Biosensor

Abstract

The invention discloses a biosensor, and belongs to the technical field of electrochemical detection. The biosensor comprises a substrate, a conductive layer and an insulating layer, and is characterized in that the conductive layer is formed on the substrate; the insulating layer comprises an upper part and a lower part which are arranged at an interval and are respectively formed at the upper end and the lower end of the conducting layer, and the interval between the upper part and the lower part enables the area of the conducting layer to be exposed to form a first electrode. According to the biosensor, the sizes of the working electrode, the counter electrode, the reference electrode and other electrodes can be controlled according to needs.

Description

[0001] This application is a divisional application. This invention is a divisional application of "Biosensor and Manufacturing Method Thereof". The original application number is 2025110725369, the application date is August 1, 2025, and the invention title is "Biosensor and Manufacturing Method Thereof". Technical Field

[0002] This invention belongs to the field of electrochemical detection technology, and specifically relates to a biosensor. Background Technology

[0003] In existing biosensors used for electrochemical detection of analyte concentrations (such as glucose, ketone bodies, hemoglobin, etc.) in samples, multiple electrodes are typically placed on one surface (upper or lower surface) of an insulating substrate, such as two electrodes (working electrode and reference electrode) and three electrodes (working electrode, counter electrode, and reference electrode), or at least one electrode is placed on each of two opposite surfaces (upper and lower surfaces) of the insulating substrate. For example, the working electrode is placed on the upper surface and the counter electrode is placed on the lower surface, or the working electrode and the counter electrode are placed on the upper surface and the reference electrode is placed on the lower surface.

[0004] In continuous analyte monitoring systems, at least a portion of the biosensor is inserted into a blood vessel, subcutaneous tissue, or other site in the test subject for analyte detection. To enable the stacking of multiple electrodes on a single surface of an insulating substrate and to prevent electrical conduction between adjacent electrodes, an insulating layer is required to separate adjacent electrodes, thereby achieving a stacked arrangement of conductive layers (such as the conductive layer containing the working electrode, the conductive layer containing the reference electrode, and the conductive layer containing the counter electrode) and insulating layers. This stacking arrangement is achieved through screen printing during the biosensor manufacturing process.

[0005] In continuous analyte monitoring systems, whether using two electrodes (working and counter electrodes), three electrodes (working, counter, and reference electrodes), or more, the dimensions of each electrode are extremely precise, for example, approximately 0.4 mm × 5 mm. This places even higher demands on the printing process to achieve the layer-by-layer stacking of conductive and insulating layers. Given that at least a portion of the biosensor is placed inside the patient before detection, high accuracy in analyte detection results is required.

[0006] No matter how precise the printing press, the printing process utilizes different screens to print conductive and insulating layers. Each screen prints one conductive layer and another one insulating layer, with different screens used for different conductive and insulating layers. The screen for printing the insulating layer contains a patterned area of ​​a specific shape. During screen printing, the insulating material passes through the mesh of this patterned area to create this specific shaped insulating layer. However, when the conductive and insulating layers are stacked, adjacent screens must be overprinted, which can lead to overprinting discrepancies. This results in deviations between adjacent conductive and insulating layers, causing differences in electrode dimensions on different card sizes and reducing consistency. For example, on some cards, if the insulating layer covering the conductive layer containing the working electrode shifts upwards, the working electrode size increases (3.2mm in length); on other cards, if the insulating layer shifts downwards, the working electrode size decreases (2.8mm in length). When the enzyme solution used to detect the analyte is fixed on the working electrode by spotting or dipping, changes in the size of the working electrode can cause deviations in the amount of enzyme solution fixed on the working electrode, resulting in poor consistency and accuracy of the test results and the occurrence of test errors. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, the present invention aims to design and provide a technical solution for a biosensor. By optimizing the screen design to compensate for the misregistration problem in the printing process and fixing the size of the electrodes, the influence of the electrodes on the test results in the analyte testing of the continuous analyte monitoring system can be greatly improved, thereby greatly improving the linearity, accuracy and bias of the test results.

[0008] The problem solved by this invention can be achieved through the following specific technical solutions: The biosensor includes a substrate, a conductive layer, and an insulating layer. The conductive layer is formed on the substrate. The insulating layer includes an upper portion and a lower portion spaced apart and formed at the upper and lower ends of the conductive layer, respectively. The gap between the upper and lower portions exposes a region of the conductive layer to form a first electrode.

[0009] Furthermore, a second conductive layer is formed on the other side surface of the substrate, and the second insulating layer includes a second upper portion and a second lower portion spaced apart and formed at the upper and lower ends of the second conductive layer, respectively. The gap between the second upper portion and the second lower portion exposes a region of the second conductive layer to form a second electrode.

[0010] Furthermore, a second conductive layer is formed on the upper part of the insulating layer. The second insulating layer includes a second upper part and a second lower part that are spaced apart and formed at the upper and lower ends of the second conductive layer, respectively. The gap between the second upper part and the second lower part exposes a region of the second conductive layer to form a second electrode.

[0011] Furthermore, it also includes a third conductive layer and a third insulating layer. The third conductive layer is formed on the second upper portion or the substrate. The third insulating layer includes a third upper portion and a third lower portion that are spaced apart and formed at the upper and lower ends of the third conductive layer, respectively. The gap between the third upper portion and the third lower portion exposes a region of the third conductive layer to form a third electrode.

[0012] Furthermore, at least one electrode is provided with the reagents required for the reaction.

[0013] Furthermore, the area of ​​the conductive layer not covered by the insulating layer forms a contact point.

[0014] Furthermore, the first electrode is the working electrode, and the second and third electrodes are the counter electrode or the reference electrode, respectively.

[0015] Furthermore, the size range of the working electrode is 1.0 mm to 3.5 mm, the size range of the reference electrode is 0.5 mm to 1.0 mm, and the size range of the counter electrode is 1.0 mm to 4.0 mm.

[0016] Furthermore, the biosensor is used for glucose detection.

[0017] Furthermore, the reagent includes glucose oxidase.

[0018] A biosensor includes a substrate, a first conductive layer, a first insulating layer, a second conductive layer, and a second insulating layer. The first conductive layer is formed on one side surface of the substrate. The first insulating layer includes a first upper portion and a first lower portion spaced apart and formed at the upper and lower ends of the first conductive layer, respectively. The space between the first upper portion and the first lower portion exposes a region of the first conductive layer to form a first electrode. The second conductive layer is formed on the other side surface of the substrate or on the first upper portion. The second insulating layer includes a second upper portion and a second lower portion spaced apart and formed at the upper and lower ends of the second conductive layer, respectively. The space between the second upper portion and the second lower portion exposes a region of the second conductive layer to form a second electrode.

[0019] Furthermore, the area of ​​the first insulating layer not covering the distal end of the first conductive layer forms a first contact, and the area of ​​the second insulating layer not covering the distal end of the second conductive layer forms a second contact.

[0020] Furthermore, it also includes a third conductive layer and a third insulating layer. The third conductive layer is formed on the substrate or the second upper portion. The third insulating layer includes a third upper portion and a third lower portion that are spaced apart and formed at the upper and lower ends of the third conductive layer, respectively. The gap between the third upper portion and the third lower portion exposes a region of the third conductive layer to form a third electrode.

[0021] Furthermore, the area at the distal end of the third conductive layer not covered by the third insulating layer forms the third contact.

[0022] Furthermore, the first electrode is the working electrode, and the second and third electrodes are the counter electrode or the reference electrode, respectively.

[0023] Furthermore, the size range of the working electrode is 1.0 mm to 3.5 mm, the size range of the reference electrode is 0.5 mm to 1.0 mm, and the size range of the counter electrode is 1.0 mm to 4.0 mm.

[0024] Specifically, the proximal end is the end where the biosensor enters the body of the test subject, and the distal end is the end where the biosensor does not enter the body of the test subject.

[0025] A method for manufacturing a biosensor includes screen printing, enzyme fixation, and cutting, wherein the screen printing specifically includes the following steps: 1) Take a substrate and screen print conductive carbon ink onto one side surface of the substrate to form the first conductive layer; 2) Using a screen with two patterned portions and the two patterned portions maintaining a first fixed distance d1, insulating ink is screen-printed onto the first conductive layer to form a first upper part and a first lower part of the first insulating layer. The first upper part of the first insulating layer covers the upper end of the first conductive layer, and the first lower part covers the lower end of the first conductive layer. The gap between the first upper part and the first lower part exposes a region of the first conductive layer to form a first electrode. 3) Conductive carbon ink is screen-printed onto the other side surface of the substrate or onto the first upper part of the first insulating layer to form a first conductive layer; 4) Using a screen with two patterned portions and the two patterned portions maintaining a second fixed distance d2, insulating ink is screen-printed onto the second conductive layer to form a second upper part and a second lower part of the second insulating layer. The second upper part of the second insulating layer covers the upper end of the second conductive layer, and the second lower part covers the lower end of the second conductive layer. The gap between the second upper part and the second lower part exposes a region of the second conductive layer to form a second electrode. 5) Following the steps above, conductive layers and insulating layers are stacked sequentially on both sides of the substrate to form multiple electrodes.

[0026] Furthermore, the first electrode is a working electrode, and the first fixed distance is 1.0 mm to 3.5 mm; the second electrode is a counter electrode or a reference electrode. If the second electrode is a reference electrode, the second fixed distance is 0.5 mm to 1.0 mm; if the second electrode is a counter electrode, the second fixed distance is 1.0 mm to 4.0 mm.

[0027] Furthermore, the first insulating layer does not completely cover the far end of the first conductive layer, thus leaving an exposed area at the far end of the first conductive layer to form a first contact; the second insulating layer does not completely cover the far end of the second conductive layer, thus leaving an exposed area at the far end of the second conductive layer to form a second contact; and so on, the Nth insulating layer does not completely cover the far end of the Nth conductive layer, thus leaving an exposed area at the far end of the Nth conductive layer to form an Nth contact, where N is an integer greater than or equal to 1.

[0028] The method for fixing electrode size in this invention is applicable to different types of biosensors, such as implantable biosensors that continuously monitor analytes and traditional non-implantable biosensors.

[0029] Compared with the prior art, the present invention has the following advantages: (1) The biosensor of this application can not only use the insulating layer to solve the conduction when adjacent electrodes are connected, and realize the stacking arrangement of the conductive layer and the insulating layer, but also control the size of the working electrode, counter electrode and reference electrode as needed; it can be applied to control the size of other electrodes such as counter electrode and reference electrode, so that the electrodes remain unchanged during the manufacturing process.

[0030] (2) After extensive research and verification, this invention has found that by optimizing the screen design to compensate for the misregistration problem in the printing process and fixing the size of the electrode, the influence of the electrode on the test results can be greatly improved in the analysis of the continuous analyte monitoring system, so that the linearity, accuracy and deviation of the test results can be greatly improved. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the front and back assembly of the biosensor in Embodiment 1 of the present invention. The portions extending from both sides of the insulating layer in the diagram have not yet been cut. Figure 2 This is a schematic diagram of the front and back assembly of the biosensor in Embodiment 1 of the present invention. The portions extending from both sides of the insulating layer in the diagram have been cut off. Figure 3 This is an exploded view of the biosensor in Embodiment 1 of the present invention. The portions extending from both sides of the insulating layer in the figure have been cut off. Figure 4 This is a cross-sectional view of the proximal end of the biosensor in Embodiment 1 of the present invention; Figure 5This is a cross-sectional view of the proximal end of the biosensor in Embodiment 2 of the present invention; Figure 6 This is a schematic diagram of the front and back assembly of the biosensor in Embodiment 2 of the present invention. The portions extending from both sides of the insulating layer in the diagram have not yet been cut off. Figure 7 This is a cross-sectional view of the proximal end of the biosensor in Embodiment 3 of the present invention; Figure 8 This is a cross-sectional view of the proximal end of the biosensor in Embodiment 4 of the present invention; Figure 9 This is a cross-sectional view of the proximal end of the biosensor in Embodiment 5 of the present invention; Figure 10 This is a standard curve plotted during the testing process of this invention, with glucose concentration as the abscissa and the measured output current as the ordinate. Figure 11 This is a correlation graph between the theoretical glucose concentration of this invention and the actual glucose concentration in PBS buffer; Figure 12 This is a standard curve plotted with glucose concentration as the abscissa and the measured output current as the ordinate during the comparative test of this invention. Figure 13 This is a graph showing the correlation between the theoretical value of glucose concentration in the comparative proportion of this invention and the actual value of glucose concentration in PBS buffer.

[0032] In the figure: 1-Biosensor, 2-First conductive layer, 21-First electrode, 22-First contact, 3-First insulating layer, 31-First upper part, 32-First lower part, 4-Second conductive layer, 41-Second electrode, 42-Second contact, 5-Second insulating layer, 51-Second upper part, 52-Second lower part, 6-Substrate, 61-Upper surface, 62-Lower surface, 7-Third conductive layer, 71-Third electrode, 72-Third contact, 8-Third insulating layer, 81-Third upper part, 82-Third lower part. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] Example 1

[0035] like Figures 1-4As shown, a biosensor 1 for electrochemically detecting the concentration of analytes (such as glucose, ketone bodies, hemoglobin, etc.) in a sample includes an insulating substrate 6, a first conductive layer 2 disposed on the substrate 6, a first insulating layer 3 disposed on the first conductive layer 2, a second conductive layer 4 disposed on the first insulating layer 3, and a second insulating layer 5 disposed on the second conductive layer 4. The substrate 6 is inverted L-shaped, but other geometric shapes are also possible.

[0036] The first conductive layer 2 is applied to the upper surface 61 of the substrate 6 by means of screen printing or other methods. The first conductive layer 2 covers the entire upper surface 61 of the substrate 6 (i.e., the first conductive layer 2 extends through the entire length of the substrate 6 from the lower end to the upper end of the substrate 6, and extends through the entire width of the substrate 6 from one side to the other opposite side). Of course, the width and length of the first conductive layer 2 can also be less than the entire length and width of the substrate 6.

[0037] An insulating material is printed onto a first conductive layer 2 using a screen printing plate with two patterned portions (a first patterned portion and a second patterned portion) maintained at a fixed distance d1. This forms a first insulating layer 3. The first insulating layer 3 consists of a first upper portion 31 and a first lower portion 32. The first upper portion 31 is printed on the upper end of the first conductive layer 2, and the insulating material can be printed onto the first upper portion 31 of the first insulating layer 3 through the mesh openings of the first patterned portion of the screen printing plate. The first lower portion 32 is printed on the lower end of the first conductive layer 2, and the insulating material can be printed onto the first lower portion 32 of the first insulating layer 3 through the mesh openings of the second patterned portion of the screen printing plate. The first upper portion 31 and the first lower portion 32 of the first insulating layer 3 are separated by a certain distance d1, which is equal to the distance between the first patterned portion and the second patterned portion in the screen printing plate used to manufacture the first insulating layer 3. The gap between the first upper portion 31 and the first lower portion 32 of the first insulating layer 3 results in the formation of the first electrode 21 in the exposed area of ​​the first conductive layer 2. It can be seen that the size of the first electrode 21 depends on the distance between the upper portion 31 and the lower portion 32 of the first insulating layer 3, which essentially depends on the first fixed distance between the first pattern portion and the second pattern portion in the screen used to manufacture the first insulating layer 3.

[0038] Therefore, when manufacturing the biosensor 1, even if there is a misregistration between the screen used to manufacture the first conductive layer 2 and the screen used to manufacture the first insulating layer 3, the size of the first electrode 21 remains unchanged because the first fixed distance between the first pattern portion and the second pattern portion in the screen used to manufacture the first insulating layer 3 remains unchanged.

[0039] When printing the first insulating layer 3, in order to fully cover the first conductive layer 2 and prevent adjacent conductive layers from conducting, the first insulating layer 3 can cover both sides of the first conductive layer 2 and then extend outwards by a certain distance. The portion of the first insulating layer 3 extending outwards relative to the sides of the first conductive layer 2 can be cut off by laser cutting or other methods. During testing, the proximal end of the first conductive layer 2 needs to be inserted into the body of the test object. The upper part of the first insulating layer 3 does not completely cover the distal end of the first conductive layer 2, thus leaving an exposed area at the distal end of the first conductive layer 2. This exposed area functions as the first contact point 22.

[0040] The second conductive layer 4 is formed by covering the first insulating layer 3 with conductive material through methods such as screen printing. Using a screen with two patterned portions (a first patterned portion and a second patterned portion) maintained at a second fixed distance d2, insulating material is printed onto the second conductive layer 4 through screen printing, thus forming the second insulating layer 5. The second insulating layer 5 comprises a second upper portion 51 and a second lower portion 52. The second upper portion 51 of the second insulating layer 5 is printed at the upper end of the second conductive layer 4, and the insulating material can be printed through the mesh openings of the first patterned portion of the screen used to manufacture the second insulating layer 5. The second lower portion 52 of the second insulating layer 5 is printed at the lower end of the second conductive layer 4, and the insulating material can be printed through the mesh openings of the second patterned portion of the screen used to manufacture the second insulating layer 5. The upper portion 51 and the lower portion 52 of the second insulating layer 5 are separated by a certain distance d2, which is equal to the second fixed distance between the first patterned portion and the second patterned portion in the screen used to manufacture the second insulating layer 5. The gap between the second upper portion 51 and the second lower portion 52 of the second insulating layer 5 results in the formation of the second electrode 41 in the exposed area of ​​the second conductive layer 4. Therefore, the size of the second electrode 41 depends on the distance between the second upper portion 51 and the second lower portion 52 of the second insulating layer 5, which essentially depends on the second fixed distance between the first pattern portion and the second pattern portion in the screen printing plate used to manufacture the second insulating layer 5.

[0041] Therefore, even if there is a misregistration between the screen used to manufacture the second conductive layer 4 and the screen used to manufacture the second insulating layer 5 when manufacturing the biosensor 1, the size of the second electrode 41 remains unchanged because the distance between the first pattern portion and the second pattern portion in the screen used to manufacture the second insulating layer 5 remains unchanged.

[0042] When printing the second insulating layer 5, in order to fully cover the second conductive layer 4 and prevent adjacent conductive layers from conducting, the second insulating layer 5 can cover both sides of the second conductive layer 4 and then extend outwards by a certain distance. The portion of the second insulating layer 5 extending relative to the sides of the second conductive layer 4 can be cut off by means of laser cutting or the like. During testing, the proximal end of the second conductive layer 4 is close to the test object. The second upper part 51 of the second insulating layer 5 does not completely cover the distal end of the second conductive layer 4, thus leaving an exposed area at the distal end of the second conductive layer 4. This exposed area functions as the second contact 42.

[0043] A third conductive layer 7 and a third insulating layer 8 are sequentially stacked on the lower surface 62 of the substrate 6. The third conductive layer 7 is formed by covering the lower surface 62 of the substrate 6 with conductive material through methods such as screen printing. Using a screen with two patterned portions (i.e., a first patterned portion and a second patterned portion) and these two patterned portions maintaining a third fixed distance d3, insulating material is printed onto the third conductive layer 7 through methods such as screen printing to form the third insulating layer 8. The third insulating layer 8 consists of two parts: a third upper portion 81 and a third lower portion 82. The third upper portion 81 is printed at the upper end of the third conductive layer 7, and the insulating material can be printed into the third upper portion 81 through the mesh of the first patterned portion of the screen used to manufacture the third insulating layer 8. The third lower portion 82 is printed at the lower end of the third conductive layer 7, and the insulating material can be printed into the third lower portion 82 of the third insulating layer 8 through the mesh of the second patterned portion of the screen used to manufacture the third insulating layer 8. The upper third portion 81 and the lower third portion 82 of the third insulating layer 8 are separated by a certain distance, which is equal to the third fixed distance between the first pattern portion and the second pattern portion in the screen printing plate used to manufacture the third insulating layer 8. The gap between the upper third portion 81 and the lower third portion 82 of the third insulating layer 8 results in the formation of the third electrode 71 in the exposed area of ​​the third conductive layer 7. Therefore, the size of the third electrode 71 depends on the distance between the upper third portion 81 and the lower third portion 82 of the third insulating layer 8, which is essentially determined by the third fixed distance d3 between the first pattern portion and the second pattern portion in the screen printing plate used to manufacture the third insulating layer 8.

[0044] Therefore, when manufacturing the biosensor 1, even if there is a misregistration between the screen used to manufacture the third conductive layer 7 and the screen used to manufacture the third insulating layer 8, the size of the third electrode 71 remains unchanged because the third fixed distance between the first pattern portion and the second pattern portion in the screen used to manufacture the third insulating layer 8 remains unchanged.

[0045] When printing the third insulating layer 8, in order to fully cover the third conductive layer 7 and prevent adjacent conductive layers from conducting, the third insulating layer 8 can cover both sides of the third conductive layer 7 and then extend outwards by a certain distance. The portion of the third insulating layer 8 extending relative to the sides of the third conductive layer 7 can be cut off by laser cutting or other methods. During testing, the proximal end of the third conductive layer 7 is close to the test object. The third upper part 81 of the third insulating layer 8 does not completely cover the distal end of the third conductive layer 7, thus leaving an exposed area at the distal end of the third conductive layer 7. This exposed area functions as the third contact 72.

[0046] The surface of the first electrode 21 (described here as the working electrode) needs to be coated with an enzyme solution, which includes an analyte reactant, a buffer solution, a cross-linking agent, and an electron mediator. When the analyte is glucose, the analyte reactant can be selected from glucose oxidase, PQQ-dependent glucose dehydrogenase (PQQ-GDH), FAD-dependent glucose dehydrogenase (FAD-GDH), and NAD(P)+-dependent glucose dehydrogenase (NAD(P)-GDH). The buffer solution can be selected from PBS buffer and HEPES buffer, etc., with a pH of 6.8-8.5. The cross-linking agent is a molecule containing at least two reactive groups, which couples the analyte reactant and the electron mediator to the surface of the working electrode, allowing electrons to be directly delivered to the working electrode when the analyte reacts with the analyte in the sample; the cross-linking agent can be selected from polyethylene glycol diglycidyl ether (PEGDGE), etc., with a molecular weight between approximately 200 and 600.

[0047] Example 2

[0048] like Figure 5 and Figure 6As shown, the difference between this embodiment and Embodiment 1 is that only the first conductive layer 2 and the first insulating layer 3 are stacked on the upper surface 61 of the substrate 6, while the second conductive layer 4 and the second insulating layer 5 are not stacked on the upper surface 61 of the substrate 6, but are stacked together with the third conductive layer 7 and the third insulating layer 8 on the lower surface 62 of the substrate 6. The third conductive layer 7 is formed by covering the lower surface 62 of the substrate 6 with conductive material through methods such as screen printing. Using a screen with two pattern portions (i.e., the first pattern portion and the second pattern portion) separated by a third fixed distance d3, insulating material is printed on the lower surface of the third conductive layer 7 through methods such as screen printing, thereby forming the third insulating layer 8. The third insulating layer 8 consists of two parts: a third upper part 81 and a third lower part 82. The third upper part 81 of the third insulating layer 8 is printed on the upper end of the third conductive layer 7, and is printed by passing insulating material through the mesh of the first pattern portion of the screen used to manufacture the third insulating layer 8. The third lower part 82 of the third insulating layer 8 is printed on the lower end of the third conductive layer 7, and is printed by passing insulating material through the mesh of the second pattern portion of the screen used to manufacture the third insulating layer 8. The third upper part 81 and the third lower part 82 of the third insulating layer 8 are separated by a certain distance d3, which is equal to the third fixed distance between the first pattern portion and the second pattern portion of the screen used to manufacture the third insulating layer 8. The second conductive layer 4 is formed by covering the lower surface of the third upper part 81 of the third insulating layer 8 with conductive material through methods such as screen printing. Using a screen printing stencil with two patterned portions (i.e., a first patterned portion and a second patterned portion) spaced two apart by a third fixed distance d3, insulating material is printed onto the lower surface of the second conductive layer 4 by means of screen printing or other methods, thereby forming the second insulating layer 5. The second insulating layer 5 consists of a second upper portion 51 and a second lower portion 52. The second upper portion 51 of the second insulating layer 5 is printed at the upper end of the second conductive layer 4, by allowing the insulating material to pass through the mesh openings of the first patterned portion in the screen printing stencil used to manufacture the second insulating layer 5; the second lower portion 52 of the second insulating layer 5 is printed at the lower end of the first conductive layer 2, by allowing the insulating material to pass through the mesh openings of the second patterned portion in the screen printing stencil used to manufacture the second insulating layer 5. The second upper portion 51 and the second lower portion 52 of the second insulating layer 5 are spaced a certain distance apart, which is equal to the second fixed distance between the first patterned portion and the second patterned portion in the screen printing stencil used to manufacture the second insulating layer 5. The gap between the second upper portion 51 and the second lower portion 52 of the second insulating layer 5 results in the exposed area of ​​the second conductive layer 4 forming the second electrode 41.

[0049] Similarly, the size of the first electrode 21 depends on the distance d1 between the first upper part 31 and the first lower part 32 of the first insulating layer 3, which essentially depends on the first fixed distance between the first pattern portion and the second pattern portion in the screen printing plate used to manufacture the first insulating layer 3; the size of the second electrode 41 depends on the distance between the second upper part 51 and the second lower part 52 of the second insulating layer 5, which essentially depends on the second fixed distance d2 between the first pattern portion and the second pattern portion in the screen printing plate used to manufacture the second insulating layer 5; the size of the third electrode 71 depends on the distance between the third upper part 81 and the third lower part 82 of the third insulating layer 8, which essentially depends on the third fixed distance d3 between the first pattern portion and the second pattern portion in the screen printing plate used to manufacture the third insulating layer 8.

[0050] Example 3

[0051] like Figure 7 As shown, the difference between this embodiment and Embodiment 1 is that the third conductive layer 7 and the third insulating layer 8 are also stacked sequentially on the upper surface 61 of the substrate 6, and are also stacked sequentially on the second insulating layer 5. This means that the three electrodes and the three insulating layers are located on the same surface of the substrate 6. Similarly, the size of the first electrode 21 depends on the distance between the upper portion 31 and the lower portion 32 of the first insulating layer 3, which essentially depends on the first fixed distance between the first pattern portion and the second pattern portion in a screen for manufacturing the first insulating layer 3; the size of the second electrode 41 depends on the distance between the upper portion 51 and the lower portion 52 of the second insulating layer 5, which essentially depends on the second fixed distance between the first pattern portion and the second pattern portion in a screen for manufacturing the second insulating layer 5; the size of the third electrode 71 depends on the distance between the upper portion 81 and the lower portion 82 of the third insulating layer 8, which essentially depends on the third fixed distance between the first pattern portion and the second pattern portion in a screen for manufacturing the third insulating layer 8.

[0052] Example 4

[0053] like Figure 8As shown, the difference between this embodiment and Embodiment 1 is that no conductive or insulating layers are stacked on the lower surface 62 of the substrate 6. This means that only the first conductive layer 2 and the first insulating layer 3, as well as the second conductive layer 4 and the second insulating layer 5, are stacked on the upper surface 61 of the substrate 6. Similarly, the size of the first electrode 21 depends on the distance between the upper portion 31 and the lower portion 32 of the first insulating layer 3, which essentially depends on the distance between the first pattern portion and the second pattern portion in a screen printing plate for manufacturing the first insulating layer 3; the size of the second electrode 41 depends on the distance between the upper portion 51 and the lower portion 52 of the second insulating layer 5, which essentially depends on the distance between the first pattern portion and the second pattern portion in a screen printing plate for manufacturing the second insulating layer 5.

[0054] Example 5

[0055] like Figure 9 As shown, the difference between this embodiment and embodiment 4 is that only the first conductive layer 2 and the first insulating layer 3 are stacked on the upper surface 61 of the substrate 6, while the second conductive layer 4 and the second insulating layer 5 are stacked on the lower surface 62 of the substrate 6. Similarly, the size of the first electrode 21 depends on the distance between the upper portion 31 and the lower portion 32 of the first insulating layer 3, which essentially depends on the distance between the first pattern portion and the second pattern portion in a screen for manufacturing the first insulating layer 3; the size of the second electrode 41 depends on the distance between the upper portion 51 and the lower portion 52 of the second insulating layer 5, which essentially depends on the distance between the first pattern portion and the second pattern portion in a screen for manufacturing the second insulating layer 5.

[0056] In various embodiments of the present invention and other readily conceivable variations, when the biosensor uses two electrodes for analyte detection, the first working electrode is typically the working electrode, and the second electrode is typically the counter electrode or reference electrode; when the biosensor uses three electrodes for analyte detection, the first working electrode is typically the working electrode, the second electrode is typically the counter electrode, and the third electrode is typically the reference electrode, or the first working electrode is typically the working electrode, the second electrode is typically the reference electrode, and the third electrode is typically the counter electrode; when the biosensor uses four or more electrodes for analyte detection, in addition to the first working electrode typically being the working electrode, the second electrode typically being the counter electrode or reference electrode, and the third electrode typically being the reference electrode or counter electrode, more electrodes can be provided by stacking more conductive and insulating layers on the upper or lower surface of the substrate, and the size of the provided electrodes remains unchanged during manufacturing. In the present invention, the size range of the working electrode can be 1.0–3.5 mm, the size range of the reference electrode can be 0.5–1.0 mm, and the size range of the counter electrode can be 1.0–4.0 mm.

[0057] The manufacturing process of the biosensor of the present invention will be described based on Example 1: The process for manufacturing the biosensor in the first embodiment of the present invention includes a screen printing process, an enzyme solution immobilization process, and a cutting process. In the fabricated biosensor, the first electrode 21 is the working electrode, the second electrode 41 is the reference electrode, and the third electrode 71 is the counter electrode.

[0058] In the screen printing process, in the large card, a screen is used to screen print conductive carbon ink onto the upper surface 61 of the insulating substrate 6, forming the first conductive layer 2 where the working electrode is located. Then, a screen with two patterned portions (i.e., the first patterned portion and the second patterned portion) and these two patterned portions are kept at a first fixed distance d1, is used to screen print insulating ink onto the first conductive layer 2, forming the upper and lower portions (i.e., the first upper portion 31 and the first lower portion 32) of the first insulating layer 3. The first upper portion 31 covers the upper end of the first conductive layer 2, and the first lower portion 32 covers the lower end of the first conductive layer 2. The upper part 31 and the lower part 32 are separated by a certain distance, which is equal to the distance between the first pattern portion and the second pattern portion in the screen printing for manufacturing the first insulating layer 3. This fixes the size of the first electrode 21, which can be selected from 1.5mm to 3.0mm. Meanwhile, when printing the first insulating layer 3, it does not completely cover the far end of the first conductive layer 2, leaving an exposed area at the far end of the first conductive layer 2. This exposed area can function as the first contact 22. Then, a screen is used to print conductive carbon ink or A... g / AgCl ink is screen-printed onto the first insulating layer 3 to form the second conductive layer 4. Then, using a screen with two patterned portions (i.e., the first patterned portion and the second patterned portion) maintained at a second fixed distance d2, insulating ink is screen-printed onto the second conductive layer 4 to form the upper and lower portions (i.e., the second upper portion 51 and the second lower portion 52) of the second insulating layer 5. The second upper portion 51 covers the upper end of the second conductive layer 4, and the second lower portion 52 covers the lower end of the second conductive layer 4. The gap between the second upper portion 51 and the second lower portion 52 of the second insulating layer 5 exposes the second conductive layer. Part 4, which is the second electrode 41, has a certain distance between the second upper part 51 and the second lower part 52. This distance is equal to the distance between the first pattern portion and the second pattern portion in the screen used to manufacture the second insulating layer 5. This fixes the size of the second electrode 41 (as a reference electrode) (selectable from 0.5 to 1.0 mm). At the same time, when printing the second insulating layer 5, the second insulating layer 5 does not completely cover the far end of the second conductive layer 4, thus leaving an exposed area at the far end of the second conductive layer 4. This exposed area can function as the second contact 42.

[0059] Furthermore, conductive carbon ink is screen-printed onto the lower surface 62 of the insulating substrate 6 using a screen printing plate to form a third conductive layer 7. Insulating ink is screen-printed onto the lower surface of the third conductive layer 7 using a screen printing plate with two patterned portions (i.e., a first patterned portion and a second patterned portion) maintained at a fixed distance d3, forming the upper and lower portions (i.e., the upper portion 81 and the lower portion 82) of the third insulating layer 8. The upper portion 81 of the third insulating layer 8 covers the upper end of the third conductive layer 7, and the lower portion 82 covers the lower end of the third conductive layer 7. At the lower end of layer 7, there is a certain distance between the third upper part 81 and the third lower part 82. This distance is equal to the distance between the first pattern portion and the second pattern portion in the screen used to manufacture the third insulating layer 8. This allows the size of the third electrode 71 (as the counter electrode) to be fixed (selectable from 1.0 to 4.0 mm). At the same time, when printing the third insulating layer 8, the third insulating layer 8 does not completely cover the far end of the third conductive layer 7, thus leaving an exposed area at the far end of the third conductive layer 7. This exposed area can function as the third contact 72.

[0060] Furthermore, when printing the first insulating layer 3, the first insulating layer 3 covers both sides of the first conductive layer 2 and then extends outward by a certain distance; when printing the second insulating layer 5, the second insulating layer 5 covers both sides of the second conductive layer 4 and then extends outward by a certain distance; when printing the third insulating layer 8, the third insulating layer 8 covers both sides of the third conductive layer 7 and then extends outward by a certain distance.

[0061] In the enzyme fixation process, the uniformly mixed enzyme solution is fixed onto the surface of the working electrode by spotting or dipping, and then cured at room temperature for 24 hours. After enzyme fixation, the portions extending from the first insulating layer 3 relative to the sides of the first conductive layer 2 are removed by laser cutting; the portions extending from the second insulating layer 5 relative to the sides of the second conductive layer 4 are removed by laser cutting; and the portions extending from the third insulating layer 8 relative to the sides of the third conductive layer 7 are removed by laser cutting. The enzyme solution used is prepared by adding 10 mg of glucose oxidase (GOD) and 9 mg of osmium complex [Os(Py-MIM)2(MIM)Cl] to 1 mL of purified water. 2+ 2Cl - 10 mg of crosslinking agent polyethylene glycol diglycidyl ether (molecular weight 500) was mixed evenly. The osmium complex used was prepared according to the synthesis method in Example 1 of Chinese Patent CN1620462B.

[0062] In the cutting process, the large cards, after undergoing screen printing and enzyme fixation, are cut into batches of individual single-use biosensors using a laser cutting machine. Finally, the uniformly mixed membrane solution is dip-coated onto the working electrode, counter electrode, and reference electrode surfaces of the individual biosensors and cured at room temperature for 24 hours in preparation for subsequent testing. The membrane solution was prepared by adding 80 mg of poly(4-vinylpyridine) (molecular weight 160,000) and 20 mg of polyethylene glycol diglycidyl ether (molecular weight 500) to 5 mL of 80% ethanol solution and mixing thoroughly.

[0063] Test method: A batch of biosensors manufactured using the biosensor manufacturing process of this invention were placed in a series of PBS buffer solutions (pH 7.0) containing different glucose concentrations. The test temperature was 37°C, and the operating voltage was set to 0.05V. Measurements were repeated four times for each glucose concentration of PBS buffer solution. The output current of each biosensor was measured using an electrochemical workstation (model Chi-1000C, Shanghai Chenhua Instrument Co., Ltd.). The coefficient of variation (CV) of the measured current values ​​for each glucose concentration of PBS buffer solution was then calculated. The test results are shown in Table 1. Simultaneously, a standard curve was plotted with glucose concentration on the x-axis and the measured output current on the y-axis, as shown in Table 1. Figure 10 As shown.

[0064] Table 1

[0065] As shown in Table 1, once the size of the working electrode is fixed, the current values ​​measured for each glucose concentration in PBS buffer are basically consistent, with good accuracy and low CV (CV is less than 10% at different glucose concentrations, with the highest value being only 6.6%), which is within an acceptable range.

[0066] Table 2

[0067] Given that the PBS buffer was measured four times for each glucose concentration, therefore according to Figure 10 Based on the established standard curve, the theoretical glucose concentration corresponding to each current value measured in PBS buffer for each glucose concentration in Table 1 was calculated (as shown in Table 2). Then, a correlation graph was plotted between the calculated theoretical glucose concentration on the x-axis and the actual glucose concentration in the PBS buffer on the y-axis, as shown in Table 2. Figure 11 As shown. By Figure 11 It can be seen that R 2=0.9934, R>0.99, which indicates that the correlation between the theoretical and actual values ​​of glucose concentration is good and the deviation between the theoretical and actual values ​​is small.

[0068] Comparative Example 1 The process for manufacturing the biosensor used as a control includes screen printing, enzyme fixation, and cutting.

[0069] In the screen printing process, on a large card, a screen is used to screen print conductive carbon ink onto an insulating substrate, forming the first conductive layer 2 where the working electrode is located. Then, a screen with a specific patterned portion is used to screen print insulating ink onto the first conductive layer 2, forming the first insulating layer 3. The first insulating layer 3 is a complete unit without gaps. The first insulating layer 3 does not completely cover the proximal end of the first conductive layer 2, leaving an exposed area at the proximal end of the first conductive layer 2. This exposed area serves as the first electrode, the working electrode. Simultaneously, the first insulating layer 3 does not completely cover the distal end of the first conductive layer 2, leaving an exposed area at the distal end of the first conductive layer 2. The first contact point functions in the first area. Next, conductive carbon ink or Ag / AgCl ink is screen-printed onto the first insulating layer 3 using a screen printing plate to form the second conductive layer 4. Then, insulating ink is screen-printed onto the second conductive layer 4 using another screen printing plate to form the second insulating layer 5. The second insulating layer 5 is a complete unit without gaps. The second insulating layer 5 does not completely cover the proximal end of the second conductive layer 4, leaving an exposed area at the proximal end. This exposed area serves as the second electrode, a reference electrode. Similarly, the second insulating layer 5 does not completely cover the distal end of the second conductive layer 4, leaving an exposed area at the distal end, which functions as the second contact point. Furthermore, when printing the first insulating layer 3, it covers both sides of the first conductive layer 2 and extends outwards by a certain distance; similarly, when printing the second insulating layer 5, it covers both sides of the second conductive layer 4 and extends outwards by a certain distance.

[0070] Furthermore, conductive carbon ink is screen-printed onto the lower surface 62 of the insulating substrate 6 using a screen printing plate to form a third conductive layer 7. Then, insulating ink is screen-printed onto the lower surface of the third conductive layer 7 using a screen printing plate with a patterned portion of a specific shape to form a third insulating layer 8. The third insulating layer 8 is a complete unit without any gaps in the middle. The third insulating layer 8 does not completely cover the far end of the third conductive layer 7, thus leaving an exposed area at the far end of the third conductive layer 7. This exposed area can function as the third contact 72. The third insulating layer 8 does not completely cover the near end of the third conductive layer 7, thus leaving an exposed area at the near end of the third conductive layer 7. This exposed area serves as the third electrode, which is the counter electrode.

[0071] In the enzyme fixation process, the uniformly mixed enzyme solution is fixed onto the surface of the working electrode by spotting or dipping, and then cured at room temperature for 24 hours. After enzyme fixation, the portions extending from the first insulating layer 3 relative to the sides of the first conductive layer 2 are removed by laser cutting; the portions extending from the second insulating layer 5 relative to the sides of the second conductive layer 4 are removed by laser cutting; and the portions extending from the third insulating layer 8 relative to the sides of the third conductive layer 7 are removed by laser cutting. The enzyme solution used is prepared by adding 10 mg of glucose oxidase (GOD) and 9 mg of osmium complex [Os(Py-MIM)2(MIM)Cl] to 1 mL of purified water. 2+ 2Cl - 10 mg of crosslinking agent polyethylene glycol diglycidyl ether (molecular weight 500) was mixed thoroughly.

[0072] In the cutting process, the large cards, after undergoing screen printing and enzyme fixation, are cut into batches of individual single-use biosensors using a laser cutting machine. Finally, the uniformly mixed membrane solution is dip-coated onto the working electrode, counter electrode, and reference electrode surfaces of the individual biosensors and cured at room temperature for 24 hours in preparation for subsequent testing. The membrane solution was prepared by adding 80 mg of poly(4-vinylpyridine) (molecular weight 160,000) and 20 mg of polyethylene glycol diglycidyl ether (molecular weight 500) to 5 mL of 80% ethanol solution and mixing thoroughly.

[0073] Test method: A batch of biosensors used as controls were placed in a series of PBS buffers (pH 7.0) containing different glucose concentrations. The test temperature was 37℃, and the operating voltage was set to 0.05V. Measurements were repeated four times for each glucose concentration of PBS buffer. The output current of each sensor was measured using an electrochemical workstation (model Chi-1000C, Shanghai Chenhua Instrument Co., Ltd.). The coefficient of variation (CV) of the measured current values ​​for each glucose concentration of PBS buffer was then calculated. The test results are shown in Table 3. Simultaneously, a standard curve was plotted with glucose concentration on the x-axis and the measured output current on the y-axis, as shown in Table 3. Figure 12 As shown.

[0074] Table 3

[0075] As shown in Table 3, among the control biosensors, since the working electrode size is not fixed, there are certain differences in the working electrode size of different biosensors. Therefore, for each glucose concentration of PBS buffer, the test results of different biosensors vary greatly, and the accuracy is significantly worse than that of the biosensor with fixed working electrode size manufactured in this invention. The CV is larger (the CV is about 10% at different glucose concentrations, with the highest value reaching 13.5%).

[0076] Table 4

[0077] Given that the PBS buffer was measured four times for each glucose concentration, therefore according to Figure 13 Based on the established standard curve, the theoretical glucose concentration corresponding to each current value measured in PBS buffer for each glucose concentration in Table 3 was calculated (as shown in Table 4). Then, a correlation graph was plotted between the calculated theoretical glucose concentration on the x-axis and the actual glucose concentration in the PBS buffer on the y-axis, as shown in Table 4. Figure 13 As shown. By Figure 13 It can be seen that R 2 =0.9479, R=0.9736, which is less than 0.99. This indicates that the correlation between the theoretical and actual values ​​of glucose concentration is poor, and the deviation between the theoretical and actual values ​​is large.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biosensor, comprising a substrate, a conductive layer, and an insulating layer, characterized in that, A conductive layer is formed on a substrate; an insulating layer includes an upper portion and a lower portion spaced apart and formed at the upper and lower ends of the conductive layer, respectively, and the gap between the upper and lower portions exposes a region of the conductive layer to form a first electrode.

2. The biosensor according to claim 1, characterized in that, The second conductive layer is formed on the other side surface of the substrate. The second insulating layer includes a second upper part and a second lower part that are spaced apart and formed at the upper and lower ends of the second conductive layer, respectively. The gap between the second upper part and the second lower part exposes a region of the second conductive layer to form a second electrode.

3. The biosensor according to claim 1, characterized in that, The second conductive layer is formed on the upper part of the insulating layer. The second insulating layer includes a second upper part and a second lower part that are spaced apart and formed at the upper and lower ends of the second conductive layer, respectively. The gap between the second upper part and the second lower part exposes a region of the second conductive layer to form a second electrode.

4. The biosensor according to claim 2 or 3, characterized in that, It also includes a third conductive layer and a third insulating layer. The third conductive layer is formed on the second upper portion or the substrate. The third insulating layer includes a third upper portion and a third lower portion that are spaced apart and formed at the upper and lower ends of the third conductive layer, respectively. The gap between the third upper portion and the third lower portion exposes a region of the third conductive layer to form a third electrode.

5. The biosensor according to any one of claims 1 to 4, characterized in that, At least one electrode has the reagents required for the reaction.

6. The biosensor according to any one of claims 1 to 4, characterized in that, The area at the far end of the conductive layer not covered by the insulating layer forms a contact point.

7. The biosensor according to claim 4, characterized in that, The first electrode is the working electrode, and the second and third electrodes are the counter electrode or the reference electrode, respectively.

8. The biosensor according to claim 7, characterized in that, The working electrode has a size range of 1.0 mm to 3.5 mm, the reference electrode has a size range of 0.5 mm to 1.0 mm, and the counter electrode has a size range of 1.0 mm to 4.0 mm.

9. The biosensor according to any one of claims 1 to 8, characterized in that, The biosensor is used for glucose detection.

10. The biosensor according to claim 9, characterized in that, The reagent includes glucose oxidase.

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