CN111272849B

CN111272849B - Working electrode of glucose sensor and preparation method thereof

Info

Publication number: CN111272849B
Application number: CN202010167544.2A
Authority: CN
Inventors: 王蕾; 卢路旺; 胡一帆; 韩明松
Original assignee: Shenzhen Guiji Sensing Technology Co ltd
Current assignee: Shenzhen Guiji Sensing Technology Co ltd
Priority date: 2019-08-20
Filing date: 2020-03-11
Publication date: 2024-02-27
Anticipated expiration: 2040-03-11
Also published as: CN118169207A; CN118000719A; CN111272849A; CN117783243A; CN118169206A; CN118169204A; CN118169205A; CN111307901A; CN118169208A; CN111307901B; CN111272850B; CN117783242A; CN111272850A

Abstract

The present disclosure describes a working electrode for a glucose sensor, comprising: a base layer made of an insulating material; a conductive layer disposed on the base 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 by coating a photosensitive material on a surface of the conductive layer to form a photosensitive layer and patterning the photosensitive layer; and the sensing part is formed by solidifying the glucose sensitive reagent which is dripped in the limiting groove. According to the method, the liquid drops with the same wetting angle are not required to be obtained by a complex surface treatment method, at least one limiting groove with a certain shape is formed on the surface of the working electrode by solidifying the photosensitive material, and the dripped glucose sensitive reagent is contained in the limiting groove and forms the same shape as the shape of the limiting groove, so that the consistency of the area and the shape of the sensing part in the working electrode in mass production can be conveniently controlled, and the glucose sensor with consistent technological parameters is obtained.

Description

Working electrode of glucose sensor and preparation method thereof

Technical Field

The present disclosure relates generally to the field of biosensors, and more particularly to a working electrode of a glucose sensor and a method of manufacturing the same.

Background

Diabetes is a metabolic disease characterized by hyperglycemia, and no radical treatment method exists at present, and diabetics often control the disease state by monitoring blood sugar. The means of monitoring blood glucose mainly include traditional blood glucose monitoring and dynamic blood glucose monitoring (Continuous Glucose Monitoring, CGM). Compared with traditional blood sugar monitoring, the CGM technology can monitor the blood sugar of a patient for at least 24 hours continuously, obtain the blood sugar condition of the patient all the day in real time, and effectively reflect the hypoglycemia and blood sugar fluctuation condition of the patient. Dynamic blood glucose monitoring can be achieved with glucose sensors.

Glucose sensors generally include a sensor probe and a processing device that records sensed information. When dynamic blood glucose monitoring is carried out, a sensor probe is often buried under the skin of a subject, a glucose sensitive reagent is arranged on a working electrode of the probe, and can specifically react with glucose molecules at an implantation position to generate an electric signal, and a processing device processes the electric signal to obtain a blood glucose value and the blood glucose change condition of a patient.

In order to obtain a more accurate blood glucose level, it is necessary to achieve mass calibration by providing each glucose sensor produced in mass with a uniform initial sensitivity as much as possible in the production of glucose sensors, and therefore, it is important to ensure uniformity of initial sensitivity of the glucose sensors produced in mass, and uniformity of process parameters such as area and morphology of the sensor portions. The sensing part is formed by dripping glucose sensitive reagent on the conductive layer of the working electrode and solidifying the glucose sensitive reagent.

In the existing dripping process, the control of the dripping liquid improves the surface roughness of the dripping liquid so that the liquid drops have consistent wetting angles on the surface, thereby controlling the area and the morphology of the liquid drops. However, it is difficult to make the surface roughness of the dispensed liquid completely uniform, so that it is difficult to ensure uniformity of the area and morphology of the liquid droplets each time the dispensing is performed, and it is difficult to ensure that each glucose sensor produced in mass has initial sensitivity with high uniformity.

Disclosure of Invention

The present disclosure has been made in view of the above-described circumstances, and an object thereof is to provide a working electrode for a glucose sensor and a method for manufacturing the same, which are capable of simply and conveniently controlling the uniformity of the area and the morphology of a droplet for dispensing, and which are advantageous for improving the uniformity of the sensing portions of each glucose sensor in mass production so as to improve the uniformity of the initial sensitivity of each glucose sensor in mass production.

To this end, a first aspect of the present disclosure provides a working electrode of a glucose sensor, characterized by comprising: a base layer which is made of an insulating material and has been pretreated to form a surface having a predetermined roughness; a conductive layer disposed on the base layer and having at least one limiting groove aligned in a predetermined direction of the conductive layer, the at least one limiting groove being formed on a surface of the conductive layer by coating a photosensitive material on the surface of the conductive layer to form a photosensitive layer, and removing the uncured photosensitive layer after curing the photosensitive layer in a predetermined pattern, the predetermined pattern including at least one closed pattern; and the sensing part is formed by dripping a predetermined amount of glucose-sensitive reagent into the at least one limit groove and solidifying the glucose-sensitive reagent.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, a complex surface treatment method is not required to obtain droplets with the same wetting angle, but at least one limiting groove with a certain shape is formed on the surface of the working electrode by solidifying the photosensitive material, and a predetermined amount of glucose-sensitive reagent dispensed can be accommodated in the limiting groove and form the same morphology as that of the limiting groove, so that the consistency of the area and morphology of the sensing part in the working electrode in mass production can be conveniently controlled, and the glucose sensor with consistent process parameters can be obtained.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, optionally, the at least one limiting groove is arranged linearly. Thus, the dispensing efficiency can be improved.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, the sensing portion is optionally further covered with a semipermeable membrane that controls passage of glucose molecules. Thus, the number of glucose molecules passing through the semipermeable membrane can be controlled.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, optionally, the glucose-sensitive reagent completely fills the limiting groove. Under the condition, the glucose sensitive reagent is limited in the limiting groove, so that the area and the appearance of the glucose sensitive reagent can be controlled, and the undried glucose sensitive reagent is prevented from flowing everywhere to form an irregular shape.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, optionally, the limit groove is a circular groove or an egg groove. Therefore, the glucose sensitive reagent which is convenient to drip can flow in the limit groove and fill the edge of the limit groove to form a required shape.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, optionally, the material of the conductive layer is at least one selected from the group consisting of glassy carbon, graphite, silver chloride, platinum, palladium, platinum-iridium, titanium, gold, and iridium. Thus, the conductive material can have good conductivity.

In the working electrode of the glucose sensor according to the first aspect of the present disclosure, optionally, the glucose-sensitive reagent is capable of chemically reacting with glucose, the glucose-sensitive reagent including a glucose enzyme, a metal polymer, and a cross-linking agent. Therefore, the glucose sensitive reagent can be conveniently attached to the limit groove of the conducting layer and can react with glucose specifically.

A second aspect of the present disclosure provides a method of preparing a working electrode of a glucose sensor, the working electrode comprising: a base layer which is made of an insulating material and has been pretreated to form a surface having a predetermined roughness; a conductive layer disposed on the base layer and having at least one stopper groove arranged along a predetermined direction of the conductive layer; and a sensor portion provided on the conductive layer; the preparation method is characterized by comprising the following steps: (a) Preparing a base layer having insulation properties and pretreating the base layer so that the surface thereof has a predetermined roughness; (b) Forming the conductive layer on the substrate layer, and coating a photosensitive layer with biocompatibility on the conductive layer, patterning the photosensitive layer according to a predetermined pattern to form the at least one limiting groove on the conductive layer, wherein the predetermined pattern comprises at least one closed pattern; (c) The sensing portion is formed by dispensing a predetermined amount of glucose-sensitive reagent into the at least one limiting groove in such a manner that the glucose-sensitive reagent is held within the range of the at least one limiting groove, and solidifying the glucose-sensitive reagent.

In the method for manufacturing a working electrode of a glucose sensor according to the second aspect of the present disclosure, a complex surface treatment method is not required to obtain droplets having the same wetting angle, but at least one limiting groove having a certain shape is formed on the surface of the working electrode by solidifying a photosensitive material, and a predetermined amount of glucose-sensitive reagent dispensed can be contained in the limiting groove and formed in the same morphology as that of the limiting groove, so that the consistency of the area and morphology of a sensing portion in a working electrode for mass production can be conveniently controlled, and a glucose sensor having consistent process parameters can be obtained.

In the method for manufacturing a working electrode of a glucose sensor according to the second aspect of the present disclosure, optionally, in step (b), the patterning method includes: the mask plate having the predetermined pattern is placed over the photosensitive layer by light irradiation or the photosensitive layer is irradiated with a laser source constituting the predetermined pattern to partially cure the photosensitive layer, and then the uncured photosensitive layer is removed to form the at least one limit groove. Thereby, the limit grooves having a predetermined pattern can be formed on the conductive layer using the characteristics of the photosensitive material.

In the method for manufacturing a working electrode of a glucose sensor according to the second aspect of the present disclosure, optionally, the conductive layer has a plurality of the limit grooves, and a sum of surface areas of the plurality of limit grooves is not less than 50% of a surface area of the conductive layer. This can increase the contact area between the glucose molecules in the sensor portion and the conductive layer.

Drawings

Fig. 1 is a schematic diagram showing a state of use of a glucose monitoring probe according to an embodiment of the present disclosure.

Fig. 2 is a plan view structural diagram illustrating a glucose sensor according to an embodiment of the present disclosure.

Fig. 3 is a schematic view showing a structure of the glucose monitoring probe of fig. 2 in a bent state.

Fig. 4 is a perspective view illustrating a case where a working electrode according to an embodiment of the present disclosure is not covered with a semipermeable membrane.

Fig. 5 is a plan view showing a working electrode according to an embodiment of the present disclosure.

Fig. 6 is a cross-sectional view taken along line B-B' showing the working electrode of fig. 5 covered with a semipermeable membrane.

Fig. 7 is a flowchart illustrating a method of manufacturing a working electrode according to an embodiment of the present disclosure.

Fig. 8 (a) -8 (e) are perspective views illustrating a method of manufacturing a working electrode according to an embodiment of the present disclosure.

Detailed Description

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same members are denoted by the same reference numerals, and overlapping description thereof is omitted. In addition, the drawings are schematic, and the ratio of the sizes of the components to each other, the shapes of the components, and the like may be different from actual ones.

In addition, headings and the like referred to in the following description of the disclosure are not intended to limit the disclosure or scope thereof, but rather are merely indicative of reading. Such subtitles are not to be understood as being used for segmenting the content of the article, nor should the content under the subtitle be limited only to the scope of the subtitle.

In the present disclosure, a glucose sensor may be simply referred to as a "sensor", a probe of the glucose sensor may be simply referred to as a "probe", a working electrode of the glucose sensor may be simply referred to as a "working electrode", and a preparation method of the working electrode of the glucose sensor may be simply referred to as a "preparation method". In addition, the working electrode of the glucose sensor and the preparation and dripping methods thereof are not only applicable to glucose sensors, but also applicable to monitoring electrodes of other physiological parameters, such as uric acid detection sensors for detecting uric acid, cholesterol monitoring sensors for detecting cholesterol and the like, and only the sensitive reagent in the sensing part of the working electrode needs to be replaced by corresponding enzyme and the like which specifically react with the target analyte. In addition, besides the monitoring electrode of the physiological parameter, the method for controlling the dripping morphology by forming the limit groove with the preset shape and the volume matched with the dripping amount at the dripping position in other production processes needing to control the dripping morphology is also applicable to the method for controlling the dripping morphology.

Fig. 1 is a schematic diagram showing a state of use of a glucose monitoring probe according to an embodiment of the present disclosure. Fig. 2 is a plan view structural diagram illustrating a glucose sensor according to an embodiment of the present disclosure. Fig. 3 is a schematic view showing a structure of the glucose monitoring probe of fig. 2 in a bent state.

In the present embodiment, the glucose sensor probe S may be referred to as an implantable glucose monitoring probe, a glucose monitor probe S, or a probe S.

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. The probe S of the portable glucose monitor G is implanted on the body surface to be in contact with the interstitial fluid on the body surface, so that the probe S can be used to sense a glucose concentration signal of the interstitial fluid, and the glucose concentration signal is transmitted to the electronic system S', so that a corresponding glucose concentration can be obtained.

Specifically, a portion (particularly, a sensing portion) of the glucose sensor probe S may be implanted on, for example, a body surface of a human body to be in contact with interstitial fluid in the body. In addition, another part of the glucose sensor probe S is also connected to an electronic system S' located on the body surface. In operation of the portable glucose monitor G, the glucose sensor probe S reacts with interstitial fluid in the body to generate a sensing signal (e.g., a current signal) and transmits the sensing signal to the electronic system S' of the body surface, which processes the sensing signal to obtain a glucose concentration. Although fig. 1 shows the arrangement position of the glucose sensor probe S, the present embodiment is not limited to this, and the glucose sensor probe S may be arranged in the abdomen, waist, leg, or the like, for example.

In the present embodiment, although the glucose sensor probe S directly detects glucose in the tissue fluid, the glucose concentration in the tissue fluid is strongly correlated with the glucose concentration in blood, and the glucose concentration in blood can be determined from the glucose in the tissue fluid.

In the present embodiment, the glucose sensor probe S may include a working electrode 1, a reference electrode 2, and a counter electrode 3 (see fig. 2). In some examples, the working electrode 1, the reference electrode 2, and the counter electrode 3 may each have an insulating base layer 10 (described later) as a substrate. In some examples, the base layer 10 of each of the working electrode 1, reference electrode 2, and counter electrode 3 may be a complete one substrate and be partially in three parts. The glucose sensor probe S may further include a contact 4 connected to the working electrode 1 via a lead wire, a contact 5 connected to the working electrode 2 via a lead wire, and a contact 6 connected to the reference electrode 3 via a lead wire. In some examples, glucose sensor probe S may be connected to electronic system 2 via contact 4, contact 5, and contact 6.

In the present embodiment, for convenience of explanation, the glucose sensor probe S may be divided into a connection portion Sa and an implant portion Sb (see fig. 3). The line A-A' in fig. 3 generally shows the general location of the skin when the glucose sensor probe S is implanted in the body surface of the tissue. After implantation, the implanted portion Sb is in the superficial layer of the skin and the electronic system S' is brought into close contact with the skin surface, and the connection portion Sa (see fig. 3) of the glucose sensor probe S is connected to and located on the skin surface.

When glucose sensors are produced in batches, the consistency of the process parameters of the sensors produced in the same batch is very important, and if the consistency of the process parameters is good, the sensors in the batch do not need to be independently corrected, and the whole batch of sensors are subjected to factory batch calibration. In order to achieve good uniformity of process parameters, it is necessary to control at least one of the area and morphology of the sensor portion 30 of the working electrode 1 and the film thickness and diffusion coefficient of the conductive layer 20 and the semipermeable film 40 on the sensor portion 30. The sensing portion 30 of the working electrode 1 is mainly formed by a dripping process, but in the dripping process, since the surface of the dripping cannot achieve hundred percent consistency in morphology and roughness, the dripping reagent easily flows irregularly on the surface of the dripping, so that the area and morphology of the sensing portion 30 formed by dripping the glucose-sensitive reagent 310 are uncontrollable. Therefore, in order to achieve good uniformity of process parameters, it is important to control uniformity of the area, morphology, and the like of the sensor portion 30 of the working electrode 1.

In the present embodiment, referring to fig. 4, the working electrode 1 of the glucose sensor may include: a base layer 10 made of an insulating material, which is pretreated to form a surface having a predetermined roughness; a conductive layer 20 disposed on the base layer 10 and having at least one limiting groove 210 aligned in a predetermined direction of the conductive layer 20, the at least one limiting groove 210 being formed on the surface of the conductive layer 20 by coating a photosensitive material on the surface of the conductive layer 20 to form a photosensitive layer 220, and removing the uncured photosensitive layer 220 after curing the photosensitive layer 220 in a predetermined pattern including at least one closed pattern aligned in the predetermined direction of the conductive layer 20; and a sensing part 30 formed by dripping a predetermined amount of glucose sensing reagent 310 to at least one of the limiting grooves 210 and solidifying.

According to the working electrode 1 of the glucose sensor according to the present disclosure, a complex surface treatment method is not required to obtain droplets having the same wetting angle, but at least one limiting groove 210 having a certain shape is formed on the surface of the working electrode 1 by solidifying a photosensitive material, and a predetermined amount of glucose-sensitive reagent 310 dispensed can be contained in the limiting groove 210 and form the same shape as the limiting groove 210, so that the uniformity of the area and the shape of the sensing part 30 in the working electrode 1 for mass production can be conveniently controlled, and a glucose sensor having uniform process parameters can be obtained. In addition, by changing the volume, shape, and dispensing amount of the limiting groove 210, the area, shape, and the like of the sensor portion 30 can be easily changed.

Fig. 5 is a plan view showing a working electrode according to an embodiment of the present disclosure. Fig. 6 is a cross-sectional view taken along line B-B' showing the working electrode of fig. 5 covered with a semipermeable membrane.

(base layer 10)

In the present embodiment, as described above, the working electrode 1 may include the base layer 10. In some examples, the base layer 10 may be composed of an insulating material.

In some examples, the base layer 10 may be selected from flexible insulating materials. The flexible insulating material may be at least one of Polyimide (PI), polyethylene terephthalate (PET), parylene (Parylene), silicone, polydimethylsiloxane (PDMS), polyethylene glycol (PEG) or polytetrafluoroethylene resin (Teflon), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene naphthalate (PEN), and the like. This allows the base layer 10 to have both flexibility and insulation properties, and can reduce discomfort after implantation into the body.

In other examples, the substrate layer 10 may be selected from a non-flexible insulating material. The inflexible material may generally comprise ceramic, polymethyl methacrylate (PMMA), alumina, silica, or the like. In this case, the base layer 10 may have excellent supporting properties. In addition, when the base layer 10 is a non-flexible insulating material, it may be made into a shape that is easy to implant, such as a needle-like shape, in which case the probe including the working electrode 1 can be implanted in a body surface (e.g., a shallow skin layer, etc.) without the need for an auxiliary implantation device (not shown) such as a needle aid.

In this embodiment, in some examples, the surface of the base layer 10 may be pretreated to achieve a specified roughness. Pretreatment may include polishing, plasma gas cleaning, ultrasonic cleaning, nitrogen drying, and the like. The predetermined roughness means that the range of roughness of the surface of the base layer 10 facilitates adhesion of the material forming the conductive layer 20 to the base layer 10. Thereby, the conductive layer 20 can be facilitated to be formed on the base layer 10, reducing the possibility of delamination or slipping.

In some examples, the thickness of the base layer 10 may be 100-200 μm. Thus, the base layer 10 may have good insulation and support properties.

(conductive layer 20)

In the present embodiment, as described above, the working electrode 1 may include the conductive layer 20. The conductive layer 20 may be disposed on the base layer 10. In some examples, the conductive layer 20 may be disposed on the base layer 10 by screen printing, spray printing, vacuum magnetron sputtering, evaporation, plating, or the like.

In some examples, the material of conductive layer 20 may be a metallic conductive material. The metallic conductive material may be selected from at least one of silver, platinum, gold, titanium, palladium, iridium, niobium, or an alloy thereof. This can provide the conductive layer 20 with good conductivity. In other examples, the material of the conductive layer 20 may also be a non-metallic material having conductivity. The nonmetallic material having conductivity may be selected from nonmetallic materials having conductivity such as glassy carbon, graphite, and the like.

In this embodiment, in some examples, the conductive layer 20 may be planar. Thereby, it can be facilitated to form at least one limit groove 210 on the conductive layer 20.

In this embodiment, the thickness of the conductive layer 20 may be 5-25 μm in some examples. Thus, the conductive layer 20 can be prevented from being excessively thick to affect the bending resistance of the working electrode 1.

In some examples, the conductive layer 20 may cover the entire base layer 10. In other words, the conductive layer 20 may entirely cover the base layer 10 and not be divided into a plurality of portions that are not connected. In other examples, the conductive layer 20 may cover only a portion of the base layer 10. In other words, the conductive layer 20 may entirely cover only a portion of the base layer 10 to form the conductive layer 20 of a sufficient area that at least the minimum area in which the at least one limiting groove 210 of the predetermined pattern can be formed on the conductive layer 20.

In some examples, the surface of the conductive layer 20 may be further provided with a nanoparticle layer (not shown), such as gold nanoparticles, platinum nanoparticles, and the like. In some examples, the nanoparticle layer may be porous. In some examples, the nanoparticle layer may be disposed on the surface of the conductive layer 20 by means such as electroplating, sputtering, and the like. Thus, the contact area between the enzyme in the glucose sensing reagent 310 of the sensor unit 30 and the conductive layer 20 can be increased.

In some examples, the surface of the conductive layer 20 or the surface of the limit groove 210 may further be provided with a three-dimensional network structure (not shown) of nanofibers composed of filiform nanofibers. The three-dimensional network structure of nanofibers may be formed based on the nanoparticle layer, that is, thin and long filiform nanofibers may be formed on the nanoparticles by electroplating a conductive material such as polyaniline based on the nanoparticles in the nanoparticle layer, and the plurality of filiform nanofibers cross each other to form the three-dimensional network structure of nanofibers. Thus, the adhesion amount of the glucose enzyme can be increased and the glucose enzyme has better conductivity.

In some examples, the thickness of the photoactive layer 220 may be less than the thickness of the conductive layer 20. This can form the limit groove 210 with a proper depth, and can facilitate the semipermeable membrane 40 to cover the limit groove 210 or the sensor portion 30. In other examples, the thickness of the photosensitive layer 220 may be equal to or greater than the thickness of the conductive layer 20.

In this embodiment, in some examples, the conductive layer 20 may have at least one stopper groove 210 thereon arranged along a predetermined direction of the conductive layer 20. The predetermined direction of the conductive layer 20 may be, for example, a length direction of the conductive layer 20. Thereby, the implantation area of the working electrode 1 can be optimized.

In this embodiment, in some examples, at least one limit groove 210 is formed on the surface of the conductive layer 20 by coating a photosensitive material on the surface of the conductive layer 20 to form a photosensitive layer 220, and curing the photosensitive layer 220 in a predetermined pattern and then removing the uncured photosensitive layer 220. The predetermined pattern includes at least one closed figure arranged along a predetermined direction of the conductive layer 20. In other words, the projection of each of the limiting grooves 210 on the conductive layer 20 is a closed pattern, that is, the cured photosensitive layer 220 forms the wall of the limiting groove 210, the wall of the limiting groove 210 encloses a shape of the closed pattern, and the surface of the conductive layer 20 and the wall formed by curing the photosensitive material form the limiting groove 210 with a certain volume. To which a predetermined amount (not greater than the volume of the limiting groove 210) of the glucose-sensitive reagent 310 is dispensed, the glucose-sensitive reagent 310 can be well confined in the limiting groove 210 without leaking out and diffusing from the side wall.

In other examples, the photosensitive layer 220 may not be present, and the at least one limit groove 210 may be formed by printing a predetermined pattern directly on the conductive layer 20 by, for example, mask printing or the like.

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 may be, for example, 1, 3, 5, 7. In some examples, the closed figure may be circular, oval, rectangular, triangular, or irregularly shaped.

In some examples, the size and shape of each closed figure may be uniform. In other examples, the size and shape of each closed figure may not be exactly the same. The predetermined pattern including the closed pattern of the different working electrodes 1 of the same batch should be kept uniform in order to control the uniformity of the volume of the dispensed glucose-sensitive reagent 310, the overall area of the formed sensing portion 30, the morphology, and thus the uniformity of the initial sensitivity between the different working electrodes 1 of the same batch.

In some examples, at least one closed figure on the same working electrode 1 is arranged in a straight line. Thereby, at least one stopper groove 210 arranged in a straight line can be obtained.

In some examples, the at least one limiting groove 210 is aligned linearly. This can improve the map efficiency. The stopper grooves 210 (stopper groove 210a, stopper groove 210b, stopper groove 210 c) may be arranged in other shapes such as a curve shape and a broken line shape.

In some examples, the limiting groove 210 may be formed at a substantially central position of the working electrode 1. In addition, the limit groove 210 can be a flat bottom groove _。 Thereby, the uncured glucose-sensing reagent 310 can be contained.

In this embodiment, the diameter or maximum width of the limiting groove 210 may be 100-150 μm. Thus, the sensing portion 30 has a sufficiently large area so that the glucose sensor has a high sensitivity.

In some examples, the limit slot 210 may be a circular slot or an oval slot. Thus, the dispensed glucose-sensitive reagent 310 can be facilitated to flow within the limiting groove 210 and fill the edges of the limiting groove 210 to form a desired topography. In other examples, the limit groove 210 may be a rectangular groove or an irregularly shaped groove. Thereby, the shape of the limit groove 210 can be optimized according to the design.

In some examples, when conductive layer 20 has only one spacing groove 210, the surface area of one spacing groove 210 is not less than the surface area of conductive layer 20. In some examples, the sum of the surface areas of the plurality of limit grooves 210, such as limit groove 210a, limit groove 210b, limit groove 210c (each of which is referred to as 210 in the following description of the limit groove for convenience of explanation) shown in fig. 4, may be not less than 50%, such as 50%,60%,70%,80%, 90%, or the like, of the surface area of the entire conductive layer 20. This can increase the contact area between the glucose molecules in the sensor portion 30 and the conductive layer 20, and can improve the sensitivity of the glucose sensor.

(sensor unit 30)

In the present embodiment, as described above, the working electrode 1 may include the sensor portion 30. The sensing portion 30 may be disposed on at least one of the limit grooves 210 of the conductive layer 20. The sensor portion 30 is formed by dropping a predetermined amount of glucose sensing reagent 310 into the limiting groove 210 and solidifying the same. Depending on the nature and the predetermined amount of the different glucose sensing agents 310, different sensing units 30 can be obtained.

In some examples, glucose-sensing reagent 310 completely fills limiting groove 210. In other words, the volume of the predetermined amount of glucose-sensing reagent 310 is the same as the volume of the limiting groove 210, and the surface of the sensing portion 30 is flush with the line between any two points of the upper edge of the limiting groove 210. In this case, the glucose sensor 310 is limited to the limiting groove 210, so that the area and the shape of the sensing unit 30 can be controlled, and the glucose sensor 310 is prevented from flowing everywhere, which is not dried, to form an irregular shape.

In some examples, the sensing portion 30 is slightly convex in the limit groove 210. In other words, the volume of the predetermined amount of glucose sensor reagent 310 is slightly larger than the volume of the limiting groove 210, and the surface tension force causes the liquid level of the unfixed glucose sensor reagent 310 to be slightly higher than the upper surface of the conductive layer 20, and the unfixed glucose sensor reagent is not diffused to the edge beyond the side surface of the limiting groove 210, so that the morphology of the cured sensor part 30 is greatly changed, and the uniformity of the area and morphology of the sensor part 30 can be well controlled.

In other examples, the surface of the sensing portion 30 is lower than the upper surface of the conductive layer 20, i.e., the volume of the predetermined amount of glucose-sensing reagent 310 is smaller than the volume of the limiting groove 210, and the glucose-sensing reagent 310 is entirely contained in the limiting groove 210.

In this embodiment, glucose sensing reagent 310 is capable of chemically reacting with glucose. In some examples, glucose-sensing reagent 310 can include a glucose enzyme, a metal polymer, and a cross-linking agent. Thus, glucose sensing reagent 310 can be conveniently attached to limiting groove 210 of conductive layer 20 and react specifically with glucose.

In some examples, glucose-sensing reagent 310 may be a mixed solution including an enzyme, a cationic polymer, and a redox electron mediator. For example, glucose-sensitive reagent 310 may be a mixed solution of a cationic polymer, such as glucose oxidase or dehydrogenase, with a redox electron mediator, such as ferricyanide, phenanthrenequinone or ferrocene, and a cross-linking agent.

In some examples, glucose sensing reagent 310 may be replaced according to the actual target analyte, i.e., replaced with a sensing reagent that specifically reacts with the target analyte in the body, whereby the effect of detecting the concentration of the target analyte other than glucose may be achieved. For example, glucose-sensitive reagent 310 may be replaced with a specific reaction substance corresponding to acetylcholine, amylase, bilirubin, cholesterol, chorionic gonadotrophin, creatine kinase, creatine, DNA, fructosamine, glucose, glutamine, growth hormone, ketone body, lactate, oxygen, peroxide, prostate-specific antigen, prothrombin, RNA, thyroid stimulating hormone, troponin, or the like.

In the present embodiment, the thickness of the sensing portion 30 may be about 0.1 μm to 100 μm, preferably 2 μm to 10 μm, and in some examples, the thickness of the sensing portion 30 may be 10 μm. In this case, the thickness of the sensing portion 30 is controlled within a certain degree, so that the problems that the adhesion force of the glucose sensing reagent 310 is reduced, the material is dropped in the body, insufficient reaction is caused by too little glucose enzyme in the glucose sensing reagent 310, and normal glucose concentration information cannot be fed back are avoided.

In some examples, the glucose in glucose-sensing reagent 310 may be attached to and immobilized on the surface of working electrode 1 by physical adsorption, covalent cross-linking, or entrapment.

In some examples, the glucose in glucose-sensing reagent 310 may be attached in a three-dimensional network of nanofibers (not shown) formed from conductive materials such as polyaniline and the like. This can increase the amount of enzyme attached to the sensor unit 30 and provide good electric signal transmission performance.

In some examples, carbon nanotubes may also be included in glucose-sensing reagent 310, with a mass percent of carbon nanotubes ranging from 5 to 10%. This can increase adhesion of the glucose enzyme and promote progress of the specific reaction. In other examples, graphene, porous titanium dioxide, or a conductive organic salt may also be added to glucose-sensing reagent 310. Thus, the reaction of the glucose can be promoted more effectively.

(semipermeable membrane 40)

In the present embodiment, as described above, the working electrode 1 may further include the semipermeable membrane 40. In some examples, the semi-permeable membrane 40 may be disposed outside of the entire working electrode 1, i.e., covering the entire surface of the working electrode 1 including the conductive layer 20 and the sensing portion 30. This can provide a good diffusion control effect.

In other examples, the semi-permeable membrane 40 may be disposed only on the sensing portion 30, i.e., cover only all of the sensing portion 30. This can reduce the use of raw materials.

In this embodiment, in some examples, the semipermeable membrane 40 includes a diffusion control layer (not shown) that controls 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 in a certain ratio, and when glucose molecules react with the glucose of the sensor unit 30, the glucose of the sensor unit 30 is in an excessive state, and the glucose concentration becomes the only factor limiting the current of the working electrode 1, so that the linear range of the glucose sensor when monitoring the glucose concentration can be widened.

In some examples, the semipermeable membrane 40 further includes an anti-tamper layer (not shown) laminated to the diffusion control layer. In other examples, the diffusion-control layer may also be disposed outside the tamper-resistant layer. In the semipermeable membrane 40, the diffusion controlling layer can control the diffusion of glucose molecules, and the anti-interference layer can prevent the diffusion of non-glucose substances. In this case, it is possible to reduce the tissue fluid or blood component passing through the semipermeable membrane 40 first and then to block the interfering substance outside the semipermeable membrane 40 through the interference preventing layer. Common interferents may include uric acid, ascorbic acid, acetaminophen, and the like, which are ubiquitous in the body.

In some examples, the semipermeable membrane 40 may be a diffusion-controlling material that is biocompatible. This can lengthen the time required for the sensor probe to be used after implantation in the body. In the present embodiment, the sensor portion 30 having a controllable area and morphology can be obtained, and thus a glucose sensor having consistent process parameters can be obtained.

The following describes the preparation method of the working electrode 1 of the glucose sensor with reference to fig. 7 and 8, and the description of specific parameters, materials, and morphology of the base layer 10, the conductive layer 20, the sensing portion 30, and the semipermeable membrane 40 is referred to in the above corresponding parts.

Fig. 7 is a flowchart illustrating a method of manufacturing a working electrode according to an embodiment of the present disclosure. Fig. 8 is a perspective view illustrating a method of manufacturing a working electrode according to an embodiment of the present disclosure.

In the present embodiment, referring to fig. 7, the working electrode 1 of the glucose sensor may include: a base layer 10 made of an insulating material, which is pretreated to form a surface having a predetermined roughness; a conductive layer 20 disposed on the base layer 10 and having at least one stopper groove 210 arranged along a predetermined direction of the conductive layer 20; and a sensor portion 30 provided on the conductive layer 20. The method of manufacturing the working electrode 1 may include the steps of: (a) Preparing a base layer 10 having insulation properties and pretreating the base layer 10 so that the surface thereof has a predetermined roughness (S10); (b) Forming a conductive layer 20 on a base layer 10 (S201), and coating a photosensitive layer 220 having biocompatibility on the conductive layer 20, 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) A predetermined amount of glucose sensor reagent 310 is dispensed to the at least one limiting groove 210 such that the glucose sensor reagent 310 is held within the range of the at least one limiting groove 210, and the glucose sensor reagent 310 is solidified to form the sensing unit 30 (S30).

In addition, in some examples, the manufacturing method of the working electrode 1 may further include (see fig. 7) step (d) of externally arranging a semipermeable membrane 40 that controls passage of glucose molecules at the sensing portion 30 (step S40). Thus, the number of glucose molecules passing through the semipermeable membrane 40 can be controlled, and the glucose enzyme in the sensor unit 30 can be in an excess state during the reaction.

In the method for manufacturing the working electrode 1 of the glucose sensor according to the present embodiment, a complicated surface treatment method is not required to obtain droplets having the same wetting angle, but at least one limiting groove 210 having a certain shape is formed on the surface of the working electrode 1 by solidifying the photosensitive material on the photosensitive layer 220, and a predetermined amount of the glucose sensing agent 310 applied in a dropwise manner can be accommodated in the limiting groove 210 and formed in the same shape as the limiting groove 210, so that the uniformity of the area and the shape of the sensing portion 30 in the working electrode 1 in mass production can be conveniently controlled, and the glucose sensor having the uniform process parameters can be obtained.

In the present embodiment, in step S10, the insulating base layer 10 is prepared, and the base layer 10 is pretreated so that the surface thereof has a predetermined roughness. In some examples, the base layer 10 may be formed on a substrate, such as a glass plate, a silicon wafer, by spin-coating, or the like. In some examples, the pretreatment may include polishing, plasma gas cleaning, ultrasonic cleaning, nitrogen drying, and the like.

As described above, in step S201, in some examples, referring to fig. 8 (a), the conductive layer 20 may be disposed on the base layer 10 by screen printing, spray printing, vacuum magnetron sputtering, evaporation, plating, or the like. In other examples, the conductive layer 20 may be planar only in the area where the at least one limiting groove 210 is disposed.

In step S202, in some examples, referring to fig. 8 (b), after the conductive layer 20 is dried, a layer of photosensitive material such as photoresist (including positive photoresist, negative photoresist, reverse photoresist or double layer photoresist), SU-8, photosensitive polyimide may be spin coated or coated on the conductive layer 20 to form a photosensitive layer 220. Thereby, the photosensitive layer 220 can be partially cured by light using the characteristics of the photosensitive material to form the limit grooves 210 having a predetermined pattern on the conductive layer 20.

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

In this embodiment, in some examples, in step 202, the method of patterning may include: the photosensitive layer 220 is partially cured by irradiating a mask having a predetermined pattern placed over the photosensitive layer 220 with light or irradiating the photosensitive layer 220 with a laser source constituting a predetermined pattern, and then removing the uncured photosensitive layer 220 to form at least one limit groove 210. Thereby, the limit grooves 210 having a predetermined pattern can be formed on the conductive layer 20 using the characteristics of the photosensitive material.

In some examples, referring to fig. 8 (c), after the photosensitive layer 220 is disposed on the conductive layer 20, the photosensitive layer 220 may be irradiated with a laser light source constituting a circle or a mask having a circular pattern disposed over the photosensitive layer 220 may be irradiated for exposure and development, thereby curing the photosensitive material in the photosensitive layer 220 into the shape of the circular limiting groove 210. Next, referring to fig. 8 (d), the uncured photosensitive material may be removed using a lift-off process or with a dedicated reagent, such that the photosensitive layer 220 retains only the cured at least one spacing groove 210 formed on the conductive layer 20. Of course, the shape of the limit groove 210 is not limited to a circle, and in other examples, the limit groove 210 may be oval, rectangular, polygonal, irregular, or the like. The number of the limiting grooves 210 in the conductive layer 20 is not limited to 1 or 3, but may be 2, 4, 5, 7, or the like. In some examples, the plurality of limiting grooves 210 may not be limited to a linear arrangement, but may be arranged in a folded or curved or irregular shape, for example.

In this embodiment, in some examples, referring to fig. 8 (e), in step S30, a predetermined amount of glucose sensing reagent 310 may be dropped to at least one of the limiting grooves 210 such that the glucose sensing reagent 310 is held within the range of the at least one of the limiting grooves 210, and the glucose sensing reagent 310 is solidified to form the sensing portion 30. Thus, the sensing part 30 with the preset shape and area can be formed in the at least one limit groove 210 on the conductive layer 20, which is beneficial to controlling the consistency of the area and shape of the sensing part 30 in the working electrode 1 in mass production, and obtaining the glucose sensor with consistent process parameters.

In step S30, in some examples, the glucose-sensing reagent 310 may be cross-linked and cured under normal temperature conditions. In some examples, the glucose-sensitive reagent 310 may be cross-linked cured in air at ambient temperature (e.g., 25 ℃ ±5 ℃), preferably for more than 40 hours, e.g., 48 hours. Thus, the glucose sensor 310 can be stably fixed in the limiting groove 210 and the water vapor in the air contributes to the stable crosslinking. In some examples, the cross-linking curing may be performed in a nitrogen cabinet at ambient temperature. This can prevent the reaction with the reactive gas in the environment during the curing process. In some examples, after the crosslinking curing is completed, the working electrode 1 may be stored in a low humidity environment, for example, in a nitrogen cabinet at normal temperature. Thus, the enzyme activity can be maintained.

In this embodiment, referring to fig. 8, in some examples, the amount of the drip liquid that is dispensed by the drip device each time is first pre-adjusted, i.e., a predetermined amount of the glucose-sensitive reagent 310 is set to match the volume of the limiting groove 210 (e.g., to make the predetermined amount equal to the volume of the limiting groove 210 or slightly greater than the volume of the limiting groove 210 but still within the range of surface tension effects); the stepping distance of the micro-drip head is adjusted so that the stepping distance coincides with the pitch of the plurality of limit grooves 210 arranged in a straight line on the conductive layer 20, for example. Then, the micro-instilling head of the dripping device is moved to be aligned with the limit groove 210 of the working electrode 1, and the glucose sensitive reagent 310 is dripped into the limit groove 210 by the instilling head.

In some examples, conductive layer 20 contains alignment marks and the dispensing device has an Automated Optical Inspection (AOI) probe, in which case the dispensing device micro-drip head can be automatically aligned with the limit groove 210 of working electrode 1.

In this embodiment, in some examples, after the sensing part 30 is dried in step S40, a semipermeable membrane 40 for controlling the passage of glucose molecules may be provided outside the sensing part 30. In some examples, the semipermeable membrane 40 may be provided outside the sensing portion 30 or the entire working electrode 1 by spin coating, or dip-coating, or the like.

While the disclosure has been described in detail in connection with the drawings and examples, it is to be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure. It will be understood by those within the art that, in general, terms used in this disclosure are generally intended to be "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.).

Claims

1. A working electrode of a glucose sensor suitable for mass production is characterized in that,

comprising the following steps:

a base layer which is made of an insulating material and has been pretreated to form a surface having a predetermined roughness;

a conductive layer disposed on the base layer and having a plurality of limit grooves arranged in a predetermined direction of the conductive layer, the conductive layer integrally covering the base layer as a whole, the plurality of limit grooves being formed on a surface of the conductive layer by coating a photosensitive material on the surface of the conductive layer to form a photosensitive layer, and curing the photosensitive layer in a predetermined pattern, the predetermined pattern including a plurality of closed patterns and each of the closed patterns being uniform in size and shape, and removing the uncured photosensitive layer; and

and the sensing parts are formed by respectively dripping a preset amount of glucose sensitive reagent into the limiting grooves and solidifying the glucose sensitive reagent.

2. The working electrode of claim 1 wherein,

the plurality of limit grooves are arranged in a straight line.

3. The working electrode of claim 1 wherein,

the sensor part is also covered with a semipermeable membrane for controlling the passage of glucose molecules.

4. The working electrode of claim 1 wherein,

and the glucose sensitive reagent completely fills the limit groove.

5. The working electrode of claim 1 wherein,

the limiting groove is a circular groove or an egg-shaped groove.

6. The working electrode of claim 1 wherein the electrode is,

the material of the conductive layer is at least one of glassy carbon, graphite, silver chloride, platinum, palladium, platinum-iridium, titanium, gold or iridium.

7. The working electrode of claim 1 wherein the electrode is,

the surface of the conducting layer or the surface of the limit groove is provided with a nanofiber three-dimensional network structure, and the glucose sensing reagent comprises glucose, a metal polymer and a cross-linking agent.

8. A method of preparing a working electrode for a glucose sensor suitable for mass production, the working electrode comprising:

a conductive layer disposed on the base layer and having a plurality of stopper grooves arranged along a predetermined direction of the conductive layer; and

a plurality of sensing parts disposed on the conductive layer;

The preparation method is characterized by comprising the following steps:

(a) Preparing a base layer having insulation properties and pretreating the base layer so that the surface thereof has a predetermined roughness;

(b) Forming the conductive layer on the substrate layer, wherein the conductive layer integrally covers the substrate layer as a whole, and coating a photosensitive layer with biocompatibility on the conductive layer, and patterning the photosensitive layer according to a predetermined pattern to form the plurality of limit grooves on the conductive layer, wherein the predetermined pattern comprises a plurality of closed patterns, and the sizes and the shapes of the closed patterns are consistent;

(c) And a step of dispensing a predetermined amount of glucose-sensitive reagent into the plurality of limiting grooves so that the glucose-sensitive reagent is held within the plurality of limiting grooves, and a step of solidifying the glucose-sensitive reagent to form the plurality of sensing portions.

9. The method according to claim 8, wherein,

in step (b), the patterning method includes: the mask plate having the predetermined pattern is placed over the photosensitive layer by light irradiation or the photosensitive layer is irradiated with a laser source constituting the predetermined pattern to partially cure the photosensitive layer, and then the uncured photosensitive layer is removed to form the plurality of limit grooves.

10. The method according to claim 8, wherein,

the sum of the surface areas of the plurality of limit grooves is not less than 50% of the surface area of the conductive layer.