Enzyme spraying method of micro electrochemical biosensor and micro sensor

Abstract

The invention discloses an enzyme spraying method of a miniature electrochemical biosensor. The enzyme spraying method comprises the following steps: printing at least one substrate electrode on the upper surface of a substrate; a hollow-out position is formed in the position, corresponding to the substrate electrode, of the shielding plate; placing the baffle plate on the upper surface of the substrate; and spraying an enzyme preparation solution to the hollow positions in a spraying manner so as to form a whole reaction enzyme layer on the substrate electrode. Therefore, the enzyme preparation solution is sprayed on the substrate electrode through ultrasonic spraying or other spraying modes in combination with the shielding plate hollowed-out position placed on the substrate electrode of the substrate so as to form the whole reaction enzyme layer, and due to the fact that spraying equipment is low in manufacturing cost and low in requirement for the enzyme preparation solution, the enzyme preparation solution can be sprayed on the substrate electrode, and the spraying efficiency is greatly improved. Meanwhile, the hollowed-out position of the baffle plate can well control the area and form of the reaction enzyme layer, so that the enzyme spraying method of the micro electrochemical biosensor reduces the production cost, and ensures the consistency and stability of production at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensors, and in particular to an enzyme spraying method of a micro electrochemical biosensor and a micro sensor. Background Art

[0002] The core elements of microsensors, especially microelectrochemical biosensors, are sensor miniaturization, process consistency and mass production stability.

[0003] Micro-electrochemical biosensors usually require a small amount of biological agents to be coated on a designated area of the sensor. This process usually requires extremely sophisticated equipment and process steps to complete.

[0004] For example, consider a microscopic glucose biosensor, also known as a continuous glucose monitoring system (CGMS) sensor. CGMS sensors typically use an enzymatic method to continuously monitor glucose concentrations in the body, making batch consistency and stability crucial. One of the most crucial processes impacting CGMS sensor performance is the uniform and consistent application of a minimal amount of enzyme preparation onto each sensor's reaction electrode to achieve optimal production efficiency and yield. Uniformly coating the enzyme preparation onto the sensor's reaction electrode typically involves inkjet printing, a technique typically employed for inkjet printing. This, however, places extremely high demands not only on the nozzle precision but also on the enzyme preparation itself, requiring a particle-free, low-viscosity enzyme preparation (<20 cPs).

[0005] Therefore, the equipment price of the existing micro electrochemical biosensor is generally high, and due to the extremely high requirements for enzyme preparations, the production stability is also poor. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide an enzyme spraying method for a micro electrochemical biosensor, which can improve the production consistency and stability of the micro electrochemical biosensor while reducing the production cost.

[0007] One of the purposes of the present invention is achieved by the following technical solution: A micro electrochemical biosensor enzyme spraying method, comprising: printing at least one substrate electrode on the upper surface of the substrate; A hollow position is provided on the shielding plate at a position corresponding to the substrate electrode; placing the shielding plate on the upper surface of the substrate; The enzyme preparation solution is sprayed on the hollowed position in a spraying manner to form a whole reaction enzyme layer on the substrate electrode.

[0008] Furthermore, the method further includes vertically uncovering the shielding plate after the first time.

[0009] Furthermore, spraying the enzyme preparation solution on the hollowed position by spraying includes: Spraying the enzyme preparation solution on the hollowed position at least once to increase the thickness of the reaction enzyme layer; The time interval between each spraying is between ten seconds and ten hours.

[0010] Furthermore, it also includes that corresponding positioning holes are provided around the base layer and the shielding plate.

[0011] Furthermore, the step of providing a hollowed-out area on the shielding plate at a position corresponding to the substrate electrode comprises: The substrate electrode includes a working electrode and a conductive circuit, and a hollow position is provided on the shielding plate at a position corresponding to the working electrode; The hollowed-out area covers part or all of the surface of the working electrode.

[0012] Furthermore, the hollowing out of the shielding plate at a position corresponding to the substrate electrode comprises: The hollow positions are created by laser processing, photolithography, plasma etching or chemical etching.

[0013] Furthermore, the step of spraying the enzyme preparation solution on the hollowed-out area to form a monolithic reaction enzyme layer on the substrate electrode comprises: Spraying the enzyme preparation solution on the hollowed-out area by spraying, the area being larger than the area of the hollowed-out area, wherein the spraying includes ultrasonic spraying; The outer edge of the working electrode is cut by laser cutting to form an enzyme sensing layer with a uniform area and a whole piece.

[0014] In a second aspect, the present invention also provides a microsensor, which includes a substrate, a substrate electrode printed on the substrate, a reaction enzyme layer formed on the substrate electrode, and a membrane layer formed on the reaction enzyme layer, wherein the reaction enzyme layer is formed on the substrate electrode by the enzyme spraying method of the micro electrochemical biosensor according to any one of claims 1 to 7.

[0015] Furthermore, the substrate electrode includes a working electrode and a conductive circuit, the reaction enzyme layer is formed on the working electrode, and the hollow position covers part or all of the surface of the working electrode.

[0016] Furthermore, 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.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present application discloses a method for spraying enzymes for a micro electrochemical biosensor, which includes: printing at least one substrate electrode on the upper surface of a substrate; providing a hollow position on a shield plate at a position corresponding to the substrate electrode; placing the shield plate on the upper surface of the substrate; spraying an enzyme solution on the hollow position to form a whole reaction enzyme layer on the substrate electrode. Therefore, the present application sprays the enzyme solution onto the substrate electrode by spraying in combination with placing a hollow position of a shield plate on the substrate electrode of the substrate to form a whole reaction enzyme layer. Since the spraying equipment is low in cost and has low requirements for the enzyme solution, and the hollow position of the shield plate can well control the area and morphology of the reaction enzyme layer, the present application's method for spraying enzymes for a micro electrochemical biosensor reduces production costs while also ensuring consistency and stability in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a flow chart of the method of the present invention; Figure 2 It is a structural schematic diagram of the shielding plate and the base plate of the present invention; Figure 3 A top view of the shielding plate of the present invention; Figure 4 This is a schematic diagram of the effect of spraying the enzyme preparation solution on the substrate electrode of the present invention; Figure 5 This is another schematic diagram of the effect of spraying the enzyme preparation solution on the substrate electrode of the present invention.

[0019] The main component symbols are described as follows: 101 - shielding plate; 102 - hollow position; 103 - substrate; 104 - substrate electrode; 105 - reaction enzyme layer; 106 - positioning hole. DETAILED DESCRIPTION

[0020] In order to more clearly understand the specific technical solutions, features and advantages of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] In the description of the present invention, it should be noted that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They 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, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limiting the present invention. Implementation Method 1

[0022] like Figures 1 to 3 As shown, a micro electrochemical biosensor enzyme spraying method comprises: S1: printing at least one substrate electrode 104 on the upper surface of the substrate 103; As mentioned above, the substrate 103 of this embodiment can be a plastic film substrate, on which one or more substrate electrodes 104 are formed using screen printing. It is worth noting that, in this embodiment, the substrate electrodes 104 are grouped into a region, each of which is provided with a working electrode (not shown) and a conductive circuit (not shown), with the working electrode and the conductive circuit connected. 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.

[0023] S2: a hollow portion 102 is formed on the shielding plate 101 at a position corresponding to the substrate electrode 104; The above means setting a shielding plate 101 and setting a hollow position 102 on the shielding plate 101, such as Figures 2 to 3 As shown, the number and position of the hollowed-out areas 102 correspond to the number and position of the substrate electrodes 104. The shielding plate 101 can be made of metal, glass, or plastic or other materials that are easy to cut and hollow out.

[0024] S3: placing the shielding plate 101 on the upper surface of the substrate 103; S4 sprays the enzyme solution on the hollowed-out portion 102 by spraying to form a whole piece of reaction enzyme layer 105 on the substrate electrode 104, such as Figure 4 shown.

[0025] 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 working electrode positions of the substrate electrode 104. After the substrate 103 and the shielding plate are fixed in position, the spraying equipment is activated, and the pre-programmed spraying program is initiated. The instrument automatically begins spraying enzyme spots onto the shielding plate 101 and its cutouts 102. During enzyme spraying, the enzyme solution penetrates the cutouts 102 of the shielding plate 101, forming a uniform, uniformly sized, and well-formed enzyme spot pattern (i.e., the enzyme layer 105) on the working electrode. Therefore, the enzyme spraying method of the micro-electrochemical biosensor of 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 spraying equipment is low in cost and has low requirements for the enzyme solution. The enzyme solution does not need to be free of particles and is compatible with high viscosity (viscosity > 20 cPs). At the same time, the total area and shape of the reaction enzyme layer 105 can be very accurately controlled through the hollow position 102, thereby ensuring the final consistency of the product, improving product yield and stability, and reducing production costs.

[0026] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor further includes: S5 After the first time, the shielding plate 101 is vertically opened.

[0027] The aforementioned "after the first period" refers to waiting for the enzyme solution sprayed on the substrate electrode 104 to dry and form a stable reaction enzyme layer 105, and then simultaneously lifting the four corners of the shielding plate 101 at an angle perpendicular to the substrate 103 to remove the shielding plate 101 from the substrate 103. This ensures the integrity of the reaction enzyme layer 105.

[0028] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor is to spray the enzyme preparation solution on the shielding plate 101 by spraying, including: Spraying the enzyme solution on the shielding plate 101 at least once to increase the thickness of the reaction enzyme layer 105; The time interval between each ultrasonic spraying is between ten seconds and ten hours.

[0029] As mentioned above, in order to ensure that the thickness of the reaction enzyme layer 105 meets the requirements, the enzyme solution can be repeatedly sprayed to increase the thickness of the enzyme layer. The interval between repeated spraying of the enzyme solution can be set to ten seconds, one minute, ten minutes to ten hours, etc. The specific time can be determined according to the effect of spraying the enzyme solution. It is worth further explaining that the time interval between spraying refers to the time interval between the same substrate electrode 104 being ultrasonically sprayed with enzyme. With respect to the substrate 103, the 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 the row; then start from one side of the other row and spray to the substrate electrode 104 on the other side of the row, and so on. When it is necessary to spray the enzyme solution repeatedly, return to one side of the first row and spray to the substrate electrode 104 on the other side of the row. The shielding plate 101 can be cleaned, dried or blown dry before reuse.

[0030] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor further includes, between step S2 and step S3, Positioning holes 106 corresponding to each other are provided around the base layer and the shielding plate 101 .

[0031] As described above, since the positions of the hollowed-out portion 102 and the substrate electrode 104 need to be completely matched, the provision of the positioning hole 106 facilitates positioning of the shielding plate 101 on the substrate 103 , thereby facilitating the matching of the positions of the hollowed-out portion 102 and the substrate electrode 104 .

[0032] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor, wherein the hollowing out portion 102 is formed on the shielding plate 101 at a position corresponding to the substrate electrode 104, comprises: The substrate electrode 104 includes a working electrode and a conductive circuit, and a hollow position 102 is provided on the shielding plate 101 at a position corresponding to the working electrode; The hollow portion 102 covers part or all of the surface of the working electrode.

[0033] As mentioned above, when there are multiple substrate electrodes 104, the shape and area of the working electrode on each substrate electrode 104 are kept consistent by printing. The area of the enzyme layer 105 includes but is not limited to the area that exceeds the corresponding working electrode in whole or in part, such as Figure 5 As shown, the surface of the hollow portion 102 includes a portion that exceeds the surface of the working electrode. The working electrode can also be completely covered by the enzyme solution.

[0034] In another specific embodiment, the printed working electrode area can be larger than the actual use area to facilitate the subsequent spraying of the enzyme preparation solution. After the spraying is completed, the laser is used to cut off the excess working electrode and the irregular part of the outer edge shape of the reaction enzyme layer 105.

[0035] In this embodiment, in the enzyme spraying method of the micro electrochemical biosensor, the hollowing out 102 is formed at a position corresponding to the substrate electrode 104 on the shielding plate 101, and the hollowing out 102 includes: The hollow position 102 is opened by laser processing, photolithography, plasma etching or chemical etching.

[0036] As mentioned above, the pattern of the hollow position 102 of the shielding plate 101, that is, the pattern and area of the final reaction enzyme layer 105, includes but is not limited to any shape such as circle, oval, rectangle, star, etc. The specific pattern shape and area of the hollow position 102 can be designed and opened according to product requirements.

[0037] In this embodiment, the enzyme spraying method of the micro electrochemical biosensor, wherein the enzyme preparation solution is sprayed on the hollow portion 102 by spraying to form a monolithic reaction enzyme layer 105 on the substrate electrode 104, comprises: The area of the enzyme preparation solution sprayed on the hollow position 102 is larger than the area of the hollow position 102 by spraying, and the spraying includes ultrasonic spraying; The outer edge of the working electrode is cut by laser cutting to form an enzyme sensing layer with a uniform area and a whole piece.

[0038] To ensure that each hollowed-out area 102 is fully coated with the enzyme solution, the area of the enzyme solution sprayed on the hollowed-out area 102 must be larger than the area of the pattern of the hollowed-out area 102. That is, the area on the substrate where the enzyme solution is sprayed includes the hollowed-out area 102 and its outer edge (i.e., the hollowed-out area 102), and the area is evenly covered. To maintain the consistency of the area of the sensor's enzyme layer 105 and ensure production stability, the excess enzyme layer 105 at the outer edge of the working electrode is cut using laser cutting to form a uniform, integrated enzyme sensing layer.

[0039] That is, in order for the sprayed enzyme solution to completely cover the working electrode, the area of the hollowed-out portion 102 can be larger than the area of the working electrode. However, for production consistency, the irregular reaction enzyme layer 105 at the outer edge of the working electrode can be cut by laser cutting, and the cut reaction enzyme layer 105 becomes an enzyme sensing layer with a uniform area. In order to cover the entire hollowed-out portion 102 when spraying the enzyme solution, the area of the hollowed-out portion 102 to be sprayed must be larger than the area of the hollowed-out portion 102 itself, and the position of the enzyme solution spraying must be such that the corresponding hollowed-out portion 102 is completely covered.

[0040] In this embodiment, the spraying method may be ultrasonic spraying or other spraying methods. Implementation Method 2

[0041] A micro sensor comprises 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 film 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 according to any one of claims 1 to 7.

[0042] The above description refers to the embodiment in which a single microsensor sensing surface has a single sensing element, namely, a reaction 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 produced using a printing process, with a thickness of less than 300 μm and a tip width of less than 400 μm.

[0043] It should be noted that the continuous glucose monitoring device (system) includes the aforementioned microsensor, an electronic system, and an injection aid. The electronic system can be a mobile terminal that receives analyte data, and the microsensor is mounted on the injection aid. The microsensor achieves consistent response after implantation through consistent production process control and the use of a low operating voltage principle, including consistent current-glucose concentration response sensitivity and consistent background current at low operating voltage. To achieve consistent response sensitivity, the enzyme spraying method of the microelectrochemical biosensor described in Example 1 allows precise control of the area, shape, and thickness of the enzyme spray in the microsensor, making it suitable for mass production.

[0044] Specifically, the micro-object sensor comprises a substrate electrode 104 formed with a non-conductive material and a conductive layer. The conductive layer is disposed on and in contact with the non-conductive material. A working electrode is defined on the conductive layer, and an enzyme layer 105 is disposed on the working electrode. The sensitivity coefficient of the working electrode is less than or equal to 8%. When the thickness of the enzyme layer (enzyme layer 105) reaches a certain level, the sensitivity of the micro-sensor is related to the surface area of the enzyme layer 105, rather than its thickness.

[0045] It is noteworthy that the sensitivity of the microsensor depends on the area of the enzyme layer 105, such as the layer disposed on the working electrode surface (on the working electrode) including the analyte enzyme, or the redox mediator or the redox mediator covalently or non-covalently linked to the polymer, but is not significantly dependent on the edge effect of the sensing element. In addition, the sensitivity of the microsensor may also depend on the analyte flux flowing through a flux-limiting membrane (such as a permeable membrane control layer or membrane layer) disposed two-dimensionally on the enzyme layer 105 to the working electrode surface (such as to a flat surface). The sensitivity of the microsensor may also depend on the analyte flux passing through a flux-limiting membrane disposed three-dimensionally on the sensing element, but these are not limited here.

[0046] In this embodiment, the substrate electrode includes a working electrode and a conductive circuit, the reaction enzyme layer is formed on the working electrode, and the surface of the hollow portion covers part or all of the surface of the working electrode.

[0047] 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.

[0048] Specifically, the enzyme solution forming the reactive enzyme layer may be a glucose-responsive enzyme (e.g., glucose oxidase, glucose dehydrogenase, etc.) or a lactate-responsive enzyme (e.g., lactate oxidase). In other embodiments, the enzyme solution may also include a redox mediator and other enzyme compositions, such as quinone compounds or transition metal complexes. The liquid used in the ultrasonic spray coating process includes, but is not limited to, an enzyme solution and may also be other biological or chemical liquids or preparations.

[0049] Therefore, the types of microsensors include but are not limited to continuous glucose monitoring sensors, and can also be other microsensors, such as analyte sensors for detecting blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides, inorganic salts, cancer and other disease markers, and small chemical molecules in the body.

[0050] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for spraying enzymes for a micro electrochemical biosensor, characterized in that: include: printing at least one substrate electrode on the upper surface of the substrate; A hollow position is provided on the shielding plate at a position corresponding to the substrate electrode; placing the shielding plate on the upper surface of the substrate; The enzyme preparation solution is sprayed on the hollowed position in a spraying manner to form a whole reaction enzyme layer on the substrate electrode.

2. The enzyme spraying method of the micro electrochemical biosensor according to claim 1, characterized in that: The method further includes vertically uncovering the shielding plate after the first time.

3. The enzyme spraying method of the micro electrochemical biosensor according to claim 1, characterized in that: The method of spraying the enzyme preparation solution on the hollowed-out area comprises: Spraying the enzyme preparation solution on the hollowed position at least once to increase the thickness of the reaction enzyme layer; The time interval between each spraying is between ten seconds and ten hours.

4. The enzyme spraying method of the micro electrochemical biosensor according to claim 1, characterized in that: It also includes that corresponding positioning holes are provided around the base layer and the shielding plate.

5. The enzyme spraying method of the micro electrochemical biosensor according to claim 1, characterized in that: The step of providing a hollow position on the shielding plate at a position corresponding to the substrate electrode comprises: The substrate electrode includes a working electrode and a conductive circuit, and a hollow position is provided on the shielding plate at a position corresponding to the working electrode; The hollowed-out area covers part or all of the surface of the working electrode.

6. The enzyme spraying method of the micro electrochemical biosensor according to claim 1, characterized in that: The hollowing out position is provided on the shielding plate at a position corresponding to the substrate electrode, wherein the hollowing out position includes: The hollow positions are created by laser processing, photolithography, plasma etching or chemical etching.

7. The enzyme spraying method of the micro electrochemical biosensor according to claim 5, characterized in that: The step of spraying the enzyme preparation solution on the hollowed-out area to form a monolithic reaction enzyme layer on the substrate electrode comprises: Spraying the enzyme preparation solution on the hollowed-out area by spraying, the area being larger than the area of the hollowed-out area, wherein the spraying includes ultrasonic spraying; The outer edge of the working electrode is cut by laser cutting to form an enzyme sensing layer with a uniform area and a whole piece.

8. A micro sensor, characterized in that: The micro electrochemical biosensor comprises a substrate, a substrate electrode printed on the substrate, a reaction enzyme layer formed on the substrate electrode, and a membrane layer formed on the reaction enzyme layer. The reaction enzyme layer is formed on the substrate electrode by the enzyme spraying method of the micro electrochemical biosensor according to any one of claims 1 to 7.

9. The microsensor according to claim 8, characterized in that: The substrate electrode includes a working electrode and a conductive circuit. The reaction enzyme layer is formed on the working electrode. The hollow portion covers part or all of the surface of the working electrode.

10. The microsensor according to claim 8, characterized in that 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.

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