CN113325058A - Implantable glucose biosensor and preparation method thereof - Google Patents
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Abstract
The invention provides an implantable glucose biosensor and a preparation method thereof, belonging to the field of blood sugar testing. The implanted glucose biosensor comprises a functionalized substrate electrode and a glucose sensing membrane consisting of electrochemically activated glucose oxidase and a conductive nano material. The implanted glucose biosensor prepared by the invention is used for detecting blood sugar, improves the sensitivity, accuracy, stability, specificity and anti-interference capability of dynamic detection of glucose, prolongs the service life of a dynamic glucometer, and greatly reduces the cost of the glucose biosensor.
Description
Technical Field
The invention belongs to the field of blood sugar testing technology, and particularly relates to an implantable glucose biosensor and a preparation method thereof.
Background
As a core component of a dynamic glucose meter, the performance of a glucose biosensor directly determines the performance and lifetime of the dynamic glucose meter. Biosensors used in existing ambulatory glucose meters have been developed based on first or second generation biosensing technologies. For example, Dexcom G4, G5, and G6 of Dekang all use the first generation biosensing technology to monitor glucose, which indirectly monitors glucose by electrochemically detecting hydrogen peroxide generated during the oxidation of glucose. Because the electrochemical method for detecting hydrogen peroxide has very strict requirements on the electrode, only a few materials such as platinum, platinum alloy and the like can be used for manufacturing the sensor of the dynamic blood glucose meter, so that the cost of the sensor of the dynamic blood glucose meter is greatly increased. In addition, the electrochemical detection of the hydrogen peroxide requires a higher detection potential, so that the anti-interference capability of the dynamic blood glucose meter is greatly reduced. The dynamic glucometer developed based on the second generation biosensing technology is FreeStyle library for yapeh diabetes care. The second generation of biosensing technology is to realize direct electrochemical detection of glucose by introducing a redox mediator-redox polymer into a biosensing membrane. Through the molecular design and optimization of the redox mediator, the detection of glucose can be realized at a lower potential, so that the anti-interference capability of the dynamic glucometer is greatly improved. Since such glucose monitoring systems provide direct electrochemical detection of glucose by a redox mediator, the sensitivity is significantly improved. However, since the redox mediator is a high molecular material, the preparation of the redox mediator is difficult to be accurately controlled, and uncertainty is brought to the performance of the dynamic blood glucose meter.
In order to overcome the defects of the first and second generation biological sensing technologies, the sensitivity, accuracy, stability, specificity and anti-interference capability of glucose dynamic detection are improved, the service life of a dynamic glucometer is prolonged, and the cost of a glucose biosensor is greatly reduced. The ultra-long-life glucose biosensors of the third generation biosensing technology have been developed in response.
Disclosure of Invention
In order to solve the technical problems, the invention provides a third-generation biosensing technology, and the glucose biosensor prepared by the method not only can be used for manufacturing a high-performance glucose biosensor urgently needed by a dynamic glucometer, but also can be applied to other fields such as food industry and the like. In addition, various biosensors containing oxidoreductase can also be manufactured based on this technique.
An implantable glucose biosensor comprising a functionalized substrate electrode; and a glucose sensing membrane consisting of electrochemically activated glucose oxidase and a conductive nanomaterial; the functionalized matrix electrode comprises an aminated carbon electrode, wherein the aminated carbon electrode comprises a printed electrode containing aminated graphite, a printed electrode of aminated carbon nanotubes and an aminated graphene printed electrode, and the functionalized matrix electrode is combined with the electrochemically activated glucose oxidase and the conductive nanomaterial through a chemical cross-linking agent.
Further, the glucose sensing membrane consisting of the electrochemically activated glucose oxidase and the conductive nano material is prepared by the following method:
s1: fully mixing a metal complex with free amino with glucose oxidase, then sequentially adding carbodiimide and N-hydroxysuccinimide, fully mixing, reacting at 4 ℃ for 12-48h, dialyzing and cutting reaction liquid, and purifying to obtain modified glucose oxidase 1 with the molecular weight of 1000-30000D;
s2: mixing modified glucose oxidase 1 in S1 with 0.1-1g/mL sodium periodate solution at 20-30 ℃ for reaction for 1-5h, dialyzing and cutting reaction liquid with molecular weight of 1000-30000D, separating and purifying, adding 0.1-5mg/mL gold with free amino group and particle size of 10-100nm, fully mixing, reacting at 4 ℃ for 2-12h, adding 1-10mg/mL sodium borohydride into the solution, mixing and reacting at 4 ℃ for 1-4h, dialyzing and cutting the reaction liquid, and purifying to obtain modified glucose oxidase 2 with molecular weight of 1000-30000D;
s3: and mixing the modified glucose oxidase 2 prepared in the S2 in a PBS buffer solution and a 0.1-5% cross-linking agent solution for reaction to obtain the glucose sensing membrane containing the electrochemically activated glucose oxidase and the conductive nano material.
Further, in the metal complex in S1, the metal is at least one of copper, cobalt, iron, nickel, ruthenium, and osmium.
Further, the concentration of the metal complex of free amino in S1 is 1-10 mg/mL; the concentration of the glucose oxidase is as follows: 1-5 mg/mL; the concentration of the carbodiimide is 1-10 mmol/; the concentration of N-hydroxysuccinimide is 0.01-0.1 mmol/L.
Further, in S3, the crosslinking agent is at least one of glutaraldehyde, 1, 4-butanediol diglycidyl ether, poly (dimethylsiloxane) diglycidyl ether, tetracyclooxypropyl-4, 4-diaminodiphenylmethane, polyethylene glycol diglycidyl ether, 4- (2, 3-epoxypropoxy) -N, N-bis (2, 3-epoxypropyl) aniline, epichlorohydrin, N-methylenebisacrylamide, acetic anhydride, diglycidyl ether, or methyl suberanilate.
Further, the conductive nanomaterial includes any one of a metal nanomaterial, a conductive glass nanomaterial, a conductive polymer, nanocarbon, a carbon nanotube, fullerene, or graphene.
Further, the metal nano material is nano gold; the conductive glass nano material comprises at least one of ITO, FTO or AZO.
A method of making an implantable glucose biosensor, comprising:
(1) coating a glucose sensing membrane consisting of electrochemically activated glucose oxidase and a conductive nano material on a functionalized substrate electrode, and reacting for 5-12h at 20-30 ℃ to obtain the glucose biological sensing membrane;
(2) uniformly coating an ethanol solution of the acrylate copolymer on a glucose biosensor membrane, drying at room temperature, repeating the step for 2-6 times, and finally obtaining the implantable glucose biosensor; the coating method is any one of a dripping coating method, a spraying method, a spin coating method or a dip-coating method.
Further, the ethanol solution of the acrylate copolymer in the step (2) is prepared by the following preparation method: mixing hydrophilic monomer, hydrophobic monomer, absolute ethyl alcohol and water, removing oxygen by argon gas, then adding Na2S2O8Reacting at 50-75 deg.C for 12-24 hr, adding acetone to precipitate acrylate and vinyl ether copolymer, and solid-liquid separatingTaking the solid phase, finally drying the solid phase at 60-120 ℃ for at least 12h in vacuum, and dissolving the solid phase with ethanol to finally obtain an ethanol solution of the acrylate copolymer.
Further, the hydrophilic monomer comprises at least one of hydroxypropyl acrylate, vinyl ether, vinyl glycol and derivatives thereof or vinyl pyrrolidone; the hydrophobic monomer comprises at least one of methyl methacrylate benzene, ethylene acrylamide and derivatives thereof, and vinylpyridine and derivatives thereof.
Further, the ethanol solution of the acrylate copolymer in the step (2) may be directly added with at least one of highly biocompatible monomers including phosphorylcholine, functionalized polyethylene oxide or functionalized polypropylene oxide; the hydrophilic and highly biocompatible polymer may be added directly to the solution of the acrylate copolymer, and may include at least one of polyvinyl alcohol, polylactic acid, hyaluronic acid and its derivatives, chitosan and its derivatives, cellulose and its derivatives, alginic acid and its derivatives, polyvinyl pyridine, styrene and vinylpyridine copolymers, styrene and vinylpyrrole copolymers, and styrene and acrylamide copolymers.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) according to the invention, by adjusting the composition of the permselective membrane and the proportion of each component, such as the types and the proportions of hydrophobic and hydrophilic components in a polymer molecule, and the proportion of a hydrophobic polymer and a hydrophilic polymer in a biocompatible membrane solution, the regulation and control of oxygen and glucose can be realized simultaneously. Through detailed research and experiments, the purpose can be achieved by covering a film of acrylate copolymer on a biosensor film of electrochemically activated glucose oxidase.
(2) After the glucose biosensor is subjected to electrochemical activation chemical treatment, the glucose oxidase is completely electrochemically activated from inside to outside, and a high-efficiency electron transfer network is formed among glucose oxidase molecules.
(3) The invention discovers that the glucose biosensor is covered with the acrylate copolymer permselective membrane, so that the stability of the glucose biosensor can be obviously improved, meanwhile, the biocompatibility of the glucose biosensor is greatly improved, and further, the service life of the glucose biosensor is greatly prolonged. After 7 days of continuous testing, the current signal decayed by less than 2%, compared with the current signal of the glucose biosensor without the acrylate copolymer permselectivity membrane, which decayed by more than 60% in 7 days of continuous testing.
(4) When the surface of the glucose biosensor is covered with the acrylate copolymer film, compared with a glucose biosensor which is not covered with any film, the monitorable range of glucose is successfully expanded from 10mmol/L to 40mmol/L, the working curve of the glucose biosensor is good in linearity between 0 and 40mmol/L, the response time of the glucose biosensor to glucose is 2-3min, the glucose biosensor is a continuous glucose monitoring system with the widest linear range at present, and the glucose monitoring requirement of a diabetic patient is completely met. The current signal of the glucose is well regulated and controlled by the layer of the biocompatible film while the monitorable range of the glucose is widened. Since the detection of glucose is performed at a very low potential (-50mV to 100mV), the anti-interference ability against drugs such as acetaminophen is very significantly improved.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a cyclic voltammogram of modified glucose oxidase 1 after electrochemical activation in a test example of the present invention; wherein, curve 1 represents the cyclic voltammogram of the electrochemically activated modified glucose oxidase 1 in the PBS buffer solution, and curve 2 represents the cyclic voltammogram of the electrochemically activated modified glucose oxidase 1 added with 5mol/L glucose.
FIG. 2 is a schematic diagram showing the electron transfer pathway of the modified glucose oxidase 2 treated with nanogold according to the invention.
FIG. 3 is a cyclic voltammogram of a test example of the present invention containing 10-100nm of nanogold-treated electrochemically activated glucose oxidase 2; wherein, the curve 1 is a cyclic voltammogram of the glucose biosensor containing 10-100nm of nanogold-treated glucose oxidase in a PBS buffer solution, and the curve 2 is a cyclic voltammogram of the glucose biosensor containing 10-100nm of nanogold-treated glucose oxidase added with 10mmol/L glucose.
FIG. 4 is a glucose concentration-current curve of a glucose biosensor coated with an acrylate copolymer film according to a test example of the present invention, detection potential: 0.05V (silver/silver chloride reference electrode).
FIG. 5 is the stability in a test example of the present invention of (1) a glucose biosensor without an acrylate copolymer permselective membrane and (2) a glucose biosensor covered with an acrylate copolymer permselective membrane and a PBS buffer solution containing 20mmol/L glucose. And (3) detecting the potential: 0.05V (silver/silver chloride reference electrode).
FIG. 6 shows the results of an experiment for implanting a glucose biosensor containing electrochemically activated glucose oxidase according to a test example of the present invention; the circles in the figure indicate blood glucose measurements.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
Step 1: 5mg/mL osmium-bipyridyl complex having a free amino group and 5mg/mL glucose oxidase were thoroughly mixed, and then 5 mmol/L carbodiimide and 0.1mmol/L N-hydroxysuccinimide were added in this order, and after thorough mixing, the mixture was reacted at 4 ℃ for 12 hours. Then dialyzing by using a super filter bag, and separating and purifying to obtain electrochemically activated glucose oxidase 1; the cut molecular weight of the dialysis bag is 30000D.
Step 2: after the purified modified glucose oxidase 1 was cultured at 20 ℃ for 2.5 hours in a sodium periodate solution containing 0.5g/mL, glucose oxidase having an aldehyde-based sugar molecule was separated and purified by dialysis using an ultrafiltration bag (cut molecular weight: 30000D). Then adding 0.1mg/mL of nanogold with 10-100 nanometers of free amino groups into the purified glucose oxidase solution, reacting for 5 hours at 4 ℃ after fully mixing, then adding 5mg/mL of sodium borohydride into the solution, reacting for 2.5 hours at 4 ℃ after fully mixing, and after the reaction is finished, separating and purifying the modified glucose oxidase by using ultrafiltration bag dialysis (cutting molecular weight: 30000D) again to obtain nanogold modified electrochemically activated glucose oxidase 2;
and step 3: adding 1% glutaraldehyde into nanogold modified electrochemically activated glucose oxidase 2, coating the mixture on a printing electrode of aminated graphite, and reacting at 25 ℃ for 8 hours to obtain a glucose biosensor membrane; and then uniformly coating and dripping an ethanol solution of the acrylate copolymer on the glucose biosensor membrane by adopting a coating method, drying at room temperature, repeating the step for 3 times, and finally obtaining the implanted glucose biosensor.
The preparation method of the ethanol solution of the acrylate copolymer comprises the following steps: a, acrylate copolymer: 150mL of vinyl ether, 50mL of methyl methacrylate, 300mL of absolute ethanol and 15mL of water, and deoxygenated with argon for 40 min. Then 500mg of Na was added2S2O8And placing the mixture in a closed container to react for 12 hours at 50 ℃. The acrylate and vinyl ether copolymers were then precipitated by adding 500mL of acetone and separated by centrifugation. Dissolving in ethanol, adding 500mL of acetone for precipitation, and centrifuging again. Repeating the steps for 3 times, and finally drying the precipitate at 60 ℃ for 12 hours in vacuum.
Example 2
Step 1: 1mg/mL of osmium-biimidazole complex having a free amino group was thoroughly mixed with 1mg/mL of glucose oxidase, and then 1mmol/L of carbodiimide and 0.01 mmol/L of N-hydroxysuccinimide were added in this order, and after thorough mixing, the mixture was reacted at 4 ℃ for 12 hours. Then dialyzing by using a super filter bag (cutting molecular weight: 30000D), and separating and purifying to obtain the modified glucose oxidase 1 after electrochemical activation.
Step 2: after the purified modified glucose oxidase 1 was cultured at 30 ℃ for 1 hour in a sodium periodate solution containing 0.1g/mL, glucose oxidase having an aldehyde-based sugar molecule was separated and purified by dialysis using an ultrafiltration bag (cut molecular weight: 1000-30000D). Then adding 0.1mg/mL of 10nm nanogold with free amino groups into the purified glucose oxidase solution, reacting for 2h at 4 ℃ after fully mixing, then adding 1mg/mL of sodium borohydride into the solution, reacting for 1h at 4 ℃ after fully mixing, and after the reaction is finished, separating and purifying the modified glucose oxidase by using an ultrafiltration bag dialysis (cutting molecular weight: 30000D) to obtain the nanogold material modified glucose oxidase 2.
And step 3: mixing the nanogold material modified glucose oxidase 2 and 10% of 1, 4-butanediol diglycidyl ether solution, coating the mixture on a printing electrode of aminated graphite, and reacting for 5 hours at the temperature of 20 ℃ to obtain the glucose biosensor film; and then uniformly coating and dripping an ethanol solution of the acrylate copolymer on the glucose biosensor membrane by adopting a spraying method, drying at room temperature, repeating the step for 3 times, and finally obtaining the implanted glucose biosensor.
The preparation method of the ethanol solution of the acrylate copolymer comprises the following steps: acrylate copolymer: 20mL of hydrophilic vinylpyrrolidone, 50mL of methylmethacrylate benzene, 100mL of absolute ethanol and 5mL of water, and deoxygenated with argon for 20 min. Then 50mg of Na was added2S2O8And placing the mixture in a closed container to react for 16 hours at 50 ℃. The copolymer was then precipitated by adding 500mL of acetone and centrifuged. Dissolving in ethanol, adding 500mL of acetone for precipitation, and centrifuging again. Repeated for 4 times, and finally the precipitate is dried in vacuum at 100 ℃ for 18 h.
Example 3
Step 1: fully mixing 10mg/mL cobalt ammonia complex with free amino and 5mg/mL glucose oxidase, then sequentially adding 10mmol/L carbodiimide and 0.1mmol/L N-hydroxysuccinimide, fully mixing, and reacting at 4 ℃ for 48 h. Then dialyzing by using a super filter bag (cutting molecular weight: 30000D), and separating and purifying to obtain the modified glucose oxidase 1 after electrochemical activation.
Step 2: after culturing the purified modified glucose oxidase 1 in a sodium periodate solution containing 1g/mL at 30 ℃ for 5 hours, separating and purifying the glucose oxidase with the aldehyde-based sugar molecules by using ultrafiltration bag dialysis (cut molecular weight: 30000D). Then adding 5mg/mL of 100nm nanogold with free amino groups into the purified glucose oxidase solution, reacting for 12h at 4 ℃ after fully mixing, then adding 10mg/mL of sodium borohydride into the solution, reacting for 1h at 4 ℃ after fully mixing, and separating and purifying the modified glucose oxidase by using an ultrafiltration bag dialysis (cutting molecular weight: 30000D) after the reaction is finished to obtain the modified glucose oxidase 2.
And step 3: adding 1% glutaraldehyde into the modified glucose oxidase 2, coating the modified glucose oxidase on a printing electrode of an aminated carbon nanotube, and reacting for 12 hours at the temperature of 30 ℃ to obtain a glucose biosensor film; and then uniformly coating and dripping an ethanol solution of the acrylate copolymer on the glucose biosensor membrane by adopting a dip-coating and pulling method, drying at room temperature, repeating the step for 6 times, and finally obtaining the implanted glucose biosensor.
The preparation method of the ethanol solution of the acrylate copolymer comprises the following steps: acrylate copolymer: 300mL of vinylpyrrolidone, 100mL of styrene, 600mL of absolute ethanol and 30mL of water, and deoxygenated with argon for 60 min. Then 1000mg of Na was added2S2O8And placing the mixture in a closed container to react for 24 hours at the temperature of 75 ℃. Then 5000mL of acetone was added to precipitate the acrylate and vinyl ether copolymers and centrifuged. Dissolving with ethanol, adding 5000mL of acetone for precipitation, and centrifuging again. Repeating the steps for 3 times, and finally drying the precipitate at 120 ℃ for 20 hours in vacuum.
Examples 4 to 10
Experimental operation is the same as example 1, except that in examples 4-10, the nano-gold in step 2 is replaced by fullerene, conductive glass nanomaterial ITO, conductive polymer, nano-carbon tube, fullerene, and graphene.
Examples 11 to 18
The experimental procedure is the same as in example 1 except that in examples 11-18, phosphorylcholine, functionalized polypropylene oxide, polyvinyl alcohol, hyaluronic acid, chitosan, styrene and vinylpyridine copolymer, styrene and vinylpyrrole copolymer, and styrene and acrylamide copolymer are added to the ethanol solution of acrylate copolymer, respectively.
Test example
To verify the feasibility of the scheme in example 1 of the present invention, the following tests were performed:
(1) verifying whether the electrochemical performance micromolecules in the modified glucose oxidase 1 after electrochemical activation obtained in the step 1 are successfully bonded to the glucose oxidase, and testing as follows:
the modified electrochemically activated modified glucose oxidase 1 was first characterized using cyclic voltammetry (see FIG. 1, curve 1). It is clear from curve 1 in fig. 1 that small molecules with superior electrochemical properties have been successfully bonded to glucose oxidase after the electrochemical modification treatment. In contrast, glucose oxidase treated as described above does not have any electrochemical activity in the absence of carbodiimide/N-hydroxysuccinimide chemical cross-linking agents.
(2) Verifying that the electrochemical treatment of the modified glucose oxidase 1 obtained after the electrochemical activation in the step 1 does not have obvious influence on the catalytic activity center of the modified glucose oxidase 1.
The catalytic activity of the modified glucose oxidase 1 after electrochemical activation was thus evaluated. Therefore, the result of adding glucose to the PBS buffer solution containing the electrochemically activated modified glucose oxidase 1 and then characterizing the electrochemically activated modified glucose oxidase 1 by cyclic voltammetry is shown in curve 2 of FIG. 1, and the cyclic voltammogram of the electrochemically activated modified glucose oxidase 1 after adding glucose clearly shows a typical electrochemical catalytic process. Further experiments show that the modified glucose oxidase 1 after electrochemical activation not only maintains the catalytic oxidation performance of the modified glucose oxidase on glucose, but also improves the catalytic oxidation efficiency of the modified glucose oxidase on glucose by two orders of magnitude compared with the catalytic oxidation efficiency of natural glucose oxidase on glucose through oxygen.
The experiment results show that through the electrochemical activation treatment, an electronic channel is established from inside to outside in the glucose oxidase from the catalytic active center of the glucose oxidase to the surface of the glucose oxidase, the catalytic active center of the glucose oxidase can directly carry out very rapid electronic exchange with an electrode, and the direct electrochemistry of the glucose oxidase is successfully realized.
(2) And (4) verifying the performance of the nanogold material modified glucose oxidase obtained in the step (3).
We found that the chemically crosslinked nanogold-modified glucose oxidase still maintains their direct electrochemistry, and the nanogold-modified glucose oxidase shows good electrochemical performance on an electrode and is a typical surface electrochemical phenomenon (the peak potential difference is far less than 59mV) (see the result in FIG. 3, curve 1). When 10mmol/L glucose is added into PBS buffer solution, the cyclic voltammogram of the glucose biosensor membrane clearly shows a typical electrochemical catalysis process (FIG. 3, curve 2) similar to the behavior of the electrochemically activated glucose oxidase in the solution. The experiment results prove that the chemical activation and crosslinking do not have obvious influence on the electrochemically activated glucose oxidase, so that a way is laid for the application of the electrochemically activated glucose oxidase in a dynamic glucometer.
(3) The influence of the acrylate copolymer film coated on the surface of the glucose biosensing membrane in example 1 on the glucose biosensing performance is verified:
the experimental results are shown in FIG. 4, and it can be seen from FIG. 4 that: in FIG. 4, curve 1 represents a glucose biosensor whose surface is covered with an acrylate copolymer film, and curve 2 represents a glucose biosensor whose surface is not covered with an acrylate copolymer film; when the surface of the glucose biosensor is covered with the acrylate copolymer film, compared with the glucose biosensor which is not covered with any film, the monitorable range of glucose is successfully expanded from 10mmol/L to 40mmol/L, the working curve of the glucose biosensor is good linear between 1.0 and 40mmol/L, the response time of the glucose biosensor to glucose is 2-3 minutes, and the glucose biosensor is a continuous glucose monitoring system with the widest linear range at present and completely meets the glucose monitoring requirement of a diabetic patient. The current signal of the glucose is well regulated and controlled by the layer of the biocompatible film while the monitorable range of the glucose is widened. Since the detection of glucose is performed at a very low potential (-50-100mV), the anti-interference ability against drugs such as acetaminophen is very significantly improved.
The stability test result of the glucose biosensor is shown in FIG. 5, and it can be seen from the figure that the current signal is only attenuated by less than 2% after 7-day continuous test experiments (FIG. 5, curve 2); in contrast, the current signal of the glucose biosensor without the acrylate copolymer permselectivity membrane was attenuated by more than 60% in the continuous test over 7 days (fig. 5, curve 1). Therefore, the glucose biosensor film is covered with the acrylate copolymer permselectivity membrane, so that the stability of the glucose biosensor film can be obviously improved, meanwhile, the biocompatibility of the glucose biosensor is greatly improved, and the service life of the glucose biosensor is greatly prolonged.
(4) The glucose biosensor in example 1 of the present invention was applied to a dynamic blood glucose meter:
the results are shown in FIG. 6, from which FIG. 6 shows: the biocompatibility and stability of the glucose biosensor are remarkably improved, and the sensitivity does not obviously change in human body tests for 20 consecutive days. This is the longest working life glucose biosensor used for human monitoring so far, and more importantly, the monitored glucose concentration is highly consistent with the result of blood glucose detection (circles in the figure).
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. An implantable glucose biosensor, comprising: the biosensor comprises a functionalized substrate electrode; and a glucose sensing membrane consisting of electrochemically activated glucose oxidase and a conductive nanomaterial; the functionalized substrate electrode comprises an aminated carbon electrode, wherein the aminated carbon electrode is a printing electrode containing aminated graphite, a printing electrode of aminated carbon nanotubes, and an aminated graphene printing electrode; the functionalized matrix electrode is combined with the electrochemically activated glucose oxidase and the conductive nano material through a chemical cross-linking agent.
2. The implantable glucose biosensor of claim 1, wherein: the glucose sensing membrane consisting of the electrochemically activated glucose oxidase and the conductive nano material is prepared by the following method:
s1: fully mixing a metal complex with free amino with glucose oxidase, then sequentially adding carbodiimide and N-hydroxysuccinimide, fully mixing, reacting at 4 ℃ for 12-48h, dialyzing and cutting reaction liquid, and purifying to obtain modified glucose oxidase 1 with the molecular weight of 1000-30000D;
s2: mixing modified glucose oxidase 1 in S1 with 0.1-1g/mL sodium periodate solution at 20-30 ℃ for reaction for 1-5h, dialyzing and cutting reaction liquid with molecular weight of 1000-30000D, separating and purifying, adding 0.1-5mg/mL nanogold with free amino group and particle size of 10-100nm, fully mixing, reacting at 4 ℃ for 2-12h, adding 1-10mg/mL sodium borohydride into the solution, mixing and reacting at 4 ℃ for 1-4h, dialyzing and cutting the reaction liquid, and purifying to obtain modified glucose oxidase 2 with molecular weight of 1000-30000D;
s3: and mixing the modified glucose oxidase 2 prepared in the S2 in a PBS buffer solution and a 0.1-5% cross-linking agent solution for reaction to obtain the glucose sensing membrane of the electrochemically activated glucose oxidase and the conductive nano material.
3. The implantable glucose biosensor of claim 2, wherein: the metal in the metal complex in S1 is at least one of copper, cobalt, iron, nickel, ruthenium or osmium.
4. The implantable glucose biosensor of claim 2, wherein: the concentration of the metal complex of the free amino in S1 is 1-10 mg/mL; the concentration of the glucose oxidase is as follows: 1-5 mg/mL; the concentration of the carbodiimide is 1-10 mmol/; the concentration of N-hydroxysuccinimide is 0.01-0.1 mmol/L.
5. The implantable glucose biosensor of claim 2, wherein: the cross-linking agent in S3 is at least one of glutaraldehyde, 1, 4-butanediol diglycidyl ether, poly (dimethylsiloxane) -diglycidyl ether, tetracyclooxypropyl-4, 4-diaminodiphenylmethane, polyethylene glycol diglycidyl ether, 4- (2, 3-epoxypropoxy) -N, N-bis (2, 3-epoxypropyl) aniline, epichlorohydrin, N-methylenebisacrylamide, acetic anhydride, diglycidyl ether, or methyl suberanilate.
6. The implantable glucose biosensor of claim 1, wherein: the conductive nano material comprises any one of metal nano materials, conductive glass nano materials, conductive polymers, nano carbon, carbon nano tubes, fullerene or graphene.
7. The implantable glucose biosensor of claim 1, wherein: the metal nano material is nano gold; the conductive glass nano material comprises at least one of ITO, FTO or AZO.
8. A method of making an implantable glucose biosensor according to any one of claims 1-7, wherein: the method comprises the following steps:
(1) coating a glucose sensing membrane consisting of electrochemically activated glucose oxidase and a conductive nano material on a functionalized substrate electrode, and reacting for 5-12h at 20-30 ℃ to obtain the glucose biological sensing membrane;
(2) uniformly coating an ethanol solution of the acrylate copolymer on a glucose biosensor membrane, drying at room temperature, repeating the step for 2-6 times, and finally obtaining the implantable glucose biosensor; the coating method is any one of a dripping coating method, a spraying method, a spin coating method or a dip-coating method.
9. The method of claim 8, wherein the implantable glucose biosensor is prepared by: the ethanol solution of the acrylate copolymer in the step (2) is obtained by the following preparation method: mixing hydrophilic monomer, hydrophobic monomer, absolute ethyl alcohol and water, removing oxygen by argon gas, then adding Na2S2O8Reacting at 50-75 ℃ for 12-24h, adding acetone to precipitate the copolymer of acrylic ester and vinyl ether after the reaction is finished, carrying out solid-liquid separation to obtain a solid phase, finally carrying out vacuum drying on the solid phase at 60-120 ℃ for at least 12h, and dissolving with ethanol to finally obtain an ethanol solution of the acrylic ester copolymer.
10. The method of claim 9, wherein the implantable glucose biosensor is prepared by: the hydrophilic monomer comprises at least one of hydroxypropyl acrylate, vinyl ether, vinyl glycol and derivatives thereof or vinyl pyrrolidone; the hydrophobic monomer comprises at least one of methyl methacrylate benzene, ethylene acrylamide and derivatives thereof, and vinylpyridine and derivatives thereof.
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