Method of making working electrode of glucose monitoring probe

Abstract

The present disclosure relates to a method for manufacturing a working electrode of a glucose monitoring probe, comprising: preparing a substrate; forming a base layer on the substrate; preparing a sensing layer reagent comprising a redox polymer, a glucose enzyme, a carbon nanotube, and a cross-linking agent, adding an amino modification to the carbon nanotube to enable the redox polymer and the carbon nanotube to form a covalent bond, and combining the carbon nanotube with the glucose enzyme, wherein the working voltage required by the working electrode is reduced under the catalytic action of the carbon nanotube, and the glucose enzyme is glucose oxidase; coating the sensing layer reagent on the base layer to form a sensing layer; forming a semi-permeable membrane on the sensing layer to control the passage rate of glucose molecules; and forming a biocompatible membrane on the semi-permeable membrane. According to the present disclosure, a method for manufacturing a working electrode of a glucose monitoring probe capable of reducing the working voltage of the working electrode and improving the reaction sensitivity to glucose can be provided.

Description

[0001] This application is a divisional application of the patent application with application number 202010246108.4 and titled 2020-03-31 , filed on Grape Working electrode of a glucose monitoring probe and method of making the same . TECHNICAL FIELD

[0002] The present disclosure relates to the field of glucose monitors, and in particular to a method for manufacturing a working electrode of a glucose monitoring probe. BACKGROUND

[0003] A biosensor is an analytical device that combines biological materials, biologically derived materials, or biomimetic materials with optical, electrochemical, thermal, piezoelectric, magnetic, or micromechanical physicochemical sensors or sensing microsystems. To date, the most successful biosensor commercially applied is the amperometric enzyme glucose sensor. The market share of the amperometric enzyme glucose sensor almost accounts for 85% of the global market today. The amperometric enzyme glucose sensor is used for detecting diabetes, and the greater its market share, the more people suffer from diabetes.

[0004] Diabetes is a series of metabolic disorder syndromes of sugar, protein, fat, water and electrolytes, etc., which is caused by genetic factors, immune function disorders, microbial infections and their toxins, etc. affecting the body to cause pancreatic islet function to decline, insulin resistance, etc. If diabetes is not well controlled, it may cause some complications, such as ketoacidosis, lactic acidosis, chronic renal failure and retinopathy. With the increasing incidence of diabetes, diabetes has become a public health problem worldwide.

[0005] Currently, there is no cure for diabetes, only control methods. For diabetic patients, if the patient can monitor glucose in real time and continuously on a daily basis, it can reduce and reduce the occurrence of complications such as hypoglycemia and hyperglycemia in insulin-dependent diabetic patients.

[0006] Generally, the monitoring of glucose needs to be achieved by a glucose detector in an amperometric enzyme glucose sensor. The sensing probe of the glucose detector is generally implanted in the body to monitor the glucose concentration in the interstitial fluid and the surrounding blood flow, the rate of metabolism and the rate of change of glucose concentration in the blood vessels. Studies have shown that the change in glucose concentration in the interstitial fluid is generally delayed by 2-45 minutes compared to the change in glucose concentration in the blood, with an average delay of about 6.7 minutes. However, when the glucose concentration in the blood begins to decrease, the glucose concentration in the interstitial fluid decreases before the glucose concentration in the blood, indicating that the decrease in glucose concentration in the interstitial fluid can predict the impending hypoglycemia.

[0007] With the development of technology, various portable glucose detectors have entered people's eyes, especially some implantable continuous glucose monitoring devices, which are favored by diabetic patients and major hospitals. However, the service life of the implantable continuous glucose detector is often not long, and is easily affected by the body's immune response and other impurities in the blood, which reduces the sensitivity. Therefore, how to better construct the detection device, prolong the service life of the glucose detector sensor probe and reduce the influence of other factors has become the biggest problem at present. SUMMARY

[0008] The present disclosure is made in view of the above, and aims to provide a working electrode of a glucose monitoring probe and a method of manufacturing the same, which prolongs the service life of the probe, reduces interference, and improves the reaction sensitivity to glucose.

[0009] To this end, an aspect of the present disclosure provides a working electrode of a glucose monitoring probe, characterized in that the working electrode comprises: a base layer disposed on a flexible substrate; a sensing layer formed on the base layer by coating a sensing layer reagent, capable of chemically reacting with glucose in blood or interstitial fluid, the sensing layer reagent including a metal polymer, a glucose enzyme, a carbon nanotube, and a cross-linking agent, the carbon nanotube adsorbing the metal polymer and the glucose enzyme, and the carbon nanotube being modified with an amino group to form a covalent bond with the metal polymer and the glucose enzyme; a semi-permeable membrane formed on the sensing layer, controlling the passage rate of glucose molecules; and a biocompatible membrane formed on the semi-permeable membrane.

[0010] In the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the sensing layer contains carbon nanotubes. In this case, the catalytic effect of the carbon nanotubes on the glucose reaction reduces the working voltage required for the normal operation of the working electrode, reducing the interference of the electrochemically active substances under high voltage with the working electrode; at the same time, it improves the reaction sensitivity of the probe to glucose, and also increases the linear range of the response of the probe to glucose, prolonging the service life of the probe.

[0011] In addition, in the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the mass percentage of the carbon nanotubes in the sensing layer reagent is 1 to 50%. Thus, the metal polymer in the sensing layer reagent can more easily form a covalent bond with the carbon nanotubes.

[0012] In addition, in the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the semi-permeable membrane optionally comprises a diffusion control layer for controlling diffusion of glucose molecules. In this case, the proportion of glucose components in tissue fluid or blood entering the semi-permeable membrane is blocked, avoiding excessive glucose molecules from reacting with the working electrode, thereby reducing the service life of the glucose monitoring probe.

[0013] In addition, in the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the semi-permeable membrane optionally comprises an anti-interference layer for blocking non-glucose substances. In this case, other components in tissue fluid or blood entering the semi-permeable membrane are blocked, avoiding other electroactive substances that can also generate current from affecting the working electrode, thereby causing inaccurate glucose detection results.

[0014] In addition, in the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the thickness of the sensing layer is 0.1 μm to 100 μm. In this way, sufficient glucose enzyme can be provided under the premise of sufficient reaction and firm adhesion.

[0015] Another aspect of the present disclosure provides a glucose monitoring probe, characterized in that it comprises a working electrode, a counter electrode and a reference electrode arranged in a dispersed manner, the working electrode comprising: a base layer disposed on a flexible substrate; a sensing layer formed on the base layer by coating a sensing layer reagent, capable of chemically reacting with glucose in blood or tissue fluid, the sensing layer reagent comprising a redox polymer, a glucose enzyme, a carbon nanotube and a cross-linking agent, the carbon nanotube adsorbing the redox polymer and the glucose enzyme, the carbon nanotube being modified with an amino group to enable the redox polymer and the carbon nanotube to form a covalent bond and combine with the glucose enzyme; a semi-permeable membrane formed on the sensing layer, the semi-permeable membrane comprising an anti-interference layer for blocking non-glucose substances and a diffusion control layer for controlling diffusion of glucose molecules; and a biocompatible membrane formed on the semi-permeable membrane.

[0016] In the glucose monitoring probe according to another aspect of the present disclosure, the sensing layer contains a redox polymer. In this case, the working voltage required for the working electrode to function normally is reduced, reducing the interference of electrochemical reactions of some electroactive substances under high voltage with the working electrode; at the same time, the reaction sensitivity of the probe to glucose is improved, the linear range of the response of the probe to glucose is also increased, and the service life of the probe is prolonged.

[0017] In addition, in the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the redox polymer in the sensing layer reagent is a metal redox polymer. In this way, the metal redox polymer can participate in the redox reaction.

[0018] In addition, in the working electrode of the glucose monitoring probe according to an aspect of the present disclosure, the metal redox polymer can be at least one selected from poly(vinyl ferrocene), quaternized poly(4-vinylpyridine) of ferricyanide, quaternized poly(l-vinylimidazole) of ferricyanide, quaternized poly(4-vinylpyridine) of ferrocyanide, quaternized poly(l-vinylimidazole) of ferrocyanide, osmium 2,2'-bipyridine complex coordination to poly(l-vinylimidazole), osmium 2,2'-bipyridine complex coordination to poly(4-vinylpyridine), cobalt 2,2'-bipyridine complex coordination to poly(l-vinylimidazole), or cobalt 2,2'-bipyridine complex coordination to poly(4-vinylpyridine). Thus, the metal redox polymer can participate in the redox reaction through a covalent bond, a coordination bond, or an ionic bond.

[0019] Another aspect of the present disclosure provides a method for manufacturing a working electrode of a glucose monitoring probe, which includes: preparing a flexible substrate; forming a base layer on the flexible substrate; preparing a sensing layer reagent including a redox polymer, a glucose enzyme, a carbon nanotube, and a cross-linking agent; coating the sensing layer reagent on the base layer to form a sensing layer; forming a semi-permeable membrane for controlling the passage of glucose molecules on the sensing layer; and forming a biocompatible membrane on the semi-permeable membrane.

[0020] In another aspect of the present disclosure, the carbon nanotube is included in the sensing layer. Thus, the working voltage of the working electrode is reduced, the interference of other factors is reduced, the reaction sensitivity of the probe to glucose is improved, the linear range of the response of the probe to glucose is increased, and the service life of the probe is prolonged.

[0021] In addition, in the method for manufacturing a working electrode of a glucose monitoring probe according to the present disclosure, the mass percentage of the carbon nanotube in the sensing layer reagent can be 1 to 50%. Thus, the reaction of the glucose enzyme can be better promoted.

[0022] According to the present disclosure, a working electrode of a glucose monitoring probe and a method for manufacturing the same can be provided, which prolong the service life of the probe, reduce the interference, and improve the reaction sensitivity to glucose. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 FIG. 1 is a schematic view showing the use state of a glucose monitoring probe according to an embodiment of the present disclosure.

[0024] Figure 2 FIG. 2 is a structural schematic view of a glucose monitoring probe according to an embodiment of the present disclosure.

[0025] Figure 3 FIG. 3 is a structural schematic view of a glucose monitoring probe according to another embodiment of the present disclosure.Figure 2 A structural diagram showing a glucose monitoring probe according to an embodiment of the present disclosure in a bent state.

[0026] Figure 4 A structural diagram showing a working electrode of a glucose monitoring probe according to an embodiment of the present disclosure.

[0027] Figure 5 A diagram showing a glucose monitoring probe according to an embodiment of the present disclosure in which a carbon nanotube adsorbs glucose oxidase.

[0028] Figure 6 A diagram showing a glucose monitoring probe according to an embodiment of the present disclosure in which the glucose monitoring probe reacts with glucose of a tissue.

[0029] Figure 7 A structural diagram showing a semi-permeable membrane of a working electrode of a glucose monitoring probe according to an embodiment of the present disclosure.

[0030] Figure 8 A flowchart showing a method of manufacturing a working electrode of a glucose monitoring probe according to an embodiment of the present disclosure.

[0031] Figure 9 A flowchart showing a method of manufacturing a semi-permeable membrane of a working electrode of a glucose monitoring probe according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0032] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same parts are given the same reference numerals, and overlapping descriptions will be omitted. In addition, the drawings are merely schematic diagrams, and the proportions of the sizes of the components with respect to each other or the shapes of the components, etc. can be different from the actual ones.

[0033] In addition, the subheadings and the like involved in the following description of the present disclosure are not intended to limit the content or the scope of the present disclosure, and merely serve as a reading aid. Such subheadings should not be understood as dividing the content of the article, and the content under the subheadings should not be limited only within the scope of the subheadings.

[0034] Figure 1 A diagram showing a glucose monitoring probe according to an embodiment of the present disclosure in a bent state. Figure 2 A structural diagram showing a glucose monitoring probe according to an embodiment of the present disclosure. Figure 3 A diagram showing Figure 2 A structural diagram showing a glucose monitoring probe according to an embodiment of the present disclosure in a bent state.

[0035] In the present embodiment, the glucose monitoring probe 1 can also be referred to as an implantable glucose monitoring probe 1, a probe 1 of a glucose monitoring meter, or a probe 1.

[0036] In the present embodiment, the portable glucose monitoring meter G can include the glucose monitoring probe 1 and the electronic system 2 connected to the glucose monitoring probe 1. By implanting the glucose monitoring probe 1 of the portable glucose monitoring meter G into a human body, for example, a body surface of a human body, the glucose monitoring probe 1 comes into contact with interstitial fluid or blood of the body surface, so that a sensing signal related to a glucose concentration of the interstitial fluid can be sensed by the glucose monitoring probe 1, and a corresponding glucose concentration can be obtained by transmitting the glucose concentration signal to the electronic system 2.

[0037] Specifically, a part of the glucose monitoring probe 1 (in particular, a sensing part) can be implanted into, for example, a body surface of a human body to come into contact with interstitial fluid in the body. In addition, another part of the glucose monitoring probe 1 is also connected to the electronic system 2 located outside the body surface. When the portable glucose monitoring meter G is in operation, the glucose monitoring probe 1 reacts with the interstitial fluid or blood in the body to generate a sensing signal (for example, an electric current signal), and transmits the sensing signal to the electronic system 2 of the body surface, and the electronic system 2 processes the sensing signal to obtain the glucose concentration. Although Figure 1 The position where the glucose monitoring probe 1 is arranged on the arm is shown, but the present embodiment is not limited thereto, and for example, the glucose monitoring probe 1 can also be arranged on the abdomen, the waist, the leg, or the like.

[0038] In addition, in the present embodiment, the glucose monitoring probe 1 can directly detect the glucose of the blood, or can detect the glucose of the interstitial fluid. In addition, the glucose concentration of the interstitial fluid has a strong correlation with the glucose concentration of the blood, and the glucose concentration of the blood can also be obtained through the glucose of the interstitial fluid.

[0039] In the present embodiment, the glucose monitoring probe 1 can include a substrate S, and a working electrode 10, a reference electrode 20, and a counter electrode 30 (see FIG. 1) disposed on the substrate S. Figure 2 In addition, the glucose monitoring probe 1 also includes a contact 41 connected to the working electrode 10 via a lead, a contact 42 connected to the reference electrode 20 via a lead, and a contact 43 connected to the counter electrode 30 via a lead. The contact 41, the contact 42, and the contact 43 are all electrical contacts. In some examples, the glucose monitoring probe 1 can be connected to the electronic system 2 via the contact 41, the contact 42, and the contact 43.

[0040] In some examples, the substrate S can be a flexible substrate. The flexible substrate can be made of at least one of polyethylene (PE), polypropylene (PP), polyimide (PI), polystyrene (PS), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), etc. In addition, in other examples, the flexible substrate can also be made of a metal foil, an ultra-thin glass, a single-layer inorganic thin film, a multi-layer organic thin film, a multi-layer inorganic thin film, etc.

[0041] In some examples, the substrate S can also be a non-flexible substrate. The non-flexible substrate can generally include a ceramic, alumina, silica, etc. with weak conductivity. In this case, the glucose monitoring probe 1 with the non-flexible substrate can also have a pointed end or a sharp edge, so as to be able to implant the glucose monitoring probe 1 into the body surface (e.g., the skin superficial layer, etc.) without the need for an auxiliary implantation device (not shown).

[0042] In the present embodiment, for the convenience of description, the glucose monitoring probe 1 can be divided into a connection portion la and an implantation portion lb (see Figure 3 ). Figure 3 The straight line A-A' in FIG. 1 shows the approximate position of the skin when the glucose monitoring probe 1 is implanted into the tissue body surface. The connection portion la is located outside the body surface, and the implantation portion lb is implanted into the body surface.

[0043] In addition, in some examples, the connection portion la and the implantation portion lb can both include a flexible substrate, but the present embodiment is not limited thereto, for example, only the implantation portion lb can include a flexible substrate, and the connection portion la can include a non-flexible substrate such as a rigid substrate.

[0044] In the present embodiment, the implantation portion lb of the glucose monitoring probe 1 can be provided in an auxiliary puncture needle (not shown), and the implantation portion lb is separable from the puncture needle. Specifically, the puncture needle can be pierced into the tissue (e.g., the skin superficial layer), and then the puncture needle is pulled out and separated from the implantation portion lb of the glucose monitoring probe 1, so that the implantation portion lb is left in the skin superficial layer, and the electronic system 2 is attached to the skin surface, and the connection portion la (see Figure 3 ) of the glucose monitoring probe 1 is connected to the electronic system 2 and located on the skin surface. Here, the electronic system 2 can be attached to the skin surface by an adhesive provided on the substrate S.

[0045] In some examples, the puncture needle as an auxiliary implantation can have a notch, and the implantation portion lb is placed in the notch of the puncture needle. The puncture needle can be made of stainless steel. In this case, the risk of using the puncture needle can be reduced, and the puncture needle has sufficient hardness to facilitate piercing the skin. It is beneficial for patients to use. In addition, in some examples, the puncture needle can also be made of plastic, glass or metal.

[0046] In the present embodiment, a puncture needle can be inserted into the skin using an auxiliary implantation device (not shown), such as a needle holder. In this case, the puncture depth can be preset using, for example, a needle holder, and by using the needle holder to achieve rapid and painless puncture, the user's pain can be reduced. Furthermore, one-handed operation can be facilitated by the auxiliary implantation device. However, the present embodiment is not limited thereto, and for example, as described above, when the glucose monitoring probe 1 is a rigid substrate, the glucose monitoring probe 1 can also be implanted into the skin without the aid of a puncture needle.

[0047] In the present embodiment, the depth at which the glucose monitoring probe 1 is implanted under the skin is determined in accordance with the different locations at which it is to be inserted, and when the fat layer is thick, it is implanted deeper, for example, in the abdomen of a human body, and the implantation depth can be approximately 10 mm to 15 mm. When the fat layer is thin, it is implanted shallower, for example, in the arm, and the implantation depth can be approximately 5 mm to 10 mm.

[0048] Figure 4 is a structural diagram showing the working electrode 10 of the glucose monitoring probe 1 according to the embodiment of the present disclosure. Figure 5 is a diagram showing the adsorption of glucose oxidase by carbon nanotubes of the glucose monitoring probe 1 according to the embodiment of the present disclosure. Figure 6 is a diagram showing the glucose reaction of the glucose monitoring probe 1 with tissue according to the embodiment of the present disclosure. Figure 7 is a structural diagram showing the semi-permeable membrane of the working electrode 10 of the glucose monitoring probe 1 according to the embodiment of the present disclosure.

[0049] In the present embodiment, as described above, the implanted portion 1b of the glucose monitoring probe 1 includes the working electrode 10 (see Figure 2 and Figure 3 ).

[0050] In the present embodiment, in some examples, the working electrode 10 can have a base layer 110, a sensing layer 120, a semi-permeable membrane 130, and a biocompatible membrane 140 (see Figure 4 ). In some examples, the base layer 110, the sensing layer 120, the semi-permeable membrane 130, and the biocompatible membrane 140 can be stacked in order.

[0051] In the present embodiment, the base layer 110 has electrical conductivity. In some examples, the base layer 110 can be made of at least one selected from gold, glassy carbon, graphite, silver, silver chloride, palladium, titanium, and iridium. In this case, the base layer 110 can have good electrical conductivity, and can inhibit the occurrence of an electrochemical reaction in the base layer 110, thereby improving the stability of the base layer 110.

[0052] In the present embodiment, the base layer 110 can be disposed on the substrate S by a deposition or plating method in some examples. The method of deposition can include physical vapor deposition, chemical vapor deposition, etc. in some examples. The method of plating can include electroplating, electroless plating, vacuum plating, etc. In addition, the base layer 110 can also be disposed on the substrate S by screen printing, extrusion, or electrolytic deposition, etc. in some examples.

[0053] In the present embodiment, the base layer 110 can be disposed on a flexible substrate. In this case, the flexible substrate makes the entire product light, strong against impact, and reduces the foreign body sensation after implantation. In other examples, the base layer 110 can also be disposed on a rigid substrate.

[0054] In the present embodiment, the sensing layer 120 can be formed on the base layer 110 by coating a sensing layer reagent, thereby being able to chemically react with glucose. In some examples, the sensing layer reagent can include a redox polymer, a glucose enzyme, carbon nanotubes 121, and a crosslinking agent. In this case, the carbon nanotubes 121 are contained in the sensing layer 120, and the catalytic effect of the carbon nanotubes 121 on the glucose reaction reduces the operating voltage required for the working electrode 10 to normally operate, thereby being able to reduce the interference of the electrically active substance generated by the electrochemical reaction at a high voltage with the working electrode 10.

[0055] In some examples, the redox polymer can have a covalent bond, a coordination bond, or an ionic bond. In some examples, the redox polymer can be a metal polymer that functions as a redox, i.e., a metal redox polymer. The metal polymer herein can be, for example, a metal polymer having a covalent bond, a coordination bond, or an ionic bond. In some examples, the metal polymer can be selected from at least one of poly(vinyl ferrocene), quaternized poly(4-vinylpyridine) of ferricyanide, quaternized poly(l-vinylimidazole) of ferricyanide, quaternized poly(4-vinylpyridine) of ferrocyanide, quaternized poly(l-vinylimidazole) of ferrocyanide, osmium 2,2'-bipyridine complex coordination to poly(l-vinylimidazole), osmium 2,2'-bipyridine complex coordination to poly(4-vinylpyridine), cobalt 2,2'-bipyridine complex coordination to poly(l-vinylimidazole), or cobalt 2,2'-bipyridine complex coordination to poly(4-vinylpyridine).

[0056] Generally, a carbon nanotube is mainly composed of carbon atoms arranged in a hexagonal shape into a coaxial circular tube of several to several tens of layers, and the layers maintain a fixed distance of about 0.34 nm from each other, and the diameter is generally 2 to 20 nm.

[0057] In some examples, as Figure 5As shown, the carbon nanotube 121 can be in a hollow columnar shape. Specifically, the carbon nanotube 121 can be in a cylindrical shape or an elliptical cylindrical shape, etc. In addition, the carbon nanotube 121 has a strong adsorption capacity for organic matters due to its large surface area and surface hydrophobicity.

[0058] In the sensing layer 120, since the carbon nanotube 121 can adsorb glucose oxidase and redox polymers (e.g., metal polymers), the carbon nanotube 121 can fully contact and catalyze the reaction during the glucose reaction, thereby being able to more effectively promote the glucose reaction.

[0059] In the present embodiment, the carbon nanotube 121 can be dissolved in a solvent to be added to the sensing layer reagent. Thereby, the sensing layer 120 containing the carbon nanotube 121 can be conveniently prepared.

[0060] In some examples, the mass percentage of the carbon nanotube 121 in the sensing layer reagent can be 1 to 50%. Thereby, the reaction of the glucose oxidase can be better promoted. In some examples, the mass percentage of the carbon nanotube 121 can be 5 to 10% in consideration of the purpose of better matching the coating process. Thereby, the corresponding promotion can be played while ensuring the effectiveness of the sensing layer reagent. Specifically, the mass percentage of the carbon nanotube 121 can be 5%, 6%, 7%, 8%, 9%, or 10%.

[0061] In some examples, the sensing layer reagent can form the sensing layer 120 by at least one process of spin coating, dip coating, drop coating, or spray coating, etc.

[0062] In the present embodiment, the sensing layer 120 can be a glucose oxidase sensing layer, or a glucose dehydrogenase sensing layer.

[0063] Hereinafter, the sensing layer 120 will be described in detail with reference to the following examples. Figure 6 The reaction occurring in the glucose sensing layer 120 will be described with glucose oxidase (GOx) and redox polymer (MED X (FAD) and oxidation-reduction polymer (MED red ) as examples.

[0064] For example, after the working electrode 10 is implanted into the subcutaneous tissue (e.g., the subcutaneous tissue of the arm), the glucose sensing layer 120 in the working electrode 10 contacts the body tissue fluid, and when GOx (FAD) encounters glucose in the tissue, the following reaction occurs: X

[0065] Glucose + GOx (FAD) → Gluconolactone + GOx (FADH2) … Reaction formula (I)

[0066] GOx (FADH2) + MED ox → GOx (FAD) + MED​red … Reaction (II)

[0067] MED red - e - → MED ox … Reaction (III)

[0068] As can be seen in the above reaction process, in the chemical reaction, the oxidation state of the redox polymer MED ox is reduced to MED red , and MED red is oxidized to MED ox by applying a working voltage, but if MED red cannot be quickly oxidized to MED ox , the reaction of MED ox is insufficient, so that the reaction speed of Reaction (II) and Reaction (I) is limited by the amount of MED ox , and the reaction with tissue glucose will slow down, resulting in the failure of the glucose monitoring probe 1. Therefore, by adding carbon nanotubes 121 in the sensing layer 120, under the catalytic action of the carbon nanotubes 121, the oxidation of MED red to MED ox can be greatly accelerated and at a lower voltage.

[0069] Through the above Reaction (I) to Reaction (III), the reaction with tissue glucose can continue. In addition, by using carbon nanotubes 121, the reaction (III) can be accelerated and the voltage required during the reaction can be reduced, thereby improving the sensitivity of the glucose monitoring probe 1, prolonging the service life of the glucose monitoring probe 1, and obtaining a low working voltage. In other words, through the carbon nanotubes 121, a high-sensitivity sensing signal of tissue glucose can be continuously obtained, the service life of the glucose monitoring probe 1 is prolonged, and a low working voltage is beneficial to improve the anti-interference performance.

[0070] In addition, in some examples, amino modification can also be added to the carbon nanotubes 121 in the sensing layer reagent. In this case, the metal polymer and the carbon nanotubes 121 can be tightly formed into a covalent bond, so that the glucose enzyme can be more stably combined. In addition, the addition of amino modification to the carbon nanotubes 121 can also make the redox polymer and the carbon nanotubes 121 tightly form a covalent bond.

[0071] In other examples, graphene, porous titanium dioxide or conductive organic salt can also be added to the sensing layer reagent. In this way, the reaction of the glucose enzyme can be better promoted.

[0072] In the embodiment, the glucose monitoring probe 1 is implanted into the human skin, and the glucose in the blood can be continuously sampled and converted into a corresponding current signal, which is transmitted to the electronic system 2 outside the body.

[0073] In the embodiment, the thickness of the sensing layer 120 can be about 0.1 μm to 100 μm, and preferably about 2 μm to 10 μm. In one example, the thickness of the sensing layer 120 can be 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm. In this case, by controlling the thickness of the glucose oxidase or dehydrogenase within a certain range, the adhesion caused by excessive glucose oxidase or dehydrogenase is avoided, which causes the material to fall off in the body, and the problem of insufficient reaction caused by insufficient glucose oxidase or dehydrogenase, which cannot feedback the normal glucose concentration information, is also avoided.

[0074] In the embodiment, as shown in Figure 4 and Figure 7 , the semi-permeable membrane 130 can be distributed on the sensing layer 120, that is, the semi-permeable membrane 130 can be arranged on the sensing layer 120.

[0075] In addition, in the embodiment, as shown in Figure 7 , the semi-permeable membrane 130 can include a diffusion control layer 131 and an anti-interference layer 132 stacked on the diffusion control layer 131. In the semi-permeable membrane 130, the diffusion control layer 131 can control the diffusion of glucose molecules, and the anti-interference layer 132 can block the diffusion of non-glucose substances. Therefore, the tissue fluid or blood components passing through the semi-permeable membrane 130 can be reduced first, and the interference substances can be blocked outside the semi-permeable membrane 130 by the anti-interference layer 132. Common interference substances can include uric acid, ascorbic acid, acetaminophen, etc. that exist in the body.

[0076] In other examples, not limited to Figure 7 , in the semi-permeable membrane 130, the diffusion control layer 131 can also be stacked on the anti-interference layer 132. In this case, the interference of impurities to the working electrode 10 can also be reduced, the accuracy of the detection result can be improved, and the service life of the glucose monitoring probe 1 can be prolonged.

[0077] In the embodiment, the semi-permeable membrane 130 can control the passing rate of glucose molecules, that is, the semi-permeable membrane 130 can limit the number of glucose molecules in the tissue fluid or blood reaching the sensing layer 120. Specifically, the diffusion control layer 131 of the semi-permeable membrane 130 can effectively reduce the number of glucose diffusing to the sensing layer 120 by a certain proportion.

[0078] In the present embodiment, the diffusion control layer 131 can reduce the rate of the incoming substance to 10 to 100 times, preferably to 30 to 80 times, for example, to 30, 40, 50, 60, 70, or 80 times. In this case, the amount of glucose diffused to the sensing layer 120 can be reduced, and the amount of glucose oxidase or dehydrogenase and other substances involved in the reaction can be ensured to be sufficient, while the glucose concentration becomes a main factor that limits the size of the electrode current, so that the size of the current can correctly reflect the concentration of glucose, and the linear range of the glucose monitoring probe 1 can be greatly increased.

[0079] In the present embodiment, the biocompatible membrane 140 can be provided on the semipermeable membrane 130 (see Figure 4 ).

[0080] In some examples, the biocompatible membrane 140 can be made of a plant material. The plant material can be sodium alginate, tragacanth gum, pectin, gum arabic, xanthan gum, guar gum, agar, or the like, or a natural material derivative. Among them, the natural material derivative can include a starch derivative, a cellulose derivative, or the like.

[0081] In other examples, the biocompatible membrane 140 can also be made of an artificial synthetic material. The artificial synthetic material can be a polyolefin-based material such as povidone, polyvinyl alcohol, polyisobutylene pressure-sensitive adhesive, ethylene-vinyl acetate copolymer, or the like; a polyacrylic acid-based material such as acrylic resin, carboxyvinyl-sucrose, carboxyvinyl-pentaerythritol copolymer, polyacrylate pressure-sensitive adhesive, or the like; a polyoxyethylene-based material such as polyoxyethylene fatty acid ester, polyoxyethylene-polyoxypropylene copolymer, or the like; a polyester-based material such as polylactic acid, polyglycolide-lactide, polyazelaic acid dinonyl ester, polycyanoalkyl amino ester, polyether polyurethane, or the like. Thus, the immune response of the human body to the glucose monitoring probe 1 can be suppressed, and the service life of the glucose monitoring probe 1 can be extended.

[0082] In addition, in some examples, the semipermeable membrane 130 can also have biocompatibility. Thus, the use of the biocompatible membrane 140 can be omitted, and the production cost can be reduced.

[0083] In other examples, the permeability of the formed membrane to the analyte of interest can be adjusted by a modifier. For example, the hydrophilic modifier includes polyethylene glycol, a hydroxyl group, or a polyhydroxy modifier. Thus, the biocompatibility of the membrane formed by the polymer can be increased, and the biocompatible membrane 140 can be replaced.

[0084] In the present embodiment, the biocompatible membrane 140 can cover the entire glucose monitoring probe 1. In some examples, the biocompatible membrane 140 can cover only the implanted portion 1b of the glucose monitoring probe 1 implanted in the body. Thus, the use of raw materials can be reduced.

[0085] In the present embodiment, the service life of the glucose monitoring probe 1 can be 1 day to 24 days, preferably 7 days to 14 days. In addition, as described above, by the semi-permeable membrane 130, the entry of part of the glucose molecules and the electroactive interfering substances can be limited, and the linear range of the glucose monitoring probe 1 can be effectively expanded, and the sensing layer 120 can be better reacted with the glucose oxidase or dehydrogenase, and the service life of the glucose monitoring probe 1 can be kept stable.

[0086] In addition, the glucose monitoring probe 1 can also be used in ordinary detection, such as single detection or short-time monitoring. For example, the monitoring time can be 1 hour to 24 hours or 24 hours to 36 hours.

[0087] In addition, the addition of the biocompatible membrane 140 can keep the service life of the glucose monitoring probe 1 at 1 day to 24 days, for example, can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, so that the user can select the glucose monitor G with different service life of the glucose monitoring probe 1 according to different needs (such as price, etc.).

[0088] In the present embodiment, as described above, the glucose monitoring probe 1 can also include a reference electrode 20 and a counter electrode 30 (see Figure 2 ). Specifically, as shown in Figure 3 , the implanted part 1b of the glucose monitoring probe 1 can include a reference electrode 20 and a counter electrode 30.

[0089] In the present embodiment, the glucose monitoring probe 1 implanted into the skin can perform an oxidation-reduction reaction with the glucose in the tissue fluid or blood through the glucose oxidase or dehydrogenase in the working electrode 10, and form a loop with the counter electrode 30 to generate a current signal.

[0090] In the present embodiment, the reference electrode 20 can form a known and fixed potential difference with the tissue fluid or blood. In this case, the potential difference between the working electrode 10 and the tissue fluid or blood can be measured through the potential difference formed by the reference electrode 20 and the working electrode 10, so as to accurately grasp the voltage generated by the working electrode 10. Therefore, the electronic system 2 can automatically adjust and maintain the stability of the voltage at the working electrode 10 according to the pre-set voltage value, so as to ensure that the measured current signal can accurately reflect the glucose concentration value.

[0091] In addition, in the present embodiment, the working electrode 10, the reference electrode 20 and the counter electrode 30 of the implanted part 1b adopt a dispersed arrangement method, but the embodiments of the present disclosure are not limited thereto, and can also include side-by-side (parallel) arrangement.

[0092] In addition, in the present embodiment, the glucose monitoring probe 1 is not limited to a planar probe, but can also be a linear probe, a probe having stacked electrodes or layered electrodes, and a probe having co-planar electrodes in which electrodes are arranged on the same plane.

[0093] In some examples, the reference electrode 20 can not be used when the potential difference between the working electrode 10 and the tissue fluid or blood does not fluctuate greatly.

[0094] In the present embodiment, the counter electrode 30 can be made of platinum, silver, silver chloride, palladium, titanium, or iridium. Thereby, the electrochemical reaction at the working electrode 10 can be affected without having good conductivity. However, the present embodiment is not limited thereto, and in other examples, the counter electrode 30 can also be made of at least one selected from gold, glassy carbon, graphite, silver, silver chloride, palladium, titanium, or iridium. Thereby, the influence on the working electrode 10 can be reduced while having good conductivity.

[0095] In addition, in some examples, the working electrode 10, the counter electrode 30, and the reference electrode 20 can use the same material.

[0096] In addition, in the present embodiment, the glucose monitoring probe 1 can include two or more electrodes. For example, the glucose monitoring probe 1 can include only two electrodes, the working electrode 10 and the counter electrode 30, and in addition, the glucose monitoring probe 1 can include an additional reference electrode in addition to the working electrode 10, the reference electrode 20, and the counter electrode 30. In this case, the potential difference of the working electrode 10 can be more accurately obtained, and the voltage of the working electrode 10 can be grasped, and thus a more accurate current can be obtained.

[0097] In the present embodiment, as described above, the connection portion 1a of the glucose monitoring probe 1 includes a plurality of contacts (contact points). The number of contacts is equal to the number of electrodes of the implantation portion 1b of the glucose monitoring probe 1. The contacts are connected by leads (wires) to the electrodes of the implantation portion 1b.

[0098] In the present embodiment, as shown in FIG. 1, the number of electrodes of the implantation portion 1b of the glucose monitoring probe 1 is three. Accordingly, the connection portion 1a includes three contacts (contact points), which are a contact 41, a contact 42, and a contact 43. However, the present embodiment is not limited thereto, and for example, the number of electrodes of the implantation portion 1b can be two or more than four electrodes, and accordingly, the connection portion 1a can include two or more than four contacts (contact points). Figure 3

[0099] ​In the present embodiment, the contacts 41, 42 and 43 can each be in the shape of a disc, for example, and can be formed as a pad. Alternatively, the contacts 41, 42 and 43 can also be formed as a solder point. In other examples, the contacts 41, 42 and 43 can also be in the shape of a rectangle, an ellipse or other irregular shape.

[0100] In the present embodiment, the current signal generated by the implanted portion 1b of the glucose monitoring probe 1 can be transmitted through the base layer 110 and the transmission wire to the contacts of the connecting portion 1a. That is, the implanted portion 1b of the glucose monitoring probe 1 is connected to the connecting portion 1a, and the connecting portion 1a is connected to the electronic system 2 via the plurality of contacts, and thus the current signal obtained by the working electrode 10 is transmitted to the electronic system 2 through the contacts of the connecting portion 1a for analysis. The electronic system 2 can analyze the current signal to obtain a glucose concentration signal.

[0101] In addition, in some examples, the electronic system 2 can transmit the glucose concentration signal to an external reading device by wireless communication, for example, Bluetooth, wifi, etc. The reading device (not shown) can receive the glucose concentration signal transmitted by the electronic system 2 and display the glucose concentration value. In addition, the glucose monitoring probe 1 according to the present embodiment can achieve continuous monitoring, and thus can achieve the purpose of continuously monitoring the glucose concentration value of the human body for a long time (for example, 1 day to 24 days). In addition, in some examples, the reading device can be a reader or a mobile phone APP.

[0102] In addition, in the present embodiment, the glucose monitoring probe 1 and the electronic system 2 can not need to be calibrated during in vivo use. In addition, the glucose monitoring probe 1 and the electronic system 2 can be calibrated in advance before leaving the factory. Thus, the user can eliminate the trouble of calibrating the monitoring system by finger blood on a regular basis, and reduce the potential source of reading error of the monitoring module during use.

[0103] In the present embodiment, the electronic system 2 can be made of a flexible PCB and a flexible battery. Thus, it can be closely attached to the skin, reducing the impact on the user's daily life. In some examples, the electronic system 2 can have a circular shape. In addition, in some examples, the electronic system 2 can also have a waterproof shell and a waterproof adhesive plaster, so that it can not affect the user's daily activities such as swimming or bathing when in use.

[0104] In the present embodiment, the glucose monitoring probe 1 can acquire glucose concentration in interstitial fluid or blood. However, the present embodiment is not limited thereto, and for example, by changing the sensing layer 120 on the glucose monitoring probe 1, data of other body fluid components other than glucose can also be acquired, where the body fluid components can be, for example, acetylcholine, amylase, bilirubin, cholesterol, chorionic gonadotropin, creatine kinase, creatine, creatinine, DNA, fructosamine, glucose, glutamine, growth hormone, hormones, ketone bodies, lactate, oxygen, peroxide, prostate specific antigen, prothrombin, RNA, thyroid stimulating hormone, and troponin, etc.

[0105] In other examples, the concentration of drugs in the body fluid can also be monitored, such as antibiotics (e.g., gentamicin, vancomycin, etc.), digitalis glycosides, digoxin, theophylline, and warfarin, etc.

[0106] In the present embodiment, first, the sensing layer 120 is formed on the base layer 110 of the working electrode 10, then the semi-permeable membrane 130 coating is formed on the sensing layer 120, and finally, the biocompatible membrane 140 layer is formed on the semi-permeable membrane 130 coating. In this way, the service life of the glucose monitoring probe 1 is prolonged, the interference of other factors is reduced, and the reaction speed of the glucose monitoring probe 1 to glucose is also improved.

[0107] Hereinafter, the manufacturing method of the working electrode 10 of the glucose monitoring probe 1 will be described in detail with reference to the accompanying drawings.

[0108] Figure 8 is a flow chart illustrating the manufacturing method of the working electrode 10 of the glucose monitoring probe 1 according to the embodiment of the present disclosure. Figure 9 is a flow chart illustrating the manufacturing method of the semi-permeable membrane 130 of the working electrode 10 of the glucose monitoring probe 1 according to the embodiment of the present disclosure.

[0109] In the present embodiment, the manufacturing method of the working electrode 10 of the glucose monitoring probe 1 can include (see Figure 8) : preparing a flexible substrate, and forming a base layer 110 on the flexible substrate (step S110); preparing a sensing layer 120 reagent including a redox polymer, a glucose oxidase, a carbon nanotube 121, and a cross-linking agent (step S120); coating the sensing layer reagent on the base layer 110, and forming the sensing layer 120 (step S130); forming a semi-permeable membrane 130 that controls the passage of glucose molecules on the sensing layer 120 (step S140); and forming a biocompatible membrane 140 on the semi-permeable membrane 130 (step S150). In this case, the carbon nanotube 121 is included in the sensing layer 120. Thereby, the working electrode 10 working voltage is reduced, the interference of other factors is reduced, and the reaction sensitivity of the probe to glucose is improved, and the linear range of the glucose monitoring probe 1 response to glucose can be increased, and the service life of the probe can be extended.

[0110] As described above, in step S110, a flexible substrate is prepared, and a base layer 110 is formed on the flexible substrate. In some examples, the base layer 110 can also be made by one or more of plating, evaporation, printing, or extrusion, etc.

[0111] In the present embodiment, in some examples, in step S130, the sensing layer 120 can be a glucose oxidase sensing layer, or a glucose dehydrogenase sensing layer.

[0112] In some examples, in step S110, the redox polymer can have a covalent bond, a coordination bond, or an ionic bond. In some examples, the redox polymer can be a metal polymer that functions as a redox. The metal polymer here can be, for example, a metal polymer containing a covalent bond, a coordination bond, or an ionic bond. In some examples, the metal polymer can be selected from at least one of poly(vinyl ferrocene), quaternized poly(4-vinylpyridine) of ferricyanide, quaternized poly(l-vinylimidazole) of ferricyanide, quaternized poly(4-vinylpyridine) of ferrocyanide, quaternized poly(l-vinylimidazole) of ferrocyanide, osmium 2,2'-bipyridine complex coordination to poly(l-vinylimidazole), osmium 2,2'-bipyridine complex coordination to poly(4-vinylpyridine), cobalt 2,2'-bipyridine complex coordination to poly(l-vinylimidazole), or cobalt 2,2'-bipyridine complex coordination to poly(4-vinylpyridine).

[0113] In the manufacturing method related to the present embodiment, as described above, in step S120, a sensing layer 120 reagent including a redox polymer, a glucose oxidase, a carbon nanotube 121, and a cross-linking agent is prepared. Figure 9As shown, step S140 can include forming the interference-resistant layer 132 on the sensing layer 120 (step S141) and then forming the diffusion control layer 131 on the interference-resistant layer (step S142). In this way, the tissue fluid or blood components passing through the semi-permeable membrane 130 can be first reduced by the interference-resistant layer 132, and then the interference substances can be blocked outside the semi-permeable membrane 130 by the diffusion control layer 131.

[0114] In some examples, the order of step S141 and step S142 in step S140 can be interchanged. That is, the diffusion control layer 131 can be formed on the glucose oxidase or dehydrogenase layer first (step S142), and then the interference-resistant layer 132 can be formed on the diffusion control layer 131 (step S141). In this way, the interference of impurities to the working electrode 10 can also be reduced, the detection result can be prevented from being inaccurate, and the service life of the glucose monitoring probe 1 can be prolonged.

[0115] Although the present disclosure is specifically described above in combination with the drawings and examples, it should be understood that the above description does not limit the present disclosure in any form. Those skilled in the art can make modifications and changes to the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1.A method for manufacturing a working electrode of a glucose monitoring probe, the method comprising: preparing a substrate; forming a base layer on the substrate; preparing a sensing layer reagent including a redox polymer, a glucose enzyme, a carbon nanotube, and a cross-linking agent, the carbon nanotube being modified with an amino group to form a covalent bond with the redox polymer and the glucose enzyme, and the working electrode requiring a lower working voltage due to catalytic action of the carbon nanotube; coating the sensing layer reagent on the base layer to form a sensing layer, the sensing layer being a glucose oxidase sensing layer or a glucose dehydrogenase sensing layer; forming a semi-permeable membrane on the sensing layer to control a glucose molecule passage rate; and forming a biocompatible membrane on the semi-permeable membrane. 2.The method of claim 1, wherein the carbon nanotube is added to the sensing layer reagent by being dissolved in a solvent. 3.The method of claim 1, wherein the sensing layer reagent is formed by at least one of spin coating, dip coating, drop coating, or spray coating. 4.The method of claim 1, wherein the base layer is made of at least one selected from gold, glassy carbon, graphite, silver, silver chloride, palladium, titanium, iridium. 5.The method of claim 1, wherein the base layer is disposed on the substrate by physical vapor deposition, chemical vapor deposition, electroplating, electroless plating, vacuum plating, screen printing, extrusion, or electrolytic deposition. 6.The method of claim 1, wherein the biocompatible membrane is made of a plant material, the plant material being at least one of sodium alginate, tragacanth gum, pectin, gum arabic, xanthan gum, guar gum, agar, or a natural material derivative thereof. 7.The method of claim 1, wherein the semi-permeable membrane includes an anti-interference layer and a diffusion control layer, the anti-interference layer being formed on the sensing layer and the diffusion control layer being formed on the anti-interference layer. 8.The method of claim 7, wherein the diffusion control layer has a 30 to 80-fold reduction in the passage rate of an entering substance. 9.The method of claim 8, wherein the anti-interference layer blocks an interfering substance outside the semi-permeable membrane, the interfering substance including uric acid, ascorbic acid, acetaminophen. 10.The method of claim 1, wherein the carbon nanotube has a mass percentage of 5% to 10% in the sensing layer reagent. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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