A method for preparing a glucose oxidase sensor having a porous outer membrane

Abstract

The application discloses a preparation method of a glucose oxidase sensor with a porous structure outer membrane, and steps of the method comprise the following steps: a chitosan outer membrane is prepared on the surface of a glucose oxidase sensor assembly; solid particles of salt are added into a pre-solidified PDMS solution, ultrasonic mixing is performed to obtain a salt-PDMS mixed solution, the salt-PDMS mixed solution is dropped into a groove, and air in the groove is extracted by vacuum; then the glucose oxidase sensor assembly with the chitosan outer membrane is immersed into the salt-PDMS mixed solution in the groove, and the glucose oxidase sensor assembly is taken out and solidified repeatedly for multiple times until a PDMS outer membrane with a set thickness is formed on the chitosan membrane layer; and then the salt particles in the PDMS outer membrane are dissolved by deionized water to obtain the glucose oxidase sensor with the porous structure outer membrane. The sensor prepared by the method has chitosan and PDMS with the porous structure as outer membrane materials, the preparation method is simple, and product quality control is convenient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical devices, and particularly relates to a preparation method of a glucose oxidase sensor with a porous outer membrane. BACKGROUND

[0002] Diabetes is a chronic disease, often accompanied by a series of complications such as stroke, blindness or kidney failure, and the disease is so serious that it can lead to the death of a diabetic patient, but good blood glucose control can reduce the harm of complications and greatly improve the survival period of a diabetic patient. In the process of diabetes control and treatment, blood glucose monitoring is very important. In order to correctly manage blood glucose levels and maintain balance with insulin, it is necessary to continuously (or at least frequently) monitor blood glucose, but continuous needle sampling is not only troublesome but also brings pain to patients, making patients prone to fear. With the development of technology, the concept of continuous glucose monitoring system (CGMS) is proposed. CGMS can provide continuous and comprehensive blood glucose information, and the subcutaneous implant needle type amperometric glucose biosensor is the most commonly used sensor in CGMS. The main target biological sample is blood, and interstitial fluid is also the target biological sample for glucose detection.

[0003] The subcutaneous implant needle type amperometric glucose biosensor detects glucose by using an electrochemical enzyme sensor method. The product of the enzymatic reaction of glucose oxidase and glucose acts between the electrodes to detect glucose. The sensor needs to ensure the smooth transmission of reactants such as glucose and oxygen. However, during the implantation process of the subcutaneous implant needle type amperometric glucose biosensor, material-tissue interaction, i.e. so-called biofouling, such as protein / platelet deposition, or attachment of inflammation-related cells, occurs, resulting in unpredictable behavior of the implanted glucose biosensor.

[0004] The outer membrane material is a key factor in realizing the biocompatibility of the glucose biosensor. A good biosensor outer membrane material can improve the compatibility of the tissue interface and reduce the negative impact of foreign body reactions on the sensor. The sensor outer membrane has multiple functions, mainly including: 1. limiting glucose passage; 2. accelerating oxygen mass transfer; 3. improving the biocompatibility of the sensor-tissue interface; 4. protecting the sensor and enhancing the bio-mechanical properties of the sensor. The outer membrane materials on the market currently include polyurethane (PU) and thermoplastic polyurethane (TPU), Nafion, polytetrafluoroethylene (PTFE), polycarbonate (PC), etc. However, these polymer materials are hydrophobic and have a high degree of cross-linking, which limits the uniform diffusion of glucose, oxygen and hydrogen peroxide. At the same time, the performance of PU and Nafion-based membranes is highly dependent on the polymerization conditions, resulting in high variability of sensor performance within a production batch. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a preparation method of a glucose oxidase sensor with a porous outer membrane.

[0006] The technical scheme of the present application is as follows:

[0007] The preparation method comprises the following steps:

[0008] Step S1: drop the chitosan solution onto the surface of the glucose oxidase sensor assembly, dry in a temperature box, form a chitosan outer membrane on the surface of the glucose oxidase sensor assembly, and obtain a glucose oxidase sensor assembly with a chitosan outer membrane;

[0009] Step S2: add the solid particles of salt into the pre-solidified PDMS solution, mix by ultrasonic, obtain a salt-PDMS mixed solution, drop the salt-PDMS mixed solution into a groove, and extract the air in the salt-PDMS mixed solution in the groove by vacuum;

[0010] Step S3: immerse the glucose oxidase sensor assembly with the chitosan outer membrane into the groove, immerse the glucose oxidase sensor assembly with the chitosan outer membrane in the salt-PDMS mixed solution in the groove for a period of time, then take out and solidify, form a PDMS outer membrane on the surface of the sensor; repeat the step until the thickness of the PDMS outer membrane reaches a set value, and obtain a glucose oxidase sensor with a PDMS / chitosan outer membrane;

[0011] Step S4: place the glucose oxidase sensor with the PDMS / chitosan outer membrane into deionized water, dissolve the salt particles in the PDMS outer membrane, and obtain a glucose oxidase sensor with a porous outer membrane.

[0012] Further, in an embodiment, the configuration method of the chitosan solution is as follows:

[0013] Add chitosan powder into 0.1 mmol of acetic acid, stir to dissolve the chitosan powder, and configure a 0.5% chitosan solution.

[0014] Further, in an embodiment, the salt in the salt-PDMS mixed solution is manganese salt, and the pore size of the porous structure of the PDMS outer membrane is the same as the particle size of the solid particles of the manganese salt.

[0015] Further, in an embodiment, the configuration method of the pre-solidified PDMS solution is as follows:

[0016] Mix dimethyl silicone oil and a curing agent uniformly at a weight ratio of 10:1 to obtain the pre-solidified PDMS solution, wherein the curing agent is dibutyl phthalate.

[0017] Further, in an embodiment, the configuration method of the pre-solidified PDMS solution is as follows:

[0018] Dimethyl silicone oil and curing agent are mixed evenly at a volume ratio of 10:1 to obtain the pre-cured PDMS solution, wherein the curing agent is dibutyl phthalate.

[0019] Furthermore, in one embodiment, the glucose oxidase sensor assembly includes an enzyme-coated electrode and an Ag / AgCl reference electrode.

[0020] Furthermore, in one embodiment, step S3 is repeated 5 times, and the sensor is immersed in the salt-PDMS mixture for 3 seconds each time.

[0021] Furthermore, in one embodiment, the PDMS outer film is cured at 37°C for 2 hours.

[0022] Furthermore, in one embodiment, in step S1, a micropipette is used to aspirate the chitosan solution to drop the chitosan solution onto the surface of the glucose oxidase sensor assembly, and the incubator drying temperature is 25°C.

[0023] Furthermore, in one embodiment, the preparation method further includes step S5 after step S4.

[0024] Step S5: Dry the glucose oxidase sensor with the porous outer membrane at room temperature, then transfer it to phosphate buffer and store it at 4°C.

[0025] The beneficial effects of the glucose oxidase sensor with a porous outer membrane prepared according to the above-described scheme are as follows:

[0026] 1) such as Figure 1 As shown in the electron microscopy image of the PDMS outer membrane, the PDMS prepared by the method of this application has a porous structure with uniform pore size. The porous structure of the PDMS outer membrane with uniform pore size is conducive to promoting cell inward growth and facilitating the passage of oxygen, hydrogen peroxide, and glucose, thus ensuring the stability of the sensor operation. After continuous measurement for 14 days after manufacturing, under a constant temperature of 37°C, the sensor showed only a 30% decrease in response current to changes in glucose concentration in a 12mM glucose solution, with a response time of less than 6 seconds and a sensitivity of 148 nA·L·mmol. -1 ·cm -2 Its performance is very similar to the initial performance.

[0027] 2) The method of the application is simple to operate, the pore size of the PDMS outer membrane porous structure depends on the particle size of the solid particles of the selected salt, the porosity of the porous structure depends on the amount of salt, the formation conditions of the outer membrane porous structure are clear and easy to control, the batch-to-batch sensor has high consistency, and the performance test of the prepared sensor has high accuracy, the linear coefficient reaches 0.995, which meets the requirements of in vivo glucose monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 SEM image of a glucose oxidase sensor with a porous structure outer membrane obtained by an embodiment of the application;

[0029] Figure 2 C-V graph of a glucose oxidase sensor with a porous structure outer membrane obtained by an embodiment of the application;

[0030] Figure 3 I-T characteristic graph of a glucose oxidase sensor with a porous structure outer membrane obtained by an embodiment of the application. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present application, the present application will be described in more detail below in conjunction with specific embodiments. All technical and scientific terms used in the present specification have the same meanings as those commonly understood by the skilled in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used in the present specification includes any and all combinations of one or more related listed items.

[0032] In addition, the technical features involved in the different embodiments of the application described below can be combined with each other as long as there is no conflict between them.

[0033] The present application provides an embodiment of a method for preparing a glucose oxidase sensor with a porous structure outer membrane, and the preparation method steps are as follows.

[0034] (1) Take a glucose oxidase sensor assembly including an enzyme-coated electrode and an Ag / AgCl reference electrode, and use a micropipette to draw chitosan solution, drop the chitosan solution onto the surface of the glucose oxidase sensor assembly, dry in a 25℃ incubator, form a chitosan outer membrane on the surface of the glucose oxidase sensor assembly, and obtain a glucose oxidase sensor assembly with a chitosan outer membrane.

[0035] The configuration method of the chitosan solution is to slowly add chitosan powder to 0.1 mmol of an aqueous acetic acid solution, stir and dissolve the chitosan powder, and prepare a chitosan solution with a mass fraction of 0.5%.

[0036] Chitosan solution forms a flexible network polymer on the surface of glucose oxidase sensor assembly, which has a high water content and excellent biological compatibility. In physiological solution, chitosan can reduce protein adsorption and subsequent inflammatory response. At the same time, its porous outer structure does not act as a selective barrier to glucose, making glucose diffusion more uniform, and at the same time facilitating cell adhesion in vivo.

[0037] (2), add solid particles of manganese salt to the pre-solidified PDMS solution, ultrasonic mixing, obtain salt-PDMS mixed solution; drop the salt-PDMS mixed solution into a stainless steel groove, and extract the air in the salt-PDMS mixed solution in the groove.

[0038] The English name of polydimethylsiloxane is Polydimethylsiloxane, which is a high molecular organic silicon compound, which has good biological and chemical inertness, and non-toxic and non-flammable characteristics. In this embodiment, the pre-solidified PDMS solution is obtained by uniformly mixing dimethyl silicone oil and dibutyl phthalate in a weight ratio of 10:1, and dibutyl phthalate is a curing agent for dimethyl silicone oil, which is used for subsequent molding and curing of the PDMS outer film. In another embodiment, the pre-solidified PDMS solution can also be obtained by uniformly mixing dimethyl silicone oil and DBP in a volume ratio of 10:1.

[0039] During the preparation of the salt-PDMS mixed solution, air is easy to enter the mixed solution, producing bubbles, which affects the pore size and porosity of the PEDMS outer film porous structure in the subsequent film forming process, so that the sensor outer film porous structure is uncontrollable. By vacuum extraction, the air in the salt-PDMS mixed solution in the stainless steel groove can be removed, so that the pore size and porosity of the PDMS outer film porous structure formed by the method of the application depend only on the particle size of the solid particles of manganese salt and the amount of salt.

[0040] (3), then immerse the glucose oxidase sensor assembly with chitosan outer film into the groove, so that the salt-PDMS mixed solution in the groove immerses the glucose oxidase sensor assembly with chitosan outer film 3s, and then take out 37℃ for 2 hours, to form a layer of PDMS outer film on the surface of the sensor, repeat the step 5 times to set the thickness of the PDMS outer film, and obtain a glucose oxidase sensor with PDMS / chitosan outer film.

[0041] In other embodiments, the immersion time and curing conditions of the sensor can be appropriately adjusted according to the performance design requirements of the sensor, and the number of times of repeating the operation of the step can be appropriately adjusted.

[0042] (4) Put the glucose oxidase sensor with PDMS / chitosan outer membrane into flowing deionized water for 2 hours to dissolve the manganese salt particles in the PDMS outer membrane, and the PDMS outer membrane forms a porous structure to obtain a glucose oxidase sensor with a porous structure outer membrane.

[0043] In this embodiment, the particle size of the solid particles of manganese salt is about 30 um. Since the solid particles of manganese salt are easily dissolved in water and not dissolved in organic solvents, the solid particles of manganese salt on the PDMS outer membrane are dissolved into the deionized water, thereby leaving holes on the PDMS outer membrane with a size about equal to the particle size of the solid particles of manganese salt, so that the PDMS outer membrane has a porous structure with a pore size of about 30 um. In this application, the manganese salt has color, and the color of the PDMS outer membrane can be used to determine whether the manganese salt on the PDMS outer membrane is completely dissolved, and the dissolution of the solid particles of salt can be easily observed.

[0044] In other embodiments, the particle size or type of the solid particles of salt added to the salt-PDMS mixed solution can be changed according to the different pore size requirements of the PDMS outer membrane, so that the glucose oxidase sensor with a porous structure outer membrane has different pore size of the porous structure, that is, the particle size of the solid particles of salt used in the salt-PDMS mixed solution affects the pore size of the porous structure of the PDMS outer membrane, and the pore size is about equal to the particle size of the solid particles. Since the pore size is related to the particle size of the solid particles of salt added, the consistency between the same batch of sensors is good.

[0045] In particular, the amount of salt in the salt-PDMS mixed solution affects the porosity of the porous structure of the PDMS outer membrane. Assuming that the volume of the pre-cured PDMS solution is V, the pore volume is Vpore, and the porosity = Vpore / V. Therefore, the total pore volume can be calculated according to the required porosity and the volume of the PDMS used in the experiment. In this embodiment, the pore size is about 30 um, and the volume of a single pore is calculated using the spherical volume formula, so that the total amount of manganese salt in the salt-PDMS mixed solution can be calculated.

[0046] (5) After washing with deionized water, the glucose oxidase sensor with a porous structure outer membrane needs to be dried at room temperature, then transferred to a phosphate buffer solution and stored at 4°C. The phosphate buffer solution storage makes the PDMS outer membrane and the chitosan outer membrane space filled with the phosphate buffer solution, keeps the surface of the sensor moist, prevents the outer membrane from being damaged due to drying and dehydration to protect the sensor, and facilitates subsequent subcutaneous implantation.

[0047] Chitosan has biodegradability, biocompatibility, non-toxicity, low price, and is widely used in in vivo materials, but it is too fragile during implantation to maintain its membrane structure and function, therefore, chitosan is difficult to be used alone as the outermost membrane of the implantable glucose sensor. PDMS belongs to the organic silicon family, has excellent oxygen permeability, good physical stability and strength, flexibility, good biocompatibility, and is stable enough to resist long-term immersion in the surrounding liquid environment, and can maintain the stability of its shape and performance, and is low in price. The present application develops a porous PDMS structure which can be elastically deformed under stress, reducing the risk of breakage, and used in combination with chitosan as the outer membrane of the implantable glucose sensor, which can prevent the collapse and swelling of the chitosan structure during implantation, and make the sensor have sufficient mechanical strength and a sharp tip to pierce the skin, and have flexibility and mechanical stability after insertion into the skin, allowing oxygen and water molecules to pass through. At the same time, using PDMS and chitosan as the outer membrane can reduce the foreign body reaction around the sensor, thereby improving the performance of the sensor in vivo, meeting the requirements of biocompatibility and structural stability of the outer layer of the sensor. At the same time, the preparation method of the chitosan membrane layer and the porous PDMS membrane layer in the present application is simple.

[0048] The glucose oxidase sensor with a porous outer membrane prepared according to the above method was subjected to scanning electron microscopy, and the scanning results are shown in Figure 1 The PDMS outer membrane has a porous structure, and the porous structure has uniform pore distribution. The glucose concentration detection test of the glucose oxidase sensor with a porous outer membrane prepared was performed, and it was found that the sensor had high accuracy, the linear coefficient reached 0.995, met the requirements of in vivo glucose monitoring, and had good consistency.

[0049] The glucose oxidase sensor with a porous outer membrane prepared by the method of the present application was measured continuously for 14 days after manufacture, and then measured again. The sensor responded to the change in glucose concentration with a current decay of only 30%, a response time of less than 6s, and a sensitivity of 148nA·L·mmol -1 ·cm -2 , which was very similar to the initial measurement results, therefore the glucose oxidase sensor with a porous outer membrane prepared by the method of the present application had good stability.

[0050] The electrode of the glucose oxidase sensor with a porous outer membrane prepared according to the above method was subjected to electrochemical performance test, and the results are shown in Figure 2As shown, a pair of reversible redox peaks appeared in the C-V curve of the glucose oxidase sensor with the porous structure outer membrane, and the curve symmetry was good. The high curve symmetry indicated that the electrode reversibility was good, and the glucose oxidase sensor with the porous structure outer membrane had good current response sensitivity, high efficiency of electron transfer between the electrode and the body fluid or blood, and good electrode redox reversibility.

[0051] The glucose oxidase sensor with the porous structure outer membrane prepared by the above method was tested with glucose solutions with concentrations of 0 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM and 25 mM, respectively, and the test results are shown in the following table. Figure 3 As shown, the sensor had good response between the glucose concentration of 0-20 mM, and therefore the biosensor obtained by the method of the application had a wide detection range and good detection ability under hyperglycemic conditions, and was suitable for accurate monitoring of the blood glucose concentration in the blood of a diabetic patient.

[0052] The above examples are only used to illustrate the technical solutions of the application, but not to limit the application; under the idea of the application, the technical features in the above examples or different examples can also be combined, the steps can be implemented in any order, and there are many other changes of different aspects of the application as described above, which are not provided in details for the sake of simplicity; although the application is described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the application.

Claims

1. A method for preparing a glucose oxidase sensor having a porous structure outer membrane, characterized by, The preparation method comprises the following steps, Step S1: drop the chitosan solution on the surface of the glucose oxidase sensor assembly, dry in a temperature box, form a chitosan outer membrane on the surface of the glucose oxidase sensor assembly, and obtain the glucose oxidase sensor assembly with the chitosan outer membrane; Step S2: add the solid particles of salt into the pre-cured PDMS solution, mix by ultrasonic, obtain a salt-PDMS mixed solution, drop the salt-PDMS mixed solution into a groove, and extract the air in the salt-PDMS mixed solution in the groove by vacuum; Step S3: immerse the glucose oxidase sensor assembly with the chitosan outer membrane into the groove, immerse the salt-PDMS mixed solution in the groove into the glucose oxidase sensor assembly for a period of time, then take out and cure, form a PDMS outer membrane on the surface of the sensor; repeat the step until the thickness of the PDMS outer membrane reaches a set value, and obtain the glucose oxidase sensor with a PDMS / chitosan outer membrane; Step S4: place the glucose oxidase sensor with the PDMS / chitosan outer membrane into deionized water, dissolve the salt particles in the PDMS outer membrane, and obtain the glucose oxidase sensor with a porous outer membrane.

2. The method for preparing a glucose oxidase sensor having a porous structure outer membrane according to claim 1, wherein, The salt in the salt-PDMS mixed solution is manganese salt, and the pore size of the porous structure of the PDMS outer membrane is the same as the particle size of the solid particles of the manganese salt.

3. The method of claim 1, wherein the porous outer membrane is formed by coating the glucose oxidase sensor with a solution of a polymer and a solvent, and then drying the coated glucose oxidase sensor. The configuration method of the chitosan solution is as follows: add chitosan powder into 0.1 mmol of acetic acid aqueous solution, stir to dissolve the chitosan powder, and configure 0.5% chitosan solution.

4. The method of claim 1, wherein the porous outer membrane is formed by coating the glucose oxidase sensor with a solution of a polymer and a solvent. The configuration method of the pre-cured PDMS solution is as follows: mix dimethyl silicone oil and curing agent uniformly at a weight ratio of 10:1 to obtain the pre-cured PDMS solution, wherein the curing agent is dibutyl phthalate.

5. The method of claim 1, wherein the porous outer membrane is formed by coating the glucose oxidase sensor with a solution of a polymer and a solvent. The configuration method of the pre-cured PDMS solution is as follows: mix dimethyl silicone oil and curing agent uniformly at a volume ratio of 10:1 to obtain the pre-cured PDMS solution, wherein the curing agent is dibutyl phthalate.

6. The method for preparing a glucose oxidase sensor with a porous outer membrane according to claim 1, characterized in that, The glucose oxidase sensor assembly comprises an enzyme-coated electrode and an Ag / AgCl reference electrode.

7. The method of claim 1, wherein the porous outer membrane is formed by coating the glucose oxidase sensor with a solution of polyvinyl alcohol, polyethylene glycol, or a mixture thereof. The number of repetitions of step S3 is 5, and the time for the salt-PDMS mixed solution to immerse the sensor each time is 3s.

8. The method for preparing a glucose oxidase sensor having a porous structure outer membrane according to claim 7, wherein, The curing condition of the PDMS outer membrane is 37℃ for 2h.

9. The method for preparing a glucose oxidase sensor with a porous outer membrane according to claim 1, characterized in that, In step S1, a micropipette is used to suck the chitosan solution and drop the chitosan solution on the surface of the glucose oxidase sensor assembly, and the drying temperature in the temperature box is 25℃.

10. The method for preparing a glucose oxidase sensor with a porous outer membrane according to claim 1, characterized in that, After step S4 of the preparation method, the method further comprises step S5, Step S5: dry the glucose oxidase sensor with the porous outer membrane at room temperature, then transfer to phosphate buffer solution and store at 4℃.

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