains of the film forming agent and the crosslinking density, and the enzyme film with proper film hole size, structure, uniformity and mechanical property is obtained. The enzyme is uniformly fixed in the hydrogel film network without loss and agglomeration, so that the high sensitivity, long-term service life and high precision of the sensor are ensured; the proper strength and toughness of the sensor are maintained, and the damage forces such as uncontrollable extrusion, friction and the like in the process of implantation into a human body and in the process of long-term wearing of a patient are better resisted, so that better stability is obtained.
The scheme improves the strength and toughness of the enzyme membrane and resists random external force damage in the storage, implantation and use processes; the pore-forming structure, the size and the uniformity of the enzyme membrane are regulated, the enzyme is better adapted, and the enzyme is uniformly fixed in the membrane for a long time.
Further, the enzyme is selected from one of glucose oxidase and glucose dehydrogenase, and the mass ratio of the enzyme in the enzyme solution is 1% -15%.
Further, the enzyme was glucose oxidase, and the ratio of the enzyme to the enzyme solution was 5%.
Further, the protective agent is a proteinaceous macromolecule selected from bovine serum albumin in a ratio to enzyme of 0.1:1-10: 1.
The film forming agent is hydrogel with good enzyme compatibility, and is selected from polyvinyl alcohol, polyvinyl alcohol copolymer, polypropylene glycol copolymer, polycaprolactone diol, polycaprolactone triol and other polymers with hydroxyl groups, wherein the mass ratio of the film forming agent to the enzyme solution is 0.1-10%.
Further, the first crosslinking agent is selected from one of boric acid, glutaraldehyde, glyoxal, citric acid, oxalic acid, tannic acid, fumaric acid, maleic anhydride, 2, 3-dimethylmaleic anhydride, 2, 3-diphenylmaleic anhydride, 2, 3-bis (2, 4, 5-trimethyl-3-thienyl) maleic anhydride, bromomaleic anhydride, 2, 3-dibromomaleic anhydride, and phenylsuccinic anhydride.
Further, the second crosslinking agent is a molecule with aldehyde groups, can react with amino groups, carboxyl groups, hydroxyl groups and the like in the enzyme, the protein protective agent and the film forming agent to further fix the enzyme, and is selected from one of glyoxal, glutaraldehyde, glyoxal, 1, 3-butanedialdehyde, terephthalaldehyde, 2, 3-dihydroxyterephthalaldehyde, 2, 4-hexadienal and the like, preferably glutaraldehyde.
The preparation method of the dynamic glucose sensor enzyme membrane comprises the following steps:
s1: preparing phosphate buffer solution, namely PB solution;
s2: preparing a glucose oxidase and bovine serum albumin mixed solution, and respectively marking the mixed solution as GOD and BSA;
s3: crosslinking polyvinyl alcohol with citric acid to generate a second solution;
s4: crosslinking polyvinyl alcohol with fumaric acid to generate a third solution;
s5: mixing polyvinyl alcohol with glucose oxidase, and marking as a fourth solution;
s6: glutaraldehyde cross-links glucose oxidase, bovine serum albumin and polyvinyl alcohol in sequence to obtain enzyme solution;
s7: enzyme liquid coating: repeatedly lifting the needle-shaped substrate electrode in enzyme solution for 5 times by adopting an immersion lifting process, wherein each lifting time is 3min;
s8: enzyme solution drying: and (3) placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 24 hours to obtain the enzyme membrane.
Drawings
FIG. 1 is a diagram showing the structure of a dynamic glucose sensor enzyme membrane.
Detailed Description
The following is a further detailed description of the embodiments:
the labels in the drawings of this specification include: enzyme 1, protective agent 2, film forming agent 3, first cross-linking agent 4 and second cross-linking agent 5.
An embodiment as shown in figure 1 of the drawings,
a dynamic glucose sensor enzyme membrane comprises an enzyme 1, a protective agent 2, a film forming agent 3, a first crosslinking agent 4 and a second crosslinking agent 5. Enzyme 1 is a glucose catalyst and is the most central component of the sensor. The protective agent 2 is a protein molecule for protecting the activity of the enzyme. The film former 3 is a hydrogel providing a skeletal support network for the enzyme film. The first crosslinking agent 4 comprises two or more compounds with different molecular chain lengths and flexibilities, and the first crosslinking agent 4 reacts with the film forming agent 3 to improve the mechanical property, pore-forming size and uniformity of the film. The second crosslinking agent 5 reacts with enzyme with amino, carboxyl and hydroxyl, protein protecting agent and film forming agent mainly by Schiff base principle, and further fixes the enzyme and well exposes the active site of the enzyme. Specifically, the preparation of the enzyme solution is performed in four steps: 1. mixing the enzyme 1 with a protective agent 2 to form a protective solution; 2. the film forming agent 3 reacts with the first crosslinking agent 4 to form a hydrogel solution; 3. mixing the protective solution with the hydrogel solution to form an enzyme intermediate solution; 4. the enzyme intermediate solution reacts with the second cross-linking agent 5 to form an enzyme solution.
the protective agent 2 is a protein macromolecule and has a certain effect on maintaining the enzyme activity. Selected from bovine serum albumin in a ratio to enzyme of 0.1:1-10: within the range of 1, preferably 2:1, a step of;
the film forming agent 3 is hydrogel with good compatibility with enzyme, and provides skeleton support for the enzyme film. Hydroxyl group-containing polymers selected from polyvinyl alcohol, polyvinyl alcohol copolymers, polypropylene glycol copolymers, polycaprolactone diols, polycaprolactone triols, and the like, preferably polyvinyl alcohol and polyvinyl alcohol copolymers, and the mass ratio of the polyvinyl alcohol to the polyvinyl alcohol copolymers in the enzyme solution is 0.1% -10%, preferably 2.5%.
The first crosslinking agent 4 includes two or more compounds having different molecular chain lengths and molecular chain flexibilities so as to have different crosslinking densities with the hydrogel. Selected from boric acid, glutaraldehyde, glyoxal, citric acid, oxalic acid, tannic acid, fumaric acid, maleic anhydride, 2, 3-dimethylmaleic anhydride, 2, 3-diphenylmaleic anhydride, 2, 3-bis (2, 4, 5-trimethyl-3-thienyl) maleic anhydride, bromomaleic anhydride, 2, 3-dibromomaleic anhydride, phenylsuccinic anhydride, and the like.
The second crosslinking agent 5 is a molecule having an aldehyde group, and can react with an enzyme, a protein protecting agent, an amino group, a carboxyl group, a hydroxyl group, or the like in a film-forming agent to further immobilize the enzyme. One selected from glyoxal, glutaraldehyde, adipaldehyde, 1, 3-butanedial, terephthalaldehyde, 2, 3-dihydroxyterephthalaldehyde, 2, 4-hexadienal, etc., preferably glutaraldehyde;
the protecting liquid is a mixed solution of enzyme and protein protecting agent, and can protect the activity of enzyme to a certain extent.
The film forming material is hydrogel, and the hydrogel solution is formed through blending and crosslinking. The first cross-linking agent has different molecular chain lengths and molecular chain flexibilities, and reacts at a certain temperature and under stirring conditions according to the principle of pre-cross-linking with low molecular chain length flexibility and post-cross-linking with high molecular chain length flexibility.
The enzyme intermediate solution is the product of blending the protective solution with the hydrogel solution, wherein the enzyme is well dispersed and embedded in the mesh formed by the hydrogel.
The enzyme intermediate solution reacts with the second crosslinking agent 5 to further lock the enzyme in the meshes of the film forming agent and strengthen the film forming hydrogel.
The preparation method of the dynamic glucose sensor enzyme membrane further comprises the following steps:
s1: phosphate buffer (pH 7.0) was prepared at 0.05 mol/L: 2.76g of sodium dihydrogen phosphate is weighed and dissolved in ultrapure water, and the solution is marked as A solution after being stirred uniformly and the volume is fixed to 100 mL; 7.16g of disodium hydrogen phosphate was weighed and dissolved in ultrapure water, and after stirring uniformly, the volume was fixed to 100mL, and the solution was labeled as solution B. Mixing 9.5mL of A solution and 15.5mL of B solution uniformly, and diluting to 100mL of constant volume by using ultrapure water to obtain 0.05mol/L phosphate buffer solution with pH of 7.0, wherein the phosphate buffer solution is marked as PB solution.
S2: preparing a mixed solution of glucose oxidase (hereinafter referred to as GOD) and bovine serum albumin (hereinafter referred to as BSA): GOD and BSA were removed from the refrigerator freezer and rewarmed at room temperature for 30min, then 0.020g GOD, 0.040g GOD, 0.960g PB were weighed separately and stirred under magnetic stirring at 200rpm for 10min until the solution was yellow clear and transparent, labeled solution 1.
S3: citric acid (hereinafter abbreviated as CA) crosslinked polyvinyl alcohol (hereinafter abbreviated as PVA): 0.4g of PVA was weighed into a single-necked flask containing 20g of ultrapure water. And adopting a condensing reflux mode, heating at 100 ℃ for 2 hours under magnetic stirring at 600rpm, naturally cooling, and dissolving PVA to be in a clear and transparent state. Then, 0.04g of CA was slowly added to the PVA solution with stirring, and heated at 60℃for 2 hours under 600rp magnetic stirring, labeled solution 2.
S4: fumaric acid (hereinafter abbreviated as FA) -crosslinked PVA: solution 2 was warmed to 90℃and then slowly added with stirring with 0.008g of FA, heated at 90℃for 1h with maintaining magnetic stirring at 600rpm and then cooled naturally, labeled solution 3.
S5: the PVA crosslinking solution and GOD are mixed: solution 1 was mixed with solution 3 and treated under magnetic stirring at 37℃and 400rpm for 30min, and the solution was observed to be yellow, clear and transparent, labeled solution 4.
S6: glutaraldehyde (hereinafter referred to as GA) crosslinks GOD, BSA, PVA: 0.05g of GA was weighed and dissolved in 0.95g of PB solution to give a 5% GA solution. And (3) maintaining the magnetic stirring condition at 37 ℃ and 400rpm, dropwise adding 5% GA into the solution 4 while stirring to obtain a solution, and continuously stirring for 1h to obtain the enzyme solution.
S7: enzyme liquid coating: and repeatedly lifting the needle-shaped substrate electrode in the enzyme solution for 5 times by adopting a dipping and lifting process, wherein the lifting interval is 3min each time.
S8: enzyme solution drying: and (3) placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 24 hours to obtain the enzyme membrane.
Example two
The second embodiment is different from the first embodiment in that: the manufacturing method of the dynamic glucose sensor enzyme membrane comprises the following steps:
s1: phosphate buffer (pH 7.0) was prepared at 0.05 mol/L: 2.76g of sodium dihydrogen phosphate is weighed and dissolved in ultrapure water, and the solution is marked as A solution after being stirred uniformly and the volume is fixed to 100 mL; 7.16g of disodium hydrogen phosphate was weighed and dissolved in ultrapure water, and after stirring uniformly, the volume was fixed to 100mL, and the solution was labeled as solution B. Mixing 9.5mL of A solution and 15.5mL of B solution uniformly, and diluting to 100mL of constant volume by using ultrapure water to obtain 0.05mol/L phosphate buffer solution with pH of 7.0, wherein the phosphate buffer solution is marked as PB solution.
S2: preparing a mixed solution of glucose oxidase (hereinafter referred to as GOD) and bovine serum albumin (hereinafter referred to as BSA): GOD and BSA were removed from the refrigerator freezer and rewarmed at room temperature for 30min, then 0.020g GOD, 0.040g GOD, 0.960g PB were weighed separately and stirred under magnetic stirring at 200rpm for 10min until the solution was yellow clear and transparent, labeled solution 1.
S3: maleic acid (hereinafter abbreviated as MA) crosslinked polyvinyl alcohol (hereinafter abbreviated as PVA): 0.4g of PVA was weighed into a single-necked flask containing 20g of ultrapure water. And adopting a condensing reflux mode, heating at 100 ℃ for 2 hours under magnetic stirring at 600rpm, naturally cooling, and dissolving PVA to be in a clear and transparent state. Then, 0.02g of MA was slowly added to the PVA solution with stirring, and heated at 120℃for 2 hours under 600rp magnetic stirring, labeled solution 2.
S4: boric acid (hereinafter abbreviated as BA) crosslinked PVA: solution 2 was cooled to 60℃and then slowly added with stirring with 0.008g BA, and after heating at 60℃for 1h with magnetic stirring at 600rpm, it was cooled naturally and marked as solution 3.
S5: the PVA crosslinking solution and GOD are mixed: solution 1 was mixed with solution 3 and treated under magnetic stirring at 37℃and 400rpm for 30min, and the solution was observed to be yellow, clear and transparent, labeled solution 4.
S6: glutaraldehyde (hereinafter referred to as GA) crosslinks GOD, BSA, PVA: 0.05g of GA was weighed and dissolved in 0.95g of PB solution to give a 5% GA solution. And (3) maintaining the magnetic stirring condition at 37 ℃ and 400rpm, dropwise adding 5% GA into the solution 4 while stirring to obtain a solution, and continuously stirring for 1h to obtain the enzyme solution.
S7: enzyme liquid coating: and repeatedly lifting the needle-shaped substrate electrode in the enzyme solution for 5 times by adopting a dipping and lifting process, wherein the lifting interval is 3min each time.
S8: enzyme solution drying: and (3) placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 24 hours to obtain the enzyme membrane.
And controlling the acting force among molecular chains of the film forming agent and the crosslinking density by adopting a mixed crosslinking mode to obtain the enzyme film with proper film hole size, structure, uniformity and mechanical property. The enzyme is uniformly fixed in the hydrogel film network without loss and agglomeration, so that the high sensitivity, long-term service life and high precision of the sensor are ensured; the proper strength and toughness of the sensor are maintained, and the damage forces such as uncontrollable extrusion, friction and the like in the process of implantation into a human body and in the process of long-term wearing of a patient are better resisted, so that better stability is obtained.
Test scheme and data processing mode:
1) Preparing PB buffer solutions with glucose contents of 2.5mmo/L, 5.0mmo/L, 10.0mmo/L, 20.0mmo/L and 30.0mmo/L respectively, and marking the PB buffer solutions as L1, L2, L3, L4 and L5;
2) Taking the day of the completion of the sensor drying as the starting time point, testing L1, L2, L3, L4 and L5 on days 1,3, 5, 7 and 9 respectively, controlling the indoor temperature to be constant at 25 ℃ (deviation +/-1.5 ℃), the humidity to be constant at 60%RH (deviation +/-3%RH), and repeating the test for 5 times on each buffer solution;
3) Within 9 days of continuous testing, during the non-testing period, the sensor is soaked in the L2 solution to simulate the state of continuous soaking in tissue fluid after the sensor is implanted;
2. response currents of the sensors of examples 1, 2 at different times are shown in table 1:
TABLE 1
3. According to the data of table 2, the measured current is plotted on the abscissa with concentration and the measured current is plotted on the ordinate, and a unitary linear fit is performed to obtain e=a×c+b, a representing the sensitivity of the sensor. The greater the sensitivity and the less the attenuation, the more accurate and long-term stability the sensor can maintain. The sensitivity of the sensors of examples 1, 2 at different times and their attenuation are shown in table 2:
TABLE 2
The mixed crosslinking agent composed of citric acid and fumaric acid used in example 1 was lower in response current in the sensor than the mixed crosslinking agent composed of maleic acid and boric acid used in example 2, and analyzed from the error point of view, and under the same circumstances, the response current measurement accuracy was higher and the error was smaller in example 2 due to the increase in the amount of detection current, i.e., the sensitivity of the sensor in example two was higher as shown in table 2. The sensor in example 1 is more stable, the sensitivity decays more slowly, and a proper cross-linking agent can be selected for matching according to the measuring environment and the requirements.
Example III
Embodiment three differs from embodiment one in that the method for preparing a dynamic glucose sensor enzyme membrane, step S7: enzyme liquid coating: and repeatedly lifting the needle-shaped substrate electrode in the enzyme liquid for 5 times by adopting a dipping and lifting process, and simultaneously carrying out ultrasonic oscillation on the enzyme liquid at each lifting interval of 3 minutes.
In the process of preparing the enzyme film, the uniformity of the enzyme film directly influences the measurement accuracy of the later-stage sensor; in order to prevent uneven thickness of the enzyme film, an electric field is introduced into the enzyme solution storage area before the coating process is carried out, and electrodes of active sites in the enzyme solution are differentiated under the influence of the electric field and are uniformly arranged under the influence of the electrodes, so that repeated accumulation of a plurality of active sites is reduced, and uneven thickness of the enzyme film in the later stage is avoided. In the hydrogel skeleton formed by mixing the cross-linking agent and the active site, the active site can be arranged according to the electrode direction by guiding the hydrogel skeleton, but the active site is deflected at the position (namely, the active site with inconsistent electrode can be deflected) due to the fixation of the cross-linking agent, the skeleton network can be driven to deflect simultaneously, the unification of the electrode orientation of the active site can occur temporarily, when the electric field force disappears at the later stage, the hydrogel skeleton can generate a restoring force, namely, the deflection at the earlier stage of restoration, the thickness of the enzyme film can be locally increased again by the twisted skeleton network, and the uneven condition is caused. The ultrasonic wave is introduced, the connection between the active sites differentiated by the electrodes and the mixed cross-linking agent is broken through microwave oscillation, under the condition that a skeleton network of the broken active sites is unchanged, the electrodes turn towards the non-uniform active sites, after the ultrasonic wave is removed, the active sites keep unchanged in direction under the continuous action of an electric field, are connected with the surrounding skeleton network again to form a skeleton network with the same active site direction, the problem that recovery deflection occurs in the skeleton network is reduced, and the enzyme film is more uniform.
The foregoing is merely an embodiment of the present invention, the present invention is not limited to the field to which this embodiment relates, and specific structures and features well known in the schemes are not described herein too much, so that those of ordinary skill in the art will know all of the prior art in this field and have the ability to apply the conventional experimental means before this date, and those of ordinary skill in the art may, in light of the teaching presented in this application, combine their own abilities to perfect and practice this scheme, and some typical well-known structures or well-known methods should not be an obstacle to practicing this application by those of ordinary skill in the art. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (8)
1. A dynamic glucose sensor enzyme membrane comprising: an enzyme, a protective agent, a film forming agent, a first crosslinking agent and a second crosslinking agent; the enzyme is mixed with a protective agent to form a protective solution; the film forming agent reacts with the first cross-linking agent to form a hydrogel solution; the protective solution is mixed with the hydrogel solution to form an enzyme intermediate solution; the enzyme intermediate solution reacts with a second cross-linking agent to form an enzyme solution; the enzyme solution is processed by a coating and drying process to obtain an enzyme film;
the enzyme is used for catalyzing glucose, the protective agent is used for protecting the activity of the enzyme, and the film forming agent provides a framework support network of an enzyme film; the first crosslinking agent comprises two or more compounds with different molecular chain lengths and flexibilities, and reacts with the film forming agent to improve the mechanical property, pore-forming size and uniformity of the film; the second crosslinking agent serves to immobilize the enzyme and expose the active site of the enzyme.
2. A dynamic glucose sensor enzyme membrane according to claim 1, characterized in that: the enzyme is selected from one of glucose oxidase and glucose dehydrogenase, and the mass ratio of the enzyme in the enzyme liquid is 1% -15%.
3. A dynamic glucose sensor enzyme membrane according to claim 2, characterized in that: the enzyme is glucose oxidase, and the proportion of the enzyme in the enzyme solution is 5%.
4. A dynamic glucose sensor enzyme membrane according to claim 1, characterized in that: the protective agent is a protein macromolecule selected from bovine serum albumin, and the ratio of the protective agent to enzyme is 0.1:1-10: 1.
5. A dynamic glucose sensor enzyme membrane according to claim 1, characterized in that: the film forming agent is hydrogel with good enzyme compatibility, and is selected from polyvinyl alcohol, polyvinyl alcohol copolymer, polypropylene glycol copolymer, polycaprolactone diol, polycaprolactone triol and other polymers with hydroxyl groups, wherein the mass ratio of the film forming agent to the enzyme solution is 0.1-10%.
6. A dynamic glucose sensor enzyme membrane according to claim 1, characterized in that: the first cross-linking agent is selected from one of boric acid, glutaraldehyde, glyoxal, citric acid, oxalic acid, tannic acid, fumaric acid, maleic anhydride, 2, 3-dimethyl maleic anhydride, 2, 3-diphenyl maleic anhydride, 2, 3-bis (2, 4, 5-trimethyl-3-thienyl) maleic anhydride, bromomaleic anhydride, 2, 3-dibromomaleic anhydride and phenylsuccinic anhydride.
7. A dynamic glucose sensor enzyme membrane according to claim 1, characterized in that: the second cross-linking agent is a molecule with aldehyde groups, can react with amino groups, carboxyl groups, hydroxyl groups and the like in enzymes, protein protectants and film forming agents to further immobilize the enzymes, and is selected from one of glyoxal, glutaraldehyde, glyoxal, 1, 3-butanedialdehyde, terephthalaldehyde, 2, 3-dihydroxyterephthalaldehyde, 2, 4-hexadienal and the like, preferably glutaraldehyde.
8. A preparation method of a dynamic glucose sensor enzyme membrane is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing phosphate buffer solution, namely PB solution;
s2: preparing a glucose oxidase and bovine serum albumin mixed solution, and respectively marking the mixed solution as GOD and BSA;
s3: crosslinking polyvinyl alcohol with citric acid to generate a second solution;
s4: crosslinking polyvinyl alcohol with fumaric acid to generate a third solution;
s5: mixing polyvinyl alcohol with glucose oxidase, and marking as a fourth solution;
s6: glutaraldehyde cross-links glucose oxidase, bovine serum albumin and polyvinyl alcohol in sequence to obtain enzyme solution;
s7: enzyme liquid coating: repeatedly lifting the needle-shaped substrate electrode in enzyme solution for 5 times by adopting an immersion lifting process, wherein each lifting time is 3min;
s8: enzyme solution drying: and (3) placing the coated electrode in a constant temperature and humidity box with the temperature of 37 ℃ and the humidity of 60% for 24 hours to obtain the enzyme membrane.
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| CN113584112A (en) * | 2020-04-30 | 2021-11-02 | 浙江荷清柔性电子技术有限公司 | Glucose sensor and enzyme immobilization method thereof |
| US20220151521A1 (en) * | 2020-11-18 | 2022-05-19 | Cercacor Laboratories, Inc. | Glucose sensors and methods of manufacturing |
| CN116490129A (en) * | 2020-10-06 | 2023-07-25 | 赞思健康科技有限公司 | Working wire of continuous biosensor with enzyme immobilization network |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5134057A (en) * | 1988-10-10 | 1992-07-28 | 501 Ppg Biomedical Systems, Inc. | Method of providing a substrate with a layer comprising a polyvinyl based hydrogel and a biochemically active material |
| CN113584112A (en) * | 2020-04-30 | 2021-11-02 | 浙江荷清柔性电子技术有限公司 | Glucose sensor and enzyme immobilization method thereof |
| CN116490129A (en) * | 2020-10-06 | 2023-07-25 | 赞思健康科技有限公司 | Working wire of continuous biosensor with enzyme immobilization network |
| US20220151521A1 (en) * | 2020-11-18 | 2022-05-19 | Cercacor Laboratories, Inc. | Glucose sensors and methods of manufacturing |
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