A method for spraying enzymes into a miniature electrochemical biosensor and the miniature sensor itself.
By printing substrate electrodes on the substrate and creating cutouts on the shielding plate, combined with spraying and laser cutting technologies, the problems of expensive and unstable production equipment for micro electrochemical biosensors have been solved, resulting in cost reduction and improved consistency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN ENDOK MEDICAL CO LTD
- Filing Date
- 2025-07-03
- Publication Date
- 2026-05-26
Smart Images
Figure CN120490258B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of electrochemical biosensors, and more particularly to an enzyme spraying method and a micro-sensor for a micro-electrochemical biosensor. Background Technology
[0002] The core elements of micro-sensors, especially micro-electrochemical biosensors, are sensor miniaturization, process consistency, and mass production stability.
[0003] Miniature electrochemical biosensors typically require coating a small amount of biological agent onto a designated area of the sensor, a process that usually requires extremely sophisticated equipment and manufacturing procedures.
[0004] For example, consider miniature glucose biosensors, such as continuous glucose monitoring systems (CGMS). CGMS sensors typically use enzymatic methods to continuously monitor glucose concentration in the body, making batch-to-batch consistency and stability crucial. One of the most important processes affecting CGMS sensor performance is the uniform and consistent coating of trace amounts of enzyme onto the reaction electrode of each sensor to achieve good production efficiency and yield. Current technology for uniformly coating the enzyme onto the sensor's reaction electrode generally uses inkjet printing. However, inkjet printing requires extremely high precision not only in the printhead but also in the enzyme itself, demanding a particulate-free and low-viscosity enzyme (<20 cPs).
[0005] Therefore, the equipment price of existing micro electrochemical biosensors is generally high, and the production stability is also poor due to the extremely high requirements of enzyme preparations. Summary of the Invention
[0006] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide an enzyme spraying method for micro electrochemical biosensors, which can improve the production consistency and stability of micro electrochemical biosensors, while also reducing production costs.
[0007] One of the objectives of this invention is achieved through the following technical solution:
[0008] A method for spraying enzymes in a miniature electrochemical biosensor, comprising:
[0009] At least one substrate electrode is printed on the upper surface of the substrate;
[0010] A cutout is made on the shielding plate at the position corresponding to the substrate electrode;
[0011] The shielding plate is placed on the upper surface of the substrate; an enzyme preparation solution is sprayed onto the hollowed-out areas to form a monolithic reaction enzyme layer on the substrate electrode.
[0012] Furthermore, it also includes vertically opening the cover after a certain period of time.
[0013] Furthermore, the enzyme preparation solution is sprayed onto the hollowed-out areas by spraying, including:
[0014] The enzyme preparation solution is sprayed onto the hollowed-out area at least once to increase the thickness of the reaction enzyme layer.
[0015] The time interval between each spraying is between ten seconds and ten hours.
[0016] Furthermore, it also includes corresponding positioning holes around the base layer and the shielding plate.
[0017] Furthermore, the step of creating a cutout on the shield plate at the position corresponding to the substrate electrode includes:
[0018] The substrate electrode includes a working electrode and conductive lines, and a cutout is made on the shielding plate at the position corresponding to the working electrode.
[0019] The hollowed-out area covers part or all of the surface of the working electrode.
[0020] Furthermore, in the step of creating a cutout on the shield plate at a position corresponding to the substrate electrode, the creation of the cutout includes:
[0021] Hollow areas can be created using laser processing, photolithography, plasma etching, or chemical etching.
[0022] Further, the step of spraying the enzyme preparation solution onto the hollowed-out areas by spraying to form a monolithic reaction enzyme layer on the substrate electrode includes:
[0023] The area of the enzyme preparation solution sprayed onto the hollowed-out area by spraying is larger than the area of the hollowed-out area, and the spraying includes ultrasonic spraying.
[0024] The outer edge of the working electrode is cut by laser cutting to form a uniform and monolithic enzyme sensing layer.
[0025] In a second aspect, the present invention also provides a microsensor comprising a substrate, a substrate electrode printed on the substrate, a reactive enzyme layer formed on the substrate electrode, and a membrane layer formed on the reactive enzyme layer, wherein the reactive enzyme layer is formed on the substrate electrode by the enzyme spraying method of the micro electrochemical biosensor described above.
[0026] Furthermore, the substrate electrode includes a working electrode and conductive circuitry, the enzyme layer is formed on the working electrode, and the cutout covers part or all of the surface of the working electrode.
[0027] Furthermore, the microelectrochemical biosensor is an analyte sensor for detecting small chemical molecules such as glucose, blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides, or inorganic salts.
[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0029] This application discloses a method for spraying enzyme onto a micro-electrochemical biosensor, comprising: printing at least one substrate electrode on the upper surface of a substrate; creating cutouts on a shielding plate at positions corresponding to the substrate electrode; placing the shielding plate on the upper surface of the substrate; and spraying an enzyme preparation solution onto the cutouts to form a monolithic reactive enzyme layer on the substrate electrode. Therefore, this application utilizes a spraying method, combined with placing a shielding plate with cutouts on the substrate electrode, to spray the enzyme preparation solution onto the substrate electrode to form a monolithic reactive enzyme layer. Because the spraying equipment is inexpensive and has low requirements for the enzyme preparation solution, and because the cutouts on the shielding plate can effectively control the area and morphology of the reactive enzyme layer, this method for spraying enzyme onto a micro-electrochemical biosensor reduces production costs while ensuring production consistency and stability. Attached Figure Description
[0030] Figure 1 This is a flowchart of the method of the present invention;
[0031] Figure 2 This is a schematic diagram of the structure of the shielding plate and the substrate of the present invention;
[0032] Figure 3 This is a top view of the shielding plate of the present invention;
[0033] Figure 4 This is a schematic diagram illustrating the effect of spraying an enzyme preparation solution onto a substrate electrode according to the present invention;
[0034] Figure 5 This is another schematic diagram illustrating the effect of spraying an enzyme preparation solution onto the substrate electrode of the present invention.
[0035] The symbols for the main components are explained below:
[0036] Shielding plate; 102-cutout; 103-substrate; 104-substrate electrode; 105-reactant layer; 106-positioning hole. Detailed Implementation
[0037] To better understand the specific technical solutions, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. In the description of the present invention, it should be noted that the terms "upper," "lower," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.
[0038] Implementation Method 1
[0039] like Figures 1 to 3 As shown, a method for spraying enzymes in a micro electrochemical biosensor includes:
[0040] S1 prints at least one substrate electrode 104 on the upper surface of the substrate 103;
[0041] As described above, the substrate 103 in this embodiment can be a plastic film substrate. One or more substrate electrodes 104 are fabricated on the substrate using screen printing. It is worth noting that in this embodiment, the substrate electrodes 104 form a group of regions. Each substrate electrode 104 is provided with a working electrode (not shown in the figure) and a conductive line (not shown in the figure), and the working electrode is connected to the conductive line. When there are multiple substrate electrodes 104, they can be arranged in multiple rows to improve the printing efficiency of the substrate electrodes 104 and the efficiency of enzyme spraying.
[0042] S2 A cutout 102 is made on the shielding plate 101 at a position corresponding to the substrate electrode 104;
[0043] The above refers to setting a cover plate 101, and setting a cutout position 102 on the cover plate 101, such as... Figures 2 to 3 As shown, the number and position of the cutouts 102 correspond to the number and position of the substrate electrodes 104. The shielding plate 101 can be a metal shielding plate 101, a glass shielding plate 101, or a shielding plate 101 made of other materials such as plastic, which is easy to cut and cut out.
[0044] S3 Place the shielding plate 101 on the upper surface of the substrate 103;
[0045] S4 applies an enzyme preparation solution to the hollowed-out area 102 by spraying to form a monolithic enzyme layer 105 on the substrate electrode 104, such as... Figure 4 As shown.
[0046] As described above, the shielding plate 101 is placed on the substrate 103, with the cutouts 102 of the shielding plate 101 corresponding to the positions of the working electrodes of the substrate electrode 104. After the positions of the substrate 103 and the shielding plate are fixed, the spraying equipment is started, and the pre-edited spraying program is initiated. The instrument automatically sprays enzyme onto the shielding plate 101 and its cutouts 102. During enzyme spraying, the enzyme solution passes through the cutouts 102 on the shielding plate 101, forming a uniform, consistent, appropriately sized, and regularly shaped whole enzyme spot pattern (i.e., the reaction enzyme layer 105) on the working electrode. Therefore, the enzyme spraying method of the micro electrochemical biosensor in this embodiment sprays the enzyme solution onto the cutouts 102 of the shielding plate 101, thereby spraying the enzyme solution onto the corresponding substrate electrode 104. The equipment for spraying is inexpensive and has low requirements for enzyme solutions. The enzyme solution does not need to be free of particulate matter and is compatible with high viscosity (viscosity > 20 cPs). At the same time, it can very precisely control the total area and shape of the reaction enzyme layer 105 through the cutout position 102, thereby ensuring the final consistency of the product, improving product yield and stability, and reducing production costs.
[0047] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor further includes,
[0048] S5 After waiting for the first time, vertically peel off the cover plate 101.
[0049] The above-mentioned "after the first time" refers to waiting for the enzyme preparation solution sprayed on the substrate electrode 104 to dry and form a stable reactive enzyme layer 105, after which the four corners of the shielding plate 101 are simultaneously lifted at an angle perpendicular to the substrate 103 to peel the shielding plate 101 off the substrate 103. This ensures the integrity of the reactive enzyme layer 105.
[0050] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor involves spraying an enzyme preparation solution onto the shielding plate 101 by spraying, including:
[0051] The enzyme preparation solution is sprayed onto the shielding plate 101 at least once to increase the thickness of the reactive enzyme layer 105; the time interval between each ultrasonic spraying is between ten seconds and ten hours.
[0052] To ensure the thickness of the enzyme layer 105 meets requirements, the enzyme solution can be repeatedly sprayed to increase its thickness. The interval between sprayings can be set to ten seconds, one minute, ten minutes to ten hours, depending on the effectiveness of the sprayed solution. It is worth noting that the time interval between sprayings refers to the time interval between ultrasonic spraying of the same substrate electrode 104. Relative to the substrate 103, spraying can be continuous or timed. When there are multiple rows of substrate electrodes 104 on the substrate 103, the spraying equipment can start from one side of the first row and spray to the substrate electrode 104 on the other side of that row; then start from one side of the other row and spray to the substrate electrode 104 on the other side of that row, and so on. When repeated spraying of the enzyme solution is required, the process returns to one side of the first row and sprays to the substrate electrode 104 on the other side of that row. The used shielding plate 101 can be cleaned, air-dried, or blow-dried before reuse.
[0053] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor further includes, between step S2 and step S3, the following:
[0054] The base layer and the shielding plate 101 are provided with corresponding positioning holes 106 around their perimeter.
[0055] As mentioned above, since the positions of the cutout 102 and the substrate electrode 104 need to be perfectly matched, the positioning hole 106 is provided to facilitate the positioning of the shielding plate 101 on the substrate 103, which makes it easier to match the positions of the cutout 102 and the substrate electrode 104.
[0056] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor, wherein the step of creating a cutout 102 on the shielding plate 101 at a position corresponding to the substrate electrode 104 includes:
[0057] The substrate electrode 104 includes a working electrode and conductive lines, and a cutout 102 is formed on the shielding plate 101 at a position corresponding to the working electrode.
[0058] The cutout 102 covers part or all of the surface of the working electrode.
[0059] As described above, when there are multiple substrate electrodes 104, the shape and area of the working electrode on each substrate electrode 104 are kept consistent through printing. The region of the enzyme layer 105 includes, but is not limited to, all or part of the region extending beyond the corresponding working electrode, such as... Figure 5 As shown, the surface of the cutout 102 therefore includes a portion that extends beyond the surface of the working electrode. This also allows the working electrode to be completely covered by the enzyme solution.
[0060] In another specific embodiment, the area of the printed working electrode can be larger than the actual area used, which facilitates the subsequent spraying of enzyme preparation solution. After the spraying is completed, the excess working electrode and the irregular part of the outer edge of the reaction enzyme layer 105 are removed by laser.
[0061] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor, wherein the step of creating a cutout 102 on the shielding plate 101 at a position corresponding to the substrate electrode 104, includes:
[0062] The cutout position 102 is created by laser processing, photolithography, plasma etching or chemical etching.
[0063] The above-mentioned pattern of the cutout 102 of the shielding plate 101, i.e. the pattern and area of the final reaction enzyme layer 105, includes but is not limited to any shape such as circle, ellipse, rectangle, star, etc. The specific pattern and area of the cutout 102 can be designed and opened according to product requirements.
[0064] In this embodiment, the enzyme spraying method for the micro electrochemical biosensor, wherein the enzyme preparation solution is sprayed onto the hollowed-out area 102 by spraying to form a monolithic reaction enzyme layer 105 on the substrate electrode 104, includes:
[0065] The area of the enzyme preparation solution sprayed onto the hollowed-out position 102 by spraying is larger than the area of the hollowed-out position 102, and the spraying includes ultrasonic spraying.
[0066] The outer edge of the working electrode is cut by laser cutting to form a uniform and monolithic enzyme sensing layer.
[0067] As described above, to ensure that each cutout 102 is fully coated with enzyme solution, the area of the enzyme solution sprayed at the cutout 102 must be larger than the area of the pattern of the cutout 102. That is, the area of the enzyme solution sprayed onto the substrate includes the cutout 102 and its outer edge (i.e., the cutout 102 itself), and is uniformly covered. In order to maintain the consistency of the area of the enzyme reaction layer 105 of the sensor and the production stability, the excess enzyme reaction layer 105 at the outer edge of the working electrode is cut off by laser cutting to form a uniform and integral enzyme sensing layer.
[0068] In other words, to ensure the enzyme solution completely covers the working electrode, the area of the cutout 102 can be larger than the area of the working electrode. However, for production consistency, the irregular enzyme layer 105 on the outer edge of the working electrode can be cut using laser cutting, resulting in an enzyme sensing layer of uniform area after cutting. Furthermore, to ensure complete coverage of the cutout 102 when spraying the enzyme solution, the area to be sprayed must be larger than the area of the cutout 102 itself, and the enzyme solution must be sprayed to completely cover the corresponding cutout 102.
[0069] In this embodiment, the spraying method can be ultrasonic spraying or other spraying methods.
[0070] Implementation Method 2
[0071] A microsensor includes a substrate 103, a substrate electrode 104 printed on the substrate 103, a reaction enzyme layer 105 formed on the substrate electrode 104, and a membrane layer formed on the reaction enzyme layer 105, wherein the reaction enzyme layer 105 is formed on the substrate electrode 104 by the enzyme spraying method of the micro electrochemical biosensor described above.
[0072] The above refers to the fact that in this embodiment, a single microsensor sensing surface has a sensing element, namely a reactive enzyme layer 105, the substrate 103 can be a flexible substrate 103, and the corresponding substrate electrode 104 can be a flexible electrode. The working electrode on the substrate electrode 104 is prepared by a printing process, the thickness of the working electrode is less than 300 μm, and the electrode tip width of the working electrode is less than 400 μm.
[0073] It should be noted that the continuous glucose monitoring device (system) includes the aforementioned microsensor, electronic system, and needle assist device. The electronic system can be a mobile terminal that receives analyte data, and the microsensor is mounted on the needle assist device. The microsensor achieves consistent response after implantation through consistent manufacturing process control and the use of a low operating voltage principle, including consistency in current-glucose concentration response sensitivity and consistency in background current under low operating voltage. To achieve consistent response sensitivity, the enzyme spraying method of the microelectrochemical biosensor in Example 1 is used to precisely control the area, shape, and thickness of the enzyme sprayed by the microsensor, making it suitable for mass production.
[0074] Specifically, a non-conductive material and a conductive layer are formed on the substrate electrode 104 of the micro-sensor. The conductive layer is disposed on and in contact with the non-conductive material. A working electrode is defined on the conductive layer. A reactive enzyme layer 105 is disposed on the working electrode. The sensitivity variation coefficient of the working electrode is less than or equal to 8%. When the thickness of the enzyme layer (reactive enzyme layer 105) reaches a certain level, the sensitivity of the micro-sensor is related to the surface area of the reactive enzyme layer 105 but is independent of the thickness of the reactive enzyme layer 105.
[0075] It is worth noting that the sensitivity of the microsensor depends on the area of the enzyme layer 105, such as a layer disposed on the surface of a working electrode containing the analyte enzyme (on the working electrode), or a redox mediator or a redox mediator covalently or non-covalently bonded to a polymer, but does not significantly depend on the edge effect of the sensing element. Additionally, the sensitivity of the microsensor can also depend on the analyte flow rate to the working electrode surface (e.g., to a flat surface) through a flow-limiting membrane (such as a permeation membrane control layer or membrane layer) disposed in a two-dimensional manner on the enzyme layer 105, and can also depend on the analyte flow rate through a flow-limiting membrane disposed in a three-dimensional manner on the sensing element; this is not limited here.
[0076] In this embodiment, the substrate electrode includes a working electrode and conductive circuitry, the enzyme layer is formed on the working electrode, and the surface of the cutout covers part or all of the surface of the working electrode.
[0077] In this embodiment, the micro electrochemical biosensor is an analyte sensor for detecting small chemical molecules such as glucose, blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides, or inorganic salts.
[0078] Specifically, the enzyme preparation solution forming the reaction enzyme layer can be a glucose-responsive enzyme (such as glucose oxidase, glucose dehydrogenase, etc.) or a lactate-responsive enzyme (such as lactate oxidase). In other embodiments, the enzyme preparation solution also includes redox mediators and other enzyme compositions, such as quinone compounds or transition metal complexes. The liquid used in the ultrasonic spraying process includes, but is not limited to, the enzyme preparation solution and can also be other biological or chemical liquids or preparations.
[0079] Therefore, the types of microsensors include, but are not limited to, continuous glucose monitoring sensors. They can also include other microsensors, such as sensors for detecting blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides, inorganic salts, cancer and other disease markers, and analytes of small chemical molecules in living organisms.
[0080] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.
Claims
1. A method of jetting an enzyme for a micro electrochemical biosensor, characterized by, include: At least one substrate electrode is printed on the upper surface of the substrate; A cutout is made on the shielding plate at the position corresponding to the substrate electrode; The shield is placed on the upper surface of the substrate; An enzyme preparation solution is sprayed onto the hollowed-out areas by ultrasonic spraying to form a monolithic reaction enzyme layer on the substrate electrode. After a short period of time, vertically peel off the cover. The enzyme preparation solution is sprayed onto the hollowed-out areas by ultrasonic spraying, including: The enzyme preparation solution is sprayed onto the hollowed-out area at least once by ultrasonic spraying to increase the thickness of the reaction enzyme layer. The time interval between each ultrasonic spraying is between ten seconds and ten minutes. The step of creating a cutout on the shielding plate at the position corresponding to the substrate electrode includes: The substrate electrode includes a working electrode and conductive lines, and a cutout is made on the shielding plate at the position corresponding to the working electrode. The hollowed-out area covers part or all of the surface of the working electrode; The step of spraying an enzyme preparation solution onto the hollowed-out areas by ultrasonic spraying to form a monolithic reaction enzyme layer on the substrate electrode includes: The area of the enzyme preparation solution sprayed onto the hollowed-out area by ultrasonic spraying is larger than the area of the hollowed-out area. The outer edge of the working electrode is cut by laser cutting to form a uniform and monolithic enzyme sensing layer. The substrate and the shielding plate are provided with corresponding positioning holes around their perimeters; the step of creating a cutout on the shielding plate at a position corresponding to the substrate electrode includes: Hollow areas can be created using laser processing, photolithography, plasma etching, or chemical etching.
2. A miniature sensor, characterized in that, It includes a substrate, a substrate electrode printed on the substrate, a reactive enzyme layer formed on the substrate electrode, and a membrane layer formed on the reactive enzyme layer, wherein the reactive enzyme layer is formed on the substrate electrode by the enzyme spraying method of the micro electrochemical biosensor according to claim 1.
3. The miniature sensor according to claim 2, characterized in that, The substrate electrode includes a working electrode and conductive circuits, the enzyme layer is formed on the working electrode, and the cutout covers part or all of the surface of the working electrode.