Method for the production of working electrodes for glucose sensors suitable for mass production

Abstract

The present disclosure describes a preparation method of a working electrode of a glucose sensor suitable for mass production, the preparation method comprising the steps of: preparing a substrate layer having insulation; forming a conductive layer on the substrate layer, the conductive layer entirely covering the substrate layer as a whole, forming a plurality of limiting grooves with uniform size and shape on the conductive layer; and respectively dropping a predetermined amount of glucose sensitive reagent into the plurality of limiting grooves in a manner such that the glucose sensitive reagent is retained within the plurality of limiting grooves, and curing the glucose sensitive reagent to form a plurality of sensing parts. According to the present disclosure, the area and the uniformity of the morphology of the droplets of the glucose sensor can be controlled simply and conveniently, and the preparation method of the working electrode of the glucose sensor.

Description

[0001] This application is a divisional application of the invention entitled "Working Electrode of Glucose Sensor and Preparation Method thereof", filed on March 11, 2020, with application number 202010167544.2. Technical Field

[0002] This disclosure generally relates to the field of biosensors, and more specifically to a method for preparing a working electrode for a glucose sensor suitable for mass production. Background Technology

[0003] Diabetes is a metabolic disease characterized by high blood sugar. Currently, there is no cure, and diabetic patients often manage their condition by monitoring their blood sugar. The main methods of blood sugar monitoring include traditional blood glucose monitoring and continuous glucose monitoring (CGM). Compared to traditional blood glucose monitoring, CGM technology can typically monitor a patient's blood sugar continuously for at least 24 hours, providing real-time information on the patient's blood sugar levels throughout the day, effectively reflecting hypoglycemia and blood sugar fluctuations. Continuous glucose monitoring can be achieved using glucose sensors.

[0004] A glucose sensor typically consists of a sensor probe and a processing device that records the sensor information. During continuous glucose monitoring (CGM), the sensor probe is often implanted subcutaneously. The working electrode of the probe contains a glucose-sensitive reagent that reacts specifically with glucose molecules at the implantation site to generate an electrical signal. The processing device then processes this signal to obtain the blood glucose level and the patient's blood glucose changes.

[0005] To obtain accurate blood glucose values, the production of glucose sensors requires ensuring that all sensors manufactured in a batch have a consistent initial sensitivity for batch calibration. Therefore, to guarantee consistent initial sensitivity across batches of glucose sensors, consistency in process parameters, such as the area and morphology of the sensing element, is crucial. The sensing element is formed by drop-coating a glucose-sensitive reagent onto the conductive layer of the working electrode and then curing it.

[0006] In existing drop coating processes, the control of the drop coating solution is achieved by improving the surface roughness of the drop coating, thereby ensuring that the droplets have a consistent wetting angle on the surface, and thus controlling the area and morphology of the droplets. However, it is difficult to achieve completely uniform surface roughness in drop coating, which makes it difficult to guarantee the consistency of droplet area and morphology each time drop coating is performed. Consequently, it is difficult to ensure that each glucose sensor produced in batches has a consistently high initial sensitivity. Summary of the Invention

[0007] This disclosure was made in view of the above-mentioned situation, and its purpose is to provide a working electrode of a glucose sensor that can easily and conveniently control the area and morphological consistency of the droplets and its preparation method, which is beneficial to improving the consistency of the sensing parts of various glucose sensors produced in batches, thereby improving the consistency of the initial sensitivity of various glucose sensors produced in batches.

[0008] Therefore, a first aspect of this disclosure provides a working electrode for a glucose sensor, characterized in that it comprises: a substrate layer made of an insulating material and having a pretreated surface with a predetermined roughness; a conductive layer disposed on the substrate layer and having at least one limiting groove arranged along a predetermined direction of the conductive layer, the at least one limiting groove being formed on the surface of the conductive layer by coating the conductive layer with a photosensitive material to form a photosensitive layer, curing the photosensitive layer according to a predetermined pattern, and then removing the uncured photosensitive layer, the predetermined pattern including at least one closed pattern; and a sensing portion formed by dripping a predetermined amount of glucose-sensitive reagent onto the at least one limiting groove and then curing it.

[0009] In the working electrode of the glucose sensor disclosed in the first aspect, there is no need for complex surface treatment methods to obtain droplets with the same wetting angle. Instead, at least one limiting groove with a certain shape is formed on the surface of the working electrode by curing a photosensitive material. A predetermined amount of glucose-sensitive reagent can be contained in the limiting groove and form a morphology with the same shape as the limiting groove. This makes it easy to control the consistency of the area and morphology of the sensing part in the working electrode of mass production, and obtain a glucose sensor with consistent process parameters.

[0010] In the working electrode of the glucose sensor according to the first aspect of this disclosure, optionally, the at least one limiting groove is arranged in a straight line. This improves the drop-coating efficiency.

[0011] Optionally, in the working electrode of the glucose sensor according to the first aspect of this disclosure, a semi-permeable membrane for controlling the passage of glucose molecules is further covered on the sensing part. This allows for control of the number of glucose molecules passing through the semi-permeable membrane.

[0012] In the working electrode of the glucose sensor according to the first aspect of this disclosure, optionally, the glucose-sensitive reagent completely fills the limiting groove. In this case, the glucose-sensitive reagent is confined within the limiting groove, thereby enabling control over the area and morphology of the glucose-sensitive reagent and preventing undried glucose-sensitive reagent from flowing everywhere and forming irregular shapes.

[0013] In the working electrode of the glucose sensor according to the first aspect of this disclosure, the limiting groove may optionally be a circular groove or an oval groove. This allows the glucose-sensitive reagent, which is readily applied, to flow within the limiting groove and fill its edges to form the desired morphology.

[0014] In the working electrode of the glucose sensor according to the first aspect of this disclosure, optionally, the material of the conductive layer is selected from at least one of glassy carbon, graphite, silver, silver chloride, platinum, palladium, platinum-iridium, titanium, gold, or iridium. This results in good conductivity.

[0015] In the working electrode of the glucose sensor according to the first aspect of this disclosure, optionally, the glucose-sensitive reagent is capable of chemically reacting with glucose, and the glucose-sensitive reagent includes a glucose enzyme, a metal polymer, and a cross-linking agent. Thus, the glucose-sensitive reagent can be conveniently attached to the conductive layer's limiting groove and specifically react with glucose.

[0016] A second aspect of this disclosure provides a method for fabricating a working electrode of a glucose sensor, the working electrode comprising: a substrate layer made of an insulating material and pretreated to form a surface with a predetermined roughness; a conductive layer disposed on the substrate layer and having at least one locating groove arranged along a predetermined direction of the conductive layer; and a sensing portion disposed on the conductive layer; the fabrication method being characterized by comprising the following steps: (a) preparing an insulating substrate layer and pretreating the substrate layer to give its surface a predetermined roughness; (b) forming the conductive layer on the substrate layer and coating the conductive layer with a biocompatible photosensitive layer, patterning the photosensitive layer according to a predetermined pattern to form the at least one locating groove on the conductive layer, the predetermined pattern including at least one closed pattern; (c) drop-coating a predetermined amount of glucose-sensitive reagent onto the at least one locating groove in such a manner that the glucose-sensitive reagent is held within the range of the at least one locating groove, and curing the glucose-sensitive reagent to form the sensing portion.

[0017] In the method for preparing the working electrode of the glucose sensor disclosed in the second aspect, there is no need for a complex surface treatment method to obtain droplets with the same wetting angle. Instead, at least one limiting groove with a certain shape is formed on the surface of the working electrode by curing a photosensitive material. A predetermined amount of glucose-sensitive reagent can be contained in the limiting groove and form a morphology with the same shape as the limiting groove. This allows for convenient control of the consistency of the area and morphology of the sensing part in the working electrode produced in mass production, thereby obtaining a glucose sensor with consistent process parameters.

[0018] In the method for fabricating the working electrode of the glucose sensor according to the second aspect of this disclosure, optionally, in step (b), the patterning method includes: partially curing the photosensitive layer by irradiating a mask having the predetermined pattern placed above the photosensitive layer with light or by irradiating the photosensitive layer with a laser source constituting the predetermined pattern, and then removing the uncured photosensitive layer to form the at least one locating groove. Thus, locating grooves with predetermined patterns can be formed on a conductive layer using the properties of the photosensitive material.

[0019] In the method for fabricating the working electrode of the glucose sensor according to the second aspect of this disclosure, optionally, the conductive layer has a plurality of the limiting grooves, and the sum of the surface areas of the plurality of limiting grooves is not less than 50% of the surface area of ​​the conductive layer. This increases the contact area between the glucose molecules of the sensing element and the conductive layer. Attached Figure Description

[0020] Figure 1 This is a schematic diagram showing the usage status of a glucose monitoring probe according to an embodiment of the present disclosure.

[0021] Figure 2 This is a planar structural diagram showing the glucose sensor according to an embodiment of the present disclosure.

[0022] Figure 3 It shows Figure 2 A schematic diagram of the glucose monitoring probe in a bent state.

[0023] Figure 4 This is a three-dimensional schematic diagram showing the working electrode of the embodiments of this disclosure not covered by a semi-permeable membrane.

[0024] Figure 5 This is a top view showing the working electrode involved in the embodiments of this disclosure.

[0025] Figure 6 It shows Figure 5 The cross-sectional view along line B-B' when the working electrode is covered with a semi-permeable membrane.

[0026] Figure 7 This is a flowchart illustrating a method for preparing a working electrode according to an embodiment of the present disclosure.

[0027] Figures 8(a)-8(e) This is a three-dimensional schematic diagram illustrating a method for preparing a working electrode according to an embodiment of the present disclosure. Detailed Implementation

[0028] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are used for the same components, and repeated descriptions are omitted. Furthermore, the drawings are merely schematic diagrams, and the proportions of the components or the shapes of the components may differ from actual figures.

[0029] Furthermore, the subheadings and similar terms used in the following description of this disclosure are not intended to limit the content or scope of this disclosure; they are merely intended to serve as reading prompts. Such subheadings should not be construed as dividing the content of the article, nor should the content under a subheading be limited to the scope of that subheading.

[0030] In this disclosure, the glucose sensor can be simply referred to as a "sensor," the probe of the glucose sensor can be simply referred to as a "probe," the working electrode of the glucose sensor can be simply referred to as a "working electrode," and the preparation method of the working electrode of the glucose sensor can be simply referred to as a "preparation method." Furthermore, the working electrode of the glucose sensor and its preparation and drop-coating method disclosed in this disclosure are not only applicable to glucose sensors but also to electrodes for monitoring other physiological parameters, such as uric acid detection sensors for detecting uric acid and cholesterol monitoring sensors for detecting cholesterol, simply by replacing the sensitive reagent in the sensing part of the working electrode with a corresponding enzyme that specifically reacts with its target analyte. In addition to electrodes for monitoring physiological parameters, the method of controlling the drop-coating morphology in this disclosure by forming a limiting groove of a predetermined shape with a volume matching the drop-coating amount at the drop-coating site is also applicable to other production processes requiring control of the drop-coating morphology.

[0031] Figure 1 This is a schematic diagram showing the usage status of a glucose monitoring probe according to an embodiment of the present disclosure. Figure 2 This is a planar structural diagram showing the glucose sensor according to an embodiment of the present disclosure. Figure 3 It shows Figure 2 A schematic diagram of the glucose monitoring probe in a bent state.

[0032] In this embodiment, the glucose sensor probe S may also be referred to as an implantable glucose monitoring probe, the probe S of a glucose monitor, or simply probe S.

[0033] In this embodiment, the portable glucose monitor G may include a glucose sensor probe S and an electronic system S' connected to the probe S. By implanting the probe S of the portable glucose monitor G into the body surface and bringing it into contact with the tissue fluid, the probe S can sense the glucose concentration signal of the tissue fluid. By transmitting this glucose concentration signal to the electronic system S', the corresponding glucose concentration can be obtained.

[0034] Specifically, a portion of the glucose sensor probe S (particularly the sensing part) can be implanted, for example, on the surface of the human body, and come into contact with the tissue fluid within the body. Additionally, another portion of the glucose sensor probe S is connected to an electronic system S' located on the body surface. When the portable glucose monitor G is operating, the glucose sensor probe S reacts with the tissue fluid within the body to generate a sensing signal (e.g., an electrical signal), and transmits this signal to the electronic system S' on the body surface. The electronic system S' processes the sensing signal to obtain the glucose concentration. Although... Figure 1 The location of the glucose sensor probe S is shown, but this embodiment is not limited to this. For example, the glucose sensor probe S can also be configured in the abdomen, waist, legs, etc.

[0035] In this embodiment, although the glucose sensor probe S directly detects glucose in the tissue fluid, the glucose concentration of the tissue fluid is strongly correlated with the glucose concentration of the blood, and the glucose concentration of the blood can be determined by the glucose in the tissue fluid.

[0036] In this embodiment, the glucose sensor probe S may include a working electrode 1, a reference electrode 2, and a counter electrode 3 (see...). Figure 2 In some examples, the working electrode 1, reference electrode 2, and counter electrode 3 may all have an insulating base layer 10 (described later) as a substrate. In some examples, the base layer 10 for the working electrode 1, reference electrode 2, and counter electrode 3 may be a single substrate or partially divided into three parts. Additionally, the glucose sensor probe S may include a contact 4 connected to the working electrode 1 via a lead, a contact 5 connected to the working electrode 2 via a lead, and a contact 6 connected to the reference electrode 3 via a lead. In some examples, the glucose sensor probe S may be connected to the electronic system 2 via contacts 4, 5, and 6.

[0037] In this embodiment, for ease of explanation, the glucose sensor probe S can be divided into a connecting portion Sa and an implanted portion Sb (see...). Figure 3 ). Figure 3 The straight line A-A' in the diagram roughly indicates the approximate location of the glucose sensor probe S in the skin when implanted into the tissue surface. After implantation, the implanted portion Sb is in the superficial layer of the skin, and the electronic system S' is closely attached to the skin surface. The connecting portion Sa of the glucose sensor probe S (see...) Figure 3 It is connected to the electronic system S' and located on the skin surface.

[0038] Figure 4 This is a three-dimensional schematic diagram showing the working electrode of the embodiments of this disclosure not covered by a semi-permeable membrane.

[0039] In the mass production of glucose sensors, the consistency of process parameters within the same batch is crucial. If the process parameters are consistent, it's unnecessary to calibrate each sensor individually within the batch; a factory batch calibration of the entire batch is sufficient. To achieve good process parameter consistency, it's necessary to control at least one of the following: the area and morphology of the sensing portion 30 of the working electrode 1, and the film thickness and diffusion coefficient of the conductive layer 20 and the semi-permeable membrane 40 on the sensing portion 30. The sensing portion 30 of the working electrode 1 is primarily formed through a drop-coating process. However, during drop-coating, the morphology and roughness of the drop-coated surface cannot be perfectly uniform, and the drop-coating reagent tends to flow irregularly on the surface, leading to uncontrollable area and morphology of the sensing portion 30 formed by the drop-coating of the glucose-sensitive reagent 310. Therefore, controlling the consistency of the area and morphology of the sensing portion 30 of the working electrode 1 is essential for achieving good process parameter consistency.

[0040] In this embodiment, see Figure 4 The working electrode 1 of the glucose sensor may include: a substrate 10, which is made of an insulating material and has a pre-treated surface with a specified roughness; a conductive layer 20, which is disposed on the substrate 10 and has at least one limiting groove 210 arranged along a predetermined direction of the conductive layer 20, wherein the at least one limiting groove 210 is formed on the surface of the conductive layer 20 by coating a photosensitive material onto the surface of the conductive layer 20 to form a photosensitive layer 220, and by curing the photosensitive layer 220 according to a predetermined pattern and then removing the uncured photosensitive layer 220, wherein the predetermined pattern includes at least one closed pattern arranged along the predetermined direction of the conductive layer 20; and a sensing part 30, which is formed by dripping a predetermined amount of glucose-sensitive reagent 310 onto the at least one limiting groove 210 and then curing it.

[0041] According to the working electrode 1 of the glucose sensor disclosed herein, there is no need for complex surface treatment methods to obtain droplets with the same wetting angle. Instead, at least one limiting groove 210 with a certain shape is formed on the surface of the working electrode 1 by curing a photosensitive material. A predetermined amount of glucose-sensitive reagent 310 can be contained in the limiting groove 210 and form a morphology with the same shape as the limiting groove 210. This allows for convenient control of the consistency of the area and morphology of the sensing part 30 in the working electrode 1 during mass production, resulting in a glucose sensor with consistent process parameters. Furthermore, by changing the volume, shape, and drop amount of the limiting groove 210, the area and morphology of the sensing part 30 can be easily changed.

[0042] Figure 5 This is a top view showing the working electrode involved in the embodiments of this disclosure. Figure 6 It shows Figure 5 The cross-sectional view along line B-B' when the working electrode is covered with a semi-permeable membrane.

[0043] (Basal layer 10)

[0044] In this embodiment, as described above, the working electrode 1 may include a substrate layer 10. In some examples, the substrate layer 10 may be made of an insulating material.

[0045] In some examples, the substrate 10 can be selected from flexible insulating materials. The flexible insulating material can be at least one of polyimide (PI), polyethylene terephthalate (PET), parylene, silicone resin, polydimethylsiloxane (PDMS), polyethylene glycol (PEG), or polytetrafluoroethylene resin (Teflon), polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PEN). This allows the substrate 10 to possess both flexibility and insulation, reducing discomfort after implantation.

[0046] In other examples, the base layer 10 can be made of a non-flexible insulating material. Non-flexible materials can generally include ceramics, polymethyl methacrylate (PMMA), alumina, or silica. In this case, the base layer 10 can have excellent support properties. Furthermore, when the base layer 10 is a non-flexible insulating material, it can be made into an easily implantable shape, such as a needle tip. In this case, the probe including the working electrode 1 can be implanted into the body surface (e.g., superficial skin) without the need for an auxiliary implantation device (not shown) such as a needle applicator.

[0047] In this embodiment, in some examples, the surface of the substrate 10 may be pretreated to achieve a specified roughness. Pretreatment may include polishing, plasma gas cleaning, ultrasonic cleaning, nitrogen drying, etc. The specified roughness refers to a range of surface roughness that facilitates the adhesion of the material forming the conductive layer 20 to the substrate 10. This facilitates the formation of the conductive layer 20 on the substrate 10 and reduces the possibility of delamination or slippage.

[0048] In some examples, the thickness of the substrate 10 can be 100-200 μm. Thus, the substrate 10 can have good insulation and support properties.

[0049] (Conductive layer 20)

[0050] In this embodiment, as described above, the working electrode 1 may include a conductive layer 20. The conductive layer 20 may be disposed on the substrate layer 10. In some examples, the conductive layer 20 may be disposed on the substrate layer 10 by means of screen printing, inkjet printing, vacuum magnetron sputtering, evaporation, or plating.

[0051] In some examples, the conductive layer 20 can be made of a metallic conductive material. The metallic conductive material can be selected from at least one of silver, platinum, gold, titanium, palladium, iridium, and niobium, or an alloy thereof. This allows the conductive layer 20 to have good conductivity. In other examples, the conductive layer 20 can also be made of a conductive non-metallic material. The conductive non-metallic material can be selected from conductive non-metallic materials such as glassy carbon and graphite.

[0052] In this embodiment, in some examples, the conductive layer 20 may be flat. This makes it easier to form at least one limiting groove 210 on the conductive layer 20.

[0053] In this embodiment, in some examples, the thickness of the conductive layer 20 can be 5-25 μm. Therefore, the conductive layer 20 is not so thick as to affect the bending resistance of the working electrode 1.

[0054] In some examples, the conductive layer 20 may cover the entire substrate layer 10. In other words, the conductive layer 20 may completely cover the substrate layer 10 without being divided into multiple unconnected portions. In other examples, the conductive layer 20 may only cover a portion of the substrate layer 10. In other words, the conductive layer 20 may only completely cover a portion of the substrate layer 10 to form a conductive layer 20 of sufficient area, which is the minimum area on which at least one limiting groove 210 of the predetermined pattern can be formed.

[0055] In some examples, a nanoparticle layer (not shown), such as gold nanoparticles or platinum nanoparticles, can be further disposed on the surface of the conductive layer 20. In some examples, the nanoparticle layer can be porous. In some examples, the nanoparticle layer can be disposed on the surface of the conductive layer 20 by means of electroplating, sputtering, etc. This increases the contact area between the enzyme in the glucose-sensitive reagent 310 of the sensing unit 30 and the conductive layer 20.

[0056] In some examples, the surface of the conductive layer 20 or the surface of the limiting groove 210 may be further provided with a three-dimensional nanofiber network structure (not shown) composed of filamentous nanofibers. The three-dimensional nanofiber network structure can be formed on a nanoparticle layer as a base; that is, based on the nanoparticles in the nanoparticle layer, fine and long filamentous nanofibers can be formed on the nanoparticles by means of conductive materials such as electroplating polyaniline, with several filamentous nanofibers intersecting to form a three-dimensional nanofiber network structure. This can improve the adhesion of glucosamine and provide better conductivity.

[0057] In some examples, the thickness of the photosensitive layer 220 can be less than the thickness of the conductive layer 20. This allows for the formation of a suitable depth of the limiting groove 210, and also facilitates the semi-permeable membrane 40 covering the limiting groove 210 or the sensing element 30. In other examples, the thickness of the photosensitive layer 220 can be equal to or greater than the thickness of the conductive layer 20.

[0058] In this embodiment, in some examples, the conductive layer 20 may have at least one limiting groove 210 arranged along a predetermined direction of the conductive layer 20. The predetermined direction of the conductive layer 20 may be, for example, the length direction of the conductive layer 20. This allows for optimization of the implantation area of ​​the working electrode 1.

[0059] In this embodiment, in some examples, at least one limiting groove 210 is formed on the surface of the conductive layer 20 by coating the surface of the conductive layer 20 with a photosensitive material to form a photosensitive layer 220, curing the photosensitive layer 220 according to a predetermined pattern, and then removing the uncured photosensitive layer 220. The predetermined pattern includes at least one closed pattern arranged along a predetermined direction of the conductive layer 20. In other words, the projection of each limiting groove 210 onto the conductive layer 20 is a closed pattern, that is, the cured photosensitive layer 220 forms the wall of the limiting groove 210, and the wall of the limiting groove 210 forms a closed pattern shape. The surface of the conductive layer 20 and the wall formed by the curing of the photosensitive material constitute a limiting groove 210 with a certain volume. When a predetermined amount (not greater than the volume of the limiting groove 210) of glucose-sensitive reagent 310 is dropped into it, the glucose-sensitive reagent 310 can be well confined in the limiting groove 210 without leaking or diffusing from the sidewall.

[0060] In other examples, the photosensitive layer 220 may be absent, and at least one limiting groove 210 may be formed by printing a predetermined pattern directly on the conductive layer 20, for example, by mask printing.

[0061] In some examples, the predetermined pattern includes at least one closed figure arranged along a predetermined direction. In some examples, the number of closed figures can be, for example, 1, 3, 5, or 7. In some examples, the closed figures can be circles, ovals, rectangles, triangles, or irregular shapes.

[0062] In some examples, the size and shape of each closed pattern can be identical. In other examples, the size and shape of each closed pattern may not be exactly the same. However, the predetermined pattern including the closed patterns of different working electrodes 1 in the same batch should be consistent in order to control the volume of the glucose-sensitive reagent 310 applied, the overall area of ​​the formed sensing part 30, and the consistency of the morphology, thereby facilitating the control of the consistency of the initial sensitivity among different working electrodes 1 in the same batch.

[0063] In some examples, at least one closed pattern on the same working electrode 1 is arranged in a straight line. Thus, at least one limiting groove 210 arranged in a straight line can be obtained.

[0064] In some examples, at least one limiting groove 210 is arranged in a straight line. This improves map efficiency. Alternatively, the limiting grooves 210 (limiting groove 210a, limiting groove 210b, limiting groove 210c) can also be arranged in other shapes, such as curved or polygonal.

[0065] In some examples, the limiting groove 210 can be formed approximately at the center of the working electrode 1. Alternatively, the limiting groove 210 can also be a flat-bottomed groove. 。 Therefore, it can accommodate uncured glucose-sensitive reagent 310.

[0066] In this embodiment, the diameter or maximum width of the limiting groove 210 can be 100-150 μm. Therefore, the sensing unit 30 has a sufficiently large area, enabling the glucose sensor to have high sensitivity.

[0067] In some examples, the limiting groove 210 can be a circular groove or an oval groove. This allows the glucose-sensitive reagent 310, which is dispensed, to flow within the limiting groove 210 and fill its edges to form the desired morphology. In other examples, the limiting groove 210 can be a rectangular groove or an irregularly shaped groove. This allows the morphology of the limiting groove 210 to be optimized according to the design.

[0068] In some examples, when the conductive layer 20 has only one limiting groove 210, the surface area of ​​the limiting groove 210 is not less than the surface area of ​​the conductive layer 20. In some examples, multiple limiting grooves 210, for example... Figure 4 The sum of the surface areas of the limiting grooves 210a, 210b, and 210c shown (hereinafter referred to as 210 for ease of explanation) can be no less than 50% of the total surface area of ​​the conductive layer 20, for example, 50%, 60%, 70%, 80%, or 90%. This increases the contact area between glucose molecules in the sensing unit 30 and the conductive layer 20, thereby improving the sensitivity of the glucose sensor.

[0069] (Sensing Unit 30)

[0070] In this embodiment, as described above, the working electrode 1 may include a sensing unit 30. The sensing unit 30 may be disposed on at least one limiting groove 210 of the conductive layer 20. The sensing unit 30 is formed by dripping a predetermined amount of glucose-sensitive reagent 310 into the limiting groove 210 and then curing it. Different sensing units 30 can be obtained depending on the characteristics of different glucose-sensitive reagents 310 and the predetermined amount.

[0071] In some examples, the glucose-sensitive reagent 310 completely fills the limiting groove 210. In other words, the volume of a predetermined amount of glucose-sensitive reagent 310 is the same as the volume of the limiting groove 210, and the surface of the sensing part 30 is flush with the line connecting any two points on the upper edge of the limiting groove 210. In this case, the glucose-sensitive reagent 310 is confined within the limiting groove 210, thereby controlling the area and morphology of the sensing part 30 and preventing undried glucose-sensitive reagent 310 from flowing everywhere and forming irregular shapes.

[0072] In some examples, the sensing element 30 is slightly protruding in the limiting groove 210. In other words, the volume of the predetermined amount of glucose-sensitive reagent 310 is slightly larger than the volume of the limiting groove 210. Due to the surface tension, the liquid level of the unfixed glucose-sensitive reagent 310 is slightly higher than the upper surface of the conductive layer 20, but it does not diffuse beyond the edge of the side of the limiting groove 210, resulting in a large change in the morphology of the sensing element 30 after curing. Therefore, the area and morphology consistency of the sensing element 30 can still be well controlled.

[0073] In other examples, the surface of the sensing part 30 is lower than the upper surface of the conductive layer 20, that is, the volume of the predetermined amount of glucose-sensitive reagent 310 is smaller than the volume of the limiting groove 210, and the glucose-sensitive reagent 310 is completely contained in the limiting groove 210.

[0074] In this embodiment, the glucose-sensitive reagent 310 is capable of chemically reacting with glucose. In some examples, the glucose-sensitive reagent 310 may include a glucose enzyme, a metal polymer, and a cross-linking agent. Thus, the glucose-sensitive reagent 310 can easily adhere to the limiting groove 210 of the conductive layer 20 and react specifically with glucose.

[0075] In some examples, glucose-sensitive reagent 310 may be a mixed solution comprising an enzyme, a cationic polymer, and a redox mediator. For example, glucose-sensitive reagent 310 may be a mixed solution of, for example, glucose oxidase or dehydrogenase with a cationic polymer, a redox mediator such as ferricyanide, diazonium quinone, or ferrocene, and a cross-linking agent.

[0076] In some examples, the glucose-sensitive reagent 310 can be replaced according to the actual target analyte, that is, replaced with a sensitive reagent that can specifically react with the target analyte in the body. This allows for the detection of the concentration of target analytes other than glucose. For example, the glucose-sensitive reagent 310 can be replaced with specific reactants corresponding to acetylcholine, amylase, bilirubin, cholesterol, human chorionic gonadotropin, creatine kinase, creatine, creatine anhydride, DNA, fructosamine, glucose, glutamine, growth hormone, hormones, ketone bodies, lactate, oxygen, peroxides, prostate-specific antigen, prothrombin, RNA, thyroid-stimulating hormone, and troponin.

[0077] In this embodiment, the thickness of the sensing element 30 can be approximately 0.1 μm to 100 μm, preferably 2 μm to 10 μm. In some examples, the thickness of the sensing element 30 can be 10 μm. In this case, by controlling the thickness of the sensing element 30 within a certain range, the adhesion of the glucose-sensitive reagent 310 is prevented from decreasing, thus avoiding material detachment within the body. It also avoids problems such as insufficient glucose oxidase in the glucose-sensitive reagent 310 leading to inadequate reaction and inability to provide normal glucose concentration information.

[0078] In some examples, the glucosidase in the glucose-sensitive reagent 310 can be attached to and fixed on the surface of the working electrode 1 by methods such as physical adsorption, covalent cross-linking or embedding.

[0079] In some examples, the glucose-sensitive reagent 310 can be attached to a three-dimensional nanofiber network structure (not shown) formed of a conductive material such as polyaniline. This increases the amount of enzyme attached to the sensing element 30 and provides good electrical signal transmission performance.

[0080] In some examples, the glucose-sensitive reagent 310 may also include carbon nanotubes, with a mass percentage of 5% to 10%. This increases the adhesion of the glucose enzyme and promotes the specific reaction. In other examples, graphene, porous titanium dioxide, or conductive organic salts may be added to the glucose-sensitive reagent 310. This further enhances the promotion of the glucose enzyme reaction.

[0081] (Semi-permeable membrane 40)

[0082] In this embodiment, as described above, the working electrode 1 may further include a semi-permeable membrane 40. In some examples, the semi-permeable membrane 40 may be disposed on the entire exterior of the working electrode 1, that is, covering the entire surface of the working electrode 1 including the conductive layer 20 and the sensing part 30. This provides good diffusion control.

[0083] In other examples, the semi-permeable membrane 40 may be disposed only on the sensing part 30, that is, covering only the entire sensing part 30. This reduces the amount of raw materials used.

[0084] In this embodiment, in some examples, the semipermeable membrane 40 includes a diffusion control layer (not shown) for controlling the diffusion of glucose molecules. In this case, when glucose molecules in tissue fluid or blood enter the semipermeable membrane 40, the number of glucose molecules is reduced proportionally, so that when glucose molecules react with the glucose enzyme in the sensing unit 30, the glucose enzyme in the sensing unit 30 is in an excess state. The glucose concentration becomes the only factor limiting the current of the working electrode 1, thereby expanding the linear range of the glucose sensor when monitoring glucose concentration.

[0085] In some examples, the semipermeable membrane 40 also includes an anti-interference layer (not shown) stacked on top of the diffusion control layer. In other examples, the diffusion control layer may be positioned outside the anti-interference layer. In the semipermeable membrane 40, the diffusion control layer controls the diffusion of glucose molecules, while the anti-interference layer prevents the diffusion of non-glucose substances. In this case, the amount of tissue fluid or blood passing through the semipermeable membrane 40 can be reduced first, and then the anti-interference layer can block interfering substances outside the semipermeable membrane 40. Common interfering substances may include uric acid, ascorbic acid, acetaminophen, etc., which are ubiquitous in the body.

[0086] In some examples, the semi-permeable membrane 40 can be a biocompatible diffusion control material. This extends the lifespan of the sensor probe after implantation. In this embodiment, a sensing element 30 with controllable area and morphology can be obtained, thereby enabling the production of a glucose sensor with consistent process parameters.

[0087] The following combination Figure 7 The fabrication method of the working electrode 1 of the glucose sensor is described in Figure 8. For the specific parameters, materials and morphology of the substrate layer 10, conductive layer 20, sensing part 30 and semi-permeable membrane 40, please refer to the corresponding content above.

[0088] Figure 7 Figure 8 is a flowchart illustrating a method for preparing a working electrode according to an embodiment of the present disclosure. Figure 8 is a perspective view illustrating a method for preparing a working electrode according to an embodiment of the present disclosure.

[0089] In this embodiment, see Figure 7The working electrode 1 of the glucose sensor may include: a substrate 10, which is made of an insulating material and has a surface with a specified roughness after pretreatment; a conductive layer 20 disposed on the substrate 10 and having at least one limiting groove 210 arranged along a predetermined direction of the conductive layer 20; and a sensing part 30 disposed on the conductive layer 20. The method for preparing the working electrode 1 may include the following steps: (a) preparing an insulating substrate layer 10 and pre-treating the substrate layer 10 to give its surface a specified roughness (S10); (b) forming a conductive layer 20 on the substrate layer 10 (S201), coating a biocompatible photosensitive layer 220 on the conductive layer 20, and patterning the photosensitive layer 220 according to a predetermined pattern to form at least one limiting groove on the conductive layer 20 (S202) (S201 and S202 are two decomposition steps of S20); (c) dripping a predetermined amount of glucose-sensitive reagent 310 onto at least one limiting groove 210 so that the glucose-sensitive reagent 310 is held within the range of at least one limiting groove 210, and curing the glucose-sensitive reagent 310 to form a sensing part 30 (S30).

[0090] In addition, in some examples, the method for preparing the working electrode 1 may also include (see...) Figure 7 Step (d) involves providing a semipermeable membrane 40 outside the sensing unit 30 to control the passage of glucose molecules (step S40). This allows control over the number of glucose molecules passing through the semipermeable membrane 40, ensuring that the glucose enzyme in the sensing unit 30 is in excess during the reaction.

[0091] In the preparation method of the working electrode 1 of the glucose sensor according to this embodiment, there is no need for a complex surface treatment method to obtain droplets with the same wetting angle. Instead, at least one limiting groove 210 with a certain shape is formed on the surface of the working electrode 1 by curing the photosensitive material on the photosensitive layer 220. The predetermined amount of glucose sensitive reagent 310 that is drop-coated can be accommodated in the limiting groove 210 and form a morphology with the same shape as the limiting groove 210. This makes it easy to control the consistency of the area and morphology of the sensing part 30 in the working electrode 1 of mass production, and obtain a glucose sensor with consistent process parameters.

[0092] In this embodiment, in step S10, an insulating substrate layer 10 is prepared and pretreated to give its surface a specified roughness. In some examples, the substrate layer 10 can be formed on a substrate such as a glass plate or a silicon wafer by spin coating or coating. In some examples, the pretreatment may include polishing, plasma gas cleaning, ultrasonic cleaning, nitrogen drying, etc.

[0093] As described above, in step S201, in some examples, referring to FIG8(a), the conductive layer 20 can be deposited on the substrate layer 10 by means of screen printing, inkjet printing, vacuum magnetron sputtering, evaporation, or plating. In other examples, the conductive layer 20 may be flat only in the area where at least one limiting groove 210 is provided.

[0094] In step S202, in some examples, referring to FIG8(b), after the conductive layer 20 dries, a photosensitive material of appropriate thickness, such as photoresist (including positive resist, negative resist, reverse resist, or double-layer resist), SU-8, or photosensitive polyimide, can be spin-coated or coated onto the conductive layer 20 to form a photosensitive layer 220. Thus, by utilizing the properties of the photosensitive material, the photosensitive layer 220 can be partially cured by illumination to form a limiting groove 210 with a predetermined pattern on the conductive layer 20.

[0095] In step S202, in some examples, the thickness of the photosensitive layer 220 can be 0.1-100 μm, preferably 1-20 μm. In this case, after the photosensitive layer 220 is cured according to the predetermined pattern, the resulting limiting groove 210 has a corresponding height, which is beneficial for designing the volume of the limiting groove 210 to accommodate a predetermined amount of glucose-sensitive reagent 310.

[0096] In this embodiment, in some examples, the patterning method in step 202 may include: partially curing the photosensitive layer 220 by irradiating a mask with a predetermined pattern placed above the photosensitive layer 220 with light or by irradiating the photosensitive layer 220 with a laser source constituting the predetermined pattern, and then removing the uncured photosensitive layer 220 to form at least one limiting groove 210. Thus, limiting grooves 210 with predetermined patterns can be formed on the conductive layer 20 by utilizing the properties of the photosensitive material.

[0097] In some examples, referring to Figure 8(c), after the photosensitive layer 220 is formed on the conductive layer 20, the photosensitive layer 220 can be exposed and developed using a circular laser source or by irradiating a mask with a circular pattern placed above the photosensitive layer 220, thereby curing the photosensitive material in the photosensitive layer 220 into the shape of a circular locating groove 210. Next, referring to Figure 8(d), the uncured photosensitive material can be removed using a lift-off process or a special reagent, so that the photosensitive layer 220 retains only the cured locating groove 210 formed on the conductive layer 20. Of course, the shape of the locating groove 210 is not limited to a circle; in other examples, the locating groove 210 can also be oval, rectangular, polygonal, irregular, etc. Furthermore, the number of locating grooves 210 on the conductive layer 20 is not limited to one or three; it can also be two, four, five, seven, etc. In some examples, the multiple limiting grooves 210 may not be arranged in a straight line, but may be arranged in a broken line, a curve, or an irregular shape.

[0098] In this embodiment, in some examples, in step S30, referring to FIG8(e), a predetermined amount of glucose-sensitive reagent 310 can be drop-coated into at least one limiting groove 210 to keep the glucose-sensitive reagent 310 within the range of at least one limiting groove 210, and the glucose-sensitive reagent 310 can be cured to form a sensing part 30. Thus, a sensing part 30 with a predetermined morphology and area can be formed within at least one limiting groove 210 on the conductive layer 20, which is beneficial for controlling the consistency of the area and morphology of the sensing part 30 in the working electrode 1 of mass production, and obtaining a glucose sensor with consistent process parameters.

[0099] In step S30, in some examples, the glucose-sensitive reagent 310 can be cross-linked and cured at room temperature. In some examples, the glucose-sensitive reagent 310 can be cross-linked and cured in air at room temperature (e.g., 25°C ± 5°C), preferably for 40 hours or more, for example, 48 hours. This allows the glucose-sensitive reagent 310 to be stably fixed within the limiting groove 210, and the water vapor in the air is beneficial for stable cross-linking. In some examples, cross-linking and curing can be performed in a nitrogen chamber at room temperature. This prevents reaction with reactive gases in the environment during the curing process. In some examples, after cross-linking and curing, the working electrode 1 can be stored in a low-humidity environment, such as in a nitrogen chamber at room temperature. This maintains the enzyme's activity.

[0100] In this embodiment, referring to FIG8, in some examples, the dosage of the dispensing liquid dispensed by the dispensing device each time is first pre-adjusted, that is, the predetermined amount of glucose-sensitive reagent 310 is set to match the volume of the limiting groove 210 (for example, the predetermined volume is equal to or slightly larger than the volume of the limiting groove 210 but still within the range of surface tension). The stepping distance of the micro-dispensing head is adjusted so that the stepping distance is consistent with the spacing of the plurality of limiting grooves 210 arranged in a linear pattern on the conductive layer 20. Then, the micro-dispensing head of the dispensing device is moved to align with the limiting groove 210 of the working electrode 1, and the dispensing head dispenses glucose-sensitive reagent 310 into the limiting groove 210.

[0101] In some examples, the conductive layer 20 contains alignment marks, and the dispensing apparatus has an automated optical inspection (AOI) probe, in which case the micro-dispensing head of the dispensing apparatus can be automatically aligned with the limiting groove 210 of the working electrode 1.

[0102] In this embodiment, in some examples, after the sensing part 30 dries in step S40, a semi-permeable membrane 40 for controlling the passage of glucose molecules can be provided outside the sensing part 30. In some examples, the semi-permeable membrane 40 can be provided on the sensing part 30 or outside the entire working electrode 1 by spin coating, coating, or dip-coating.

[0103] While the present disclosure has been specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the foregoing description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from its essential spirit and scope, and all such modifications and variations fall within the scope of the present disclosure. Those skilled in the art will understand that, in general, the terminology used in this disclosure is intended to be "open" terminology (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "at least having," and the term "comprising" should be interpreted as "including but not limited to," etc.).

Claims

1. A method for preparing a working electrode suitable for mass production of a glucose sensor, the working electrode comprising: A base layer, the base layer being made of an insulating material; A conductive layer disposed on the substrate layer and having a plurality of limiting grooves arranged along a predetermined direction of the conductive layer; as well as Multiple sensing units are disposed on the conductive layer; The preparation method is characterized by including the following steps: (a) Prepare an insulating base layer; (b) A conductive layer is formed on the substrate layer, the conductive layer completely covering the substrate layer as a whole, and a plurality of limiting grooves of the same size and shape are formed on the conductive layer; (c) A predetermined amount of glucose-sensitive reagent is applied to each of the plurality of limiting grooves in such a manner that the glucose-sensitive reagent is held within the range of the plurality of limiting grooves, and the glucose-sensitive reagent is cured to form the plurality of sensing parts.

2. The preparation method according to claim 1, characterized in that, Step (b) includes: coating a biocompatible photosensitive layer on the conductive layer, and patterning the photosensitive layer according to a predetermined pattern to form the plurality of locating grooves of the same size and shape on the conductive layer, the predetermined pattern including a plurality of closed patterns and each closed pattern being of the same size and shape.

3. The preparation method according to claim 2, characterized in that, In step (b), the patterning method includes: irradiating the photosensitive layer with the predetermined pattern placed above the photosensitive layer with light or irradiating the photosensitive layer with a laser source that constitutes the predetermined pattern to partially cure the photosensitive layer, and then removing the uncured photosensitive layer to form the plurality of limiting grooves.

4. The preparation method according to claim 2, characterized in that, After the photosensitive layer is deposited on the conductive layer, the photosensitive layer is irradiated with a circular laser source or with a photomask with a circular pattern placed above the photosensitive layer for exposure and development, thereby solidifying the photosensitive material in the photosensitive layer into the shape of a circular limiting groove.

5. The preparation method according to claim 4, characterized in that, The uncured photosensitive material is removed using a stripping process or a special reagent, thereby leaving the photosensitive layer with multiple cured locating grooves formed on the conductive layer.

6. The preparation method according to claim 1, characterized in that, The substrate layer is pretreated to give its surface a specified roughness. The pretreatment includes polishing, plasma gas cleaning, ultrasonic cleaning, and nitrogen drying.

7. The preparation method according to claim 1, characterized in that, The conductive layer is deposited on the substrate layer by screen printing, inkjet printing, vacuum magnetron sputtering, evaporation, or plating.

8. The preparation method according to claim 1, characterized in that, The cured photosensitive layer forms the walls of the plurality of limiting grooves, and the walls of each limiting groove form the shape of each closed pattern. The surface of the conductive layer and the walls formed by the curing of the photosensitive material constitute a limiting groove with a certain volume.

9. The preparation method according to claim 1, characterized in that, A predetermined amount of glucose-sensitive reagent is dispensed and contained in the plurality of limiting grooves, forming a morphology identical to that of the plurality of limiting grooves.

10. The preparation method according to claim 9, characterized in that, The volume of the predetermined amount of glucose-sensitive reagent is the same as the volume of the limiting groove, and the surface of the sensing part is flush with the line connecting any two points on the upper edge of the limiting groove.

Images