Biosensor and method of manufacturing the same

Abstract

The application discloses a biosensor and a manufacturing method thereof, and belongs to the technical field of electrochemical detection. The biosensor comprises 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 comprises a first upper part and a first lower part which are arranged at intervals and are formed on upper and lower ends of the first conductive layer, and the interval between the first upper part and the first lower part 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 the first upper part, and the second insulating layer comprises a second upper part and a second lower part which are arranged at intervals and are formed on upper and lower ends of the second conductive layer, and the interval between the second upper part and the second lower part exposes a region of the second conductive layer to form a second electrode. The biosensor can control the size of electrodes such as a working electrode, a counter electrode and a reference electrode according to requirements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemical detection, and particularly relates to a biosensor and a manufacturing method thereof. BACKGROUND

[0002] In the existing biosensor for electrochemically detecting the concentration of an analyte (such as glucose, ketone body, hemoglobin, etc.) in a sample, a plurality of electrodes, such as two electrodes (a working electrode and a reference electrode) and three electrodes (a working electrode, a counter electrode and a reference electrode), are usually arranged on one surface (an upper surface or a lower surface) of an insulating substrate, or at least one electrode is arranged on each of two opposite surfaces (an upper surface and a lower surface) of the insulating substrate, for example, a working electrode is arranged on the upper surface, a counter electrode is arranged on the lower surface, or a working electrode and a counter electrode are arranged on the upper surface, and a reference electrode is arranged on the lower surface.

[0003] In a continuous analyte monitoring system, at least a part of the biosensor is inserted into a blood vessel or subcutaneous tissue of a testee or other parts for analyte detection. In order to realize the stacked arrangement of a plurality of electrodes on one surface of the insulating substrate and prevent the electrical conduction between adjacent electrodes, it is necessary to isolate adjacent electrodes by an insulating layer, so as to realize the stacked arrangement of the conductive layer (such as a conductive layer where the working electrode is located, a conductive layer where the reference electrode is located, and a conductive layer where the counter electrode is located) and the insulating layer. This stacked arrangement is realized by screen printing during the manufacturing process of the biosensor.

[0004] In the continuous analyte monitoring system, whether two electrodes (a working electrode and a counter electrode) or three electrodes (a working electrode, a counter electrode and a reference electrode) or more electrodes are used, the size of each electrode is extremely fine, such as about 0.4 mm x 5 mm. This requires higher requirements for the layer-by-layer stacking of the conductive layer and the insulating layer by printing. Since at least a part of the biosensor is placed in the patient's body before detection, it is required that the accuracy of the analyte detection result must be high.

[0005] No matter how precise the printing machine is, in the printing process, different screens are used to print the conductive layer and the insulating layer, and one screen is used to print one conductive layer and another screen is used to print one insulating layer, and different screens are used for different conductive layers and different insulating layers. The screen for printing the insulating layer contains a pattern part with a specific shape, and when screen printing, the insulating material can be printed into an insulating layer with this specific shape through the mesh holes of the pattern part. However, when the conductive layer and the insulating layer are stacked, the adjacent screens need to be registered, which will cause a registration deviation, so that the adjacent conductive layer and the insulating layer will also have a certain deviation, thereby causing the electrode size on different cards to have differences, and the consistency is poor. For example, when the insulating layer for covering the conductive layer where the working electrode is located is upwardly offset on some cards, the size of the working electrode will become larger (3.2 mm long); when the insulating layer for covering the conductive layer where the working electrode is located is downwardly offset on some other cards, the size of the working electrode will become smaller (2.8 mm long). When the enzyme solution for detecting the analyte is fixed on the working electrode in the form of a dot liquid or dip coating, the size change of the working electrode will cause the number of enzyme solution fixed on the working electrode to deviate, thereby making the consistency of the test result poor, the accuracy is not good, and the test error occurs. SUMMARY

[0006] In order to make up for the shortcomings of the prior art, the purpose of the present application is to design a technical scheme of a biosensor and a manufacturing method thereof, to make up for the registration deviation problem in the printing process by optimizing the screen design, to fix the size of the electrode, and to greatly improve the influence of the electrode on the test result in the analyte test of the continuous analyte monitoring system, so that the linearity, accuracy and deviation of the test result are greatly improved.

[0007] The problems solved by the present application can be realized by the following specific technical schemes:

[0008] The biosensor comprises 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 comprises a first upper part and a first lower part arranged at intervals and formed on the upper and lower ends of the first conductive layer, respectively, and the interval between the first upper part and the first lower part exposes the 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 the first upper part, and the second insulating layer comprises a second upper part and a second lower part arranged at intervals and formed on the upper and lower ends of the second conductive layer, respectively, and the interval between the second upper part and the second lower part exposes the region of the second conductive layer to form a second electrode.

[0009] Further, the first insulating layer does not cover the region of the distal end of the first conductive layer to form a first contact, and the second insulating layer does not cover the region of the distal end of the second conductive layer to form a second contact.

[0010] Further, a third conductive layer and a third insulating layer are further included, 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 which are spaced apart and formed on the upper end and the lower end of the third conductive layer respectively, and the interval between the third upper portion and the third lower portion exposes the region of the third conductive layer to form a third electrode.

[0011] Further, the third insulating layer does not cover the region of the distal end of the third conductive layer to form a third contact.

[0012] Further, the first electrode is a working electrode, and the second electrode and the third electrode are a counter electrode or a reference electrode respectively.

[0013] Further, the size of the working electrode ranges from 1.0 mm to 3.5 mm, the size of the reference electrode ranges from 0.5 mm to 1.0 mm, and the size of the counter electrode ranges from 1.0 mm to 4.0 mm.

[0014] Specifically, the proximal end is the end of the biosensor entering the body of the test object, and the distal end is the end of the biosensor not entering the body of the test object.

[0015] A manufacturing method of a biosensor includes screen printing, enzyme liquid fixing, and cutting, and the screen printing specifically includes the following steps:

[0016] 1) Take a substrate, and screen print conductive carbon ink on one side surface of the substrate to form a first conductive layer;

[0017] 2) Screen print insulating ink on the first conductive layer by using a screen plate provided with two pattern portions which are kept at a first fixed distance d1 to form a first upper portion and a first lower portion of a first insulating layer, the first upper portion of the first insulating layer covers the upper end of the first conductive layer, and the first lower portion covers the lower end of the first conductive layer, and the interval between the first upper portion and the first lower portion exposes the region of the first conductive layer to form a first electrode;

[0018] 3) Screen print conductive carbon ink on the other side surface of the substrate or the first upper portion of the first insulating layer to form a first conductive layer;

[0019] 4) using a screen with two patterned sections and the two patterned sections maintaining a second fixed distance d2, screen printing insulating ink on 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 covering the upper end of the second conductive layer, and the second lower part of the second insulating layer covering the lower end of the second conductive layer, the interval between the second upper part and the second lower part allowing the area of the second conductive layer to be exposed to form a second electrode;

[0020] 5) sequentially stacking and arranging the conductive layers and the insulating layers on both side surfaces of the substrate to form a plurality of electrodes according to the above steps.

[0021] Further, the first electrode is a working electrode, 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.

[0022] Further, the first insulating layer does not completely cover the distal end of the first conductive layer, thereby leaving an exposed area at the distal end of the first conductive layer to form a first contact; the second insulating layer does not completely cover the distal end of the second conductive layer, thereby leaving an exposed area at the distal end of the second conductive layer to form a second contact; and so on, the Nth insulating layer does not completely cover the distal end of the Nth conductive layer, thereby leaving an exposed area at the distal end of the Nth conductive layer to form an Nth contact, N being an integer greater than or equal to 1.

[0023] The method for fixing the size of the electrode can be applied to different types of biosensors, such as implantable biosensors for continuous monitoring of analytes, traditional non-implantable biosensors, etc.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] (1) The biosensor of the present application not only solves the conduction between adjacent electrodes by using the insulating layer to achieve the stacking and arrangement of the conductive layer and the insulating layer, but also controls the size of the working electrode, the counter electrode and the reference electrode according to the needs; and can be applied to control the size of the counter electrode and the reference electrode and other electrodes, so that the electrodes remain unchanged during the manufacturing process.

[0026] (2) Through many researches and verifications, it is found that the size of the electrode can be fixed by optimizing the screen design to compensate for the overprinting deviation problem in the printing process, so that in the analyte test of the continuous analyte monitoring system, the influence of the electrode on the test result can be greatly improved, and the linearity, precision and deviation of the test result can be greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Assembled view of the front and back of the biosensor of Example 1 of the present invention, with the portions of the insulating layer extending beyond the sides not yet cut away;

[0028] Figure 2 Assembled view of the front and back of the biosensor of Example 1 of the present invention, with the portions of the insulating layer extending beyond the sides already cut away;

[0029] Figure 3 Exploded view of the biosensor of Example 1 of the present invention, with the portions of the insulating layer extending beyond the sides already cut away;

[0030] Figure 4 Cross-sectional view of the proximal end of the biosensor of Example 1 of the present invention;

[0031] Figure 5 Cross-sectional view of the proximal end of the biosensor of Example 2 of the present invention;

[0032] Figure 6 Assembled view of the front and back of the biosensor of Example 2 of the present invention, with the portions of the insulating layer extending beyond the sides not yet cut away;

[0033] Figure 7 Cross-sectional view of the proximal end of the biosensor of Example 3 of the present invention;

[0034] Figure 8 Cross-sectional view of the proximal end of the biosensor of Example 4 of the present invention;

[0035] Figure 9 Cross-sectional view of the proximal end of the biosensor of Example 5 of the present invention;

[0036] Figure 10 Standard curve plot of glucose concentration as the abscissa and measured output current as the ordinate during testing of the present invention;

[0037] Figure 11 Correlation plot of the theoretical value of glucose concentration and the true value of glucose concentration in PBS buffer of the present invention;

[0038] Figure 12 Standard curve plot of glucose concentration as the abscissa and measured output current as the ordinate during testing of the comparative example of the present invention;

[0039] Figure 13 Correlation plot of the theoretical value of glucose concentration and the true value of glucose concentration in PBS buffer of the comparative example of the present invention.

[0040] 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

[0041] 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.

[0042] Example 1

[0043] like Figures 1-4 As 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.

[0044] 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.

[0045] The first insulating layer 3 is formed by printing the insulating material on the first conductive layer 2 through screen printing or the like using a screen plate provided with two pattern portions, i.e. a first pattern portion and a second pattern portion, and the two pattern portions are kept at a first fixed distance d1. The first insulating layer 3 is composed 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 to form the first upper portion 31 of the first insulating layer 3 through the screen holes of the first pattern portion of the screen plate used to form the first insulating layer 3. The first lower portion 32 is printed on the lower end of the first conductive layer 2, and the insulating material can be printed to form the first lower portion 32 of the first insulating layer 3 through the screen holes of the second pattern portion of the screen plate used to form the first insulating layer 3. The first upper portion 31 and the first lower portion 32 of the first insulating layer 3 are kept at a distance d1, which is equal to the distance between the first pattern portion and the second pattern portion of the screen plate used to form 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 exposed area of the first conductive layer 2 forming the first electrode 21. Therefore, the size of the first electrode 21 depends on the distance between the first upper portion 31 and the first lower portion 32 of the first insulating layer 3, and essentially depends on the first fixed distance between the first pattern portion and the second pattern portion of the screen plate used to form the first insulating layer 3.

[0046] Therefore, when the biosensor 1 is manufactured, even if there is a misregistration between the screen plate used to form the first conductive layer 2 and the screen plate used to form 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 of the screen plate used to form the first insulating layer 3 remains unchanged.

[0047] In order to sufficiently cover the first conductive layer 2 and prevent the adjacent conductive layer from being conductive, the first insulating layer 3 can be extended outwardly by a certain distance after covering both sides of the first conductive layer 2 when the first insulating layer 3 is printed. The portions of the first insulating layer 3 extending outwardly relative to both sides of the first conductive layer 2 can be cut off through laser cutting or the like. In the test, the proximal end of the first conductive layer 2 needs to be inserted into the body of the test subject, and the upper portion of the first insulating layer 3 does not 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, which serves as the first contact 22.

[0048] The second conductive layer 4 is formed by covering the first insulating layer 3 with a conductive material by screen printing or the like. The second insulating layer 5 is formed by printing the insulating material on the second conductive layer 4 by screen printing or the like using a screen having two pattern portions, i.e., a first pattern portion and a second pattern portion, which are separated by a second fixed distance d2. The second insulating layer 5 includes a second upper portion 51 and a second lower portion 52. The second upper portion 51 of the second insulating layer 5 is printed on the upper end of the second conductive layer 4, and is formed by the screen holes of the first pattern portion of the screen used to form the second insulating layer 5. The second lower portion 52 of the second insulating layer 5 is printed on the lower end of the second conductive layer 4, and is formed by the screen holes of the second pattern portion of the screen used to form the second insulating layer 5. The second upper portion 51 and the second lower portion 52 of the second insulating layer 5 are separated by a distance d2, which is equal to the second fixed distance between the first pattern portion and the second pattern portion of the screen used to form 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. Thus, 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, and essentially depends on the second fixed distance between the first pattern portion and the second pattern portion of the screen used to form the second insulating layer 5.

[0049] Therefore, in the manufacture of the biosensor 1, even if there is a misregistration between the screen used to form the second conductive layer 4 and the screen used to form the second insulating layer 5, the size of the second electrode 41 remains unchanged because the distance between the first pattern portion and the second pattern portion of the screen used to form the second insulating layer 5 remains unchanged.

[0050] In the printing of the second insulating layer 5, in order to sufficiently cover the second conductive layer 4 and prevent the conduction between adjacent conductive layers, the second insulating layer 5 can be extended outwardly by a certain distance after covering both sides of the second conductive layer 4. The portions of the second insulating layer 5 extending outwardly relative to both sides of the second conductive layer 4 can be cut off by laser cutting or the like. In the test, the proximal end of the second conductive layer 4 is close to the test object. The second upper portion 51 of the second insulating layer 5 does not cover the distal end of the second conductive layer 4 completely, and thus leaves an exposed area at the distal end of the second conductive layer 4, which functions as the second contact 42.

[0051] The third conductive layer 7 is formed by applying a conductive material to the lower surface 62 of the substrate 6 by screen printing or the like. The third insulating layer 8 is formed by printing an insulating material on the third conductive layer 7 by screen printing or the like using a screen having two pattern portions, i.e., a first pattern portion and a second pattern portion, which are separated by a third fixed distance d3. The third insulating layer 8 is formed of a third upper portion 81 and a third lower portion 82. The third upper portion 81 is printed on the upper end of the third conductive layer 7, and is printed by the screen holes of the first pattern portion of the screen used to form the third insulating layer 8. The third lower portion 82 is printed on the lower end of the third conductive layer 7, and is printed by the screen holes of the second pattern portion of the screen used to form the third insulating layer 8. The third upper portion 81 and the third lower portion 82 of the third insulating layer 8 are separated by a distance equal to the third fixed distance between the first pattern portion and the second pattern portion of the screen used to form the third insulating layer 8. The gap between the third upper portion 81 and the third lower portion 82 of the third insulating layer 8 results in an exposed region of the third conductive layer 7, which forms the third electrode 71. Thus, the size of the third electrode 71 depends on the distance between the third upper portion 81 and the third lower portion 82 of the third insulating layer 8, and essentially depends on the third fixed distance d3 between the first pattern portion and the second pattern portion of the screen used to form the third insulating layer 8.

[0052] Thus, in the manufacture of the biosensor 1, even if there is a misregistration between the screen used to form the third conductive layer 7 and the screen used to form 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 of the screen used to form the third insulating layer 8 remains unchanged.

[0053] In printing the third insulating layer 8, in order to sufficiently cover the third conductive layer 7 and prevent conduction between adjacent conductive layers, the third insulating layer 8 can be printed to extend beyond the two sides of the third conductive layer 7 by a certain distance, and the portions of the third insulating layer 8 extending beyond the two sides of the third conductive layer 7 can be cut off by laser cutting or the like. In testing, the proximal end of the third conductive layer 7 is close to the test object. The third upper portion 81 of the third insulating layer 8 does not cover the distal end of the third conductive layer 7, and thus leaves an exposed region at the distal end of the third conductive layer 7, which functions as the third contact 72.

[0054] The surface of the first electrode 21 (herein, the first electrode is taken as an example of the working electrode) needs to be coated with an enzyme solution, which includes an analyte reaction enzyme, a buffer, a cross-linking agent and an electron mediator. When the analyte is glucose, the analyte reaction enzyme 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 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 can couple the analyte reaction enzyme and the electron mediator to the surface of the working electrode, so that the electrons are directly delivered to the working electrode when the analyte reaction enzyme reacts with the analyte in the sample; the cross-linking agent can be selected from polyethylene glycol diglycidyl ether (PEGDGE) with a molecular weight of about 200-600, etc.

[0055] Example 2

[0056] As 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 screen printing or the like on the lower surface 62 of the substrate 6 with a conductive material. The third insulating layer 8 is formed by screen printing or the like on the lower surface of the third conductive layer 7 with an insulating material using a screen having two pattern portions (i.e., a first pattern portion and a second pattern portion) separated by a third fixed distance d3. The third insulating layer 8 is composed of a third upper portion 81 and a third lower portion 82, the third upper portion 81 of the third insulating layer 8 is printed on the upper end of the third conductive layer 7 by allowing the insulating material to pass through the screen holes of the first pattern portion of the screen used to manufacture the third insulating layer 8, and the third lower portion 82 of the third insulating layer 8 is printed on the lower end of the third conductive layer 7 by allowing the insulating material to pass through the screen holes of the second pattern portion of the screen used to manufacture the third insulating layer 8. The third upper portion 81 and the third lower portion 82 of the third insulating layer 8 are separated by a 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 screen printing or the like on the lower surface of the third upper portion 81 of the third insulating layer 8 with a conductive material. The second insulating layer 5 is formed by screen printing or the like on the lower surface of the second conductive layer 4 with an insulating material using a screen having two pattern portions (i.e., a first pattern portion and a second pattern portion) separated by a third fixed distance d3. The second insulating layer 5 is composed 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 on the upper end of the second conductive layer 4 by allowing the insulating material to pass through the screen holes of the first pattern portion of the screen used to manufacture the second insulating layer 5, and the second lower portion 52 of the second insulating layer 5 is printed on the lower end of the first conductive layer 2 by allowing the insulating material to pass through the screen holes of the second pattern portion of the screen 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 separated by a distance, which is equal to the second fixed distance between the first pattern portion and the second pattern portion of 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 exposed area of the second conductive layer 4 forming the second electrode 41.

[0057] 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.

[0058] Example 3

[0059] 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.

[0060] Example 4

[0061] 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.

[0062] Example 5

[0063] 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.

[0064] 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.

[0065] The manufacturing process of the biosensor of the present application is described based on the first embodiment of the present application:

[0066] The manufacturing process of the biosensor of the first embodiment of the present application includes a screen printing process, an enzyme solution fixing process and a cutting process. In the manufactured 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.

[0067] In the screen printing process, in the large card, a conductive carbon ink is screen printed on the upper surface 61 of the insulating substrate 6 by using a screen plate to form the first conductive layer 2 where the working electrode is located, then an insulating ink is screen printed on the first conductive layer 2 by using a screen plate provided with two pattern portions (i.e. the first pattern portion and the second pattern portion) and the two pattern portions keep a first fixed distance d1 to form the upper and lower portions of the first insulating layer 3 (i.e. the first upper portion 31 and the first lower portion 32), the first upper portion 31 covers the upper end of the first conductive layer 2, the first lower portion 32 covers the lower end of the first conductive layer 2, and the first upper portion 31 and the first lower portion 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 plate used to manufacture the first insulating layer 3, so as to fix the size of the first electrode 21 which can be selected from 1.5mm to 3.0mm, at the same time, when the first insulating layer 3 is printed, the first insulating layer 3 does not completely cover the distal end of the first conductive layer 2, so as to leave an exposed area at the distal end of the first conductive layer 2 which can function as the first contact 22; then a conductive carbon ink or Ag / AgCl ink is screen printed on the first insulating layer 3 by using a screen plate to form the second conductive layer 4, then an insulating ink is screen printed on the second conductive layer 4 by using a screen plate provided with two pattern portions (i.e. the first pattern portion and the second pattern portion) and the two pattern portions keep a second fixed distance d2 to form the upper and lower portions of the second insulating layer 5 (i.e. the second upper portion 51 and the second lower portion 52), the second upper portion 51 covers the upper end of the second conductive layer 4, the second lower portion 52 covers the lower end of the second conductive layer 4, and the gap between the second upper portion 51 and the second lower portion 52 of the second insulating layer 5 exposes a part of the second conductive layer 4 which is the second electrode 41, and the second upper portion 51 and the second lower portion 52 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 plate used to manufacture the second insulating layer 5, so as to fix the size of the second electrode 41 (as the reference electrode) which can be selected from 0.5mm to 1.0mm, at the same time, when the second insulating layer 5 is printed, the second insulating layer 5 does not completely cover the distal end of the second conductive layer 4, so as to leave an exposed area at the distal end of the second conductive layer 4 which can function as the second contact 42.

[0068] Furthermore, the conductive carbon ink is screen-printed on the lower surface 62 of the insulating substrate 6 by using a screen plate to form the third conductive layer 7, and the insulating ink is screen-printed on the lower surface of the third conductive layer 7 by using a screen plate provided with two pattern portions (i.e. the first pattern portion and the second pattern portion) and the two pattern portions keep a third fixed distance d3 to form the upper and lower portions of the third insulating layer 8 (i.e. the third upper portion 81 and the third lower portion 82), the third upper portion 81 of the third insulating layer 8 covers the upper end of the third conductive layer 7, the third lower portion 82 covers the lower end of the third conductive layer 7, and the third upper portion 81 and the third lower portion 82 are separated by a certain distance, which is equal to the distance between the first pattern portion and the second pattern portion of the screen plate used for manufacturing the third insulating layer 8, so as to fix the size of the third electrode 71 (as the counter electrode) (which can be selected from 1.0 to 4.0 mm); at the same time, when the third insulating layer 8 is printed, the third insulating layer 8 does not completely cover the distal end of the third conductive layer 7, so as to leave an exposed area at the distal end of the third conductive layer 7, which can function as the third contact 72.

[0069] In addition, when the first insulating layer 3 is printed, the first insulating layer 3 extends outward by a certain distance after covering the two sides of the first conductive layer 2; when the second insulating layer 5 is printed, the second insulating layer 5 extends outward by a certain distance after covering the two sides of the second conductive layer 4; when the third insulating layer 8 is printed, the third insulating layer 8 extends outward by a certain distance after covering the two sides of the third conductive layer 7.

[0070] In the enzyme solution fixing process, the uniformly mixed enzyme solution is fixed on the surface of the working electrode in the form of point liquid or dip coating, and then cured at room temperature for 24 hours. After the enzyme solution is fixed, the portion of the first insulating layer 3 extending outward relative to the two sides of the first conductive layer 2 is cut off by laser cutting; the portion of the second insulating layer 5 extending outward relative to the two sides of the second conductive layer 4 is cut off by laser cutting; and the portion of the third insulating layer 8 extending outward relative to the two sides of the third conductive layer 7 is cut off by laser cutting. The enzyme solution is prepared by adding 10 mg of glucose oxidase (GOD), 9 mg of osmium complex [Os (Py-MIM) 2 (MIM) Cl] 2+ 2Cl - 10 mg of crosslinking agent polyethylene glycol diglycidyl ether (molecular weight 500) into 1 mL of purified water, and uniformly mixing, wherein the osmium complex used is prepared according to the synthesis method in Example 1 of Chinese Patent CN1620462B.

[0071] In the cutting process, the large card after the silk screen printing process and enzyme liquid fixing process is cut into a batch of independent single-person bio-sensors on a laser cutting machine. Finally, the mixed and uniform membrane liquid is covered on the surface of the working electrode, the counter electrode and the reference electrode of the single-person bio-sensor in the way of dip coating, and is cured at room temperature for 24 hours, so as to prepare for subsequent tests. The preparation method of the membrane liquid used is to add 80 mg of poly-4-vinylpyridine (molecular weight 160000) and 20 mg of polyethylene glycol diglycidyl ether (molecular weight 500) into 5 mL of 80% ethanol solution and mix uniformly.

[0072] Test method:

[0073] A batch of bio-sensors manufactured by using the bio-sensor manufacturing process of the application is placed in a series of PBS buffers (pH 7.0) containing different glucose concentrations, the test temperature is 37°C, the working voltage is set to 0.05V, for each PBS buffer with different glucose concentrations, the measurement is repeated 4 times, the output current of each bio-sensor is measured by an electrochemical workstation model chi-1000c (Shanghai Chenhua Instrument Co., Ltd.), and then the coefficient of variation (CV) of the measured current value for each PBS buffer with different glucose concentrations is calculated. The detection results are shown in Table 1. At the same time, a standard curve graph is drawn with the glucose concentration as the abscissa and the measured output current as the ordinate, as shown in Figure 10 .

[0074] Table 1

[0075]

[0076] As can be seen from Table 1, after the size of the working electrode is fixed, the measured current value for each PBS buffer with different glucose concentrations is basically consistent, the accuracy is good, and the CV is small (when the glucose concentration is different, the CV is less than 10%, and the highest value is only 6.6%), which is within the acceptable range.

[0077] Table 2

[0078]

[0079] Since each PBS buffer with different glucose concentrations is measured 4 times, according to the standard curve determined in Figure 10 , the theoretical value of the glucose concentration corresponding to each current value measured in Table 1 for each PBS buffer with different glucose concentrations is calculated (as shown in Table 2), and then a correlation graph between the calculated glucose concentration theoretical value and the true value of the glucose concentration in the PBS buffer is drawn with the calculated glucose concentration theoretical value as the abscissa and the true value of the glucose concentration in the PBS buffer as the ordinate, as shown in Figure 11 . As can be seen from Figure 11 , R2 =0.9934, R>0.99, which indicates that the correlation between the theoretical value and the true value of the glucose concentration is good, and the deviation between the theoretical value and the true value is small.

[0080] Comparative Example 1

[0081] The process of manufacturing the biosensor as a control includes a screen printing process, an enzyme solution fixing process, and a cutting process.

[0082] In the screen printing process, in a large card, a conductive carbon ink is screen printed on an insulating substrate by using a screen plate to form a first conductive layer 2 where the working electrode is located, then an insulating ink is screen printed on the first conductive layer 2 by using a screen plate with a pattern part of a specific shape to form a first insulating layer 3, the first insulating layer 3 is a complete whole without a gap in the middle, the first insulating layer 3 does not completely cover the proximal end of the first conductive layer 2, thereby leaving an exposed area at the proximal end of the first conductive layer 2, which is the first electrode as the working electrode, at the same time, the first insulating layer 3 does not completely cover the distal end of the first conductive layer 2, thereby leaving an exposed area at the distal end of the first conductive layer 2, which is the first contact to play a role; then a conductive carbon ink or Ag / AgCl ink is screen printed on the first insulating layer 3 by using a screen plate to form a second conductive layer 4, and then an insulating ink is screen printed on the second conductive layer 4 by using a screen plate to form a second insulating layer 5, the second insulating layer 5 is a complete whole without a gap in the middle; the second insulating layer 5 does not completely cover the proximal end of the second conductive layer 4, thereby leaving an exposed area at the proximal end of the second conductive layer 4, which is the second electrode as the reference electrode, and the second insulating layer 5 does not completely cover the distal end of the second conductive layer 4, thereby leaving an exposed area at the distal end of the second conductive layer 4, which can play a role as the second contact. In addition, 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.

[0083] Furthermore, a conductive carbon ink is screen-printed on the lower surface 62 of the insulating substrate 6 using a screen to form a third conductive layer 7, and then an insulating ink is screen-printed on the lower surface of the third conductive layer 7 using a screen having a pattern portion with a specific shape to form a third insulating layer 8, which is a complete whole without any gap in the middle, and does not completely cover the distal end of the third conductive layer 7, so that an exposed area is left on the distal end of the third conductive layer 7, which can function as a third contact 72; the third insulating layer 8 does not completely cover the proximal end of the third conductive layer 7, so that an exposed area is left on the proximal end of the third conductive layer 7, which is a third electrode serving as a counter electrode.

[0084] In the enzyme solution fixing process, the uniformly mixed enzyme solution is fixed on the surface of the working electrode in the form of a point liquid or dip coating, and then cured at room temperature for 24 h. After the enzyme solution is fixed, the portions of the first insulating layer 3 extending beyond both sides of the first conductive layer 2 are cut off by laser cutting; the portions of the second insulating layer 5 extending beyond both sides of the second conductive layer 4 are cut off by laser cutting; and the portions of the third insulating layer 8 extending beyond both sides of the third conductive layer 7 are cut off by laser cutting. The enzyme solution is prepared by adding 10 mg of glucose oxidase (GOD), 9 mg of osmium complex [Os (Py-MIM) 2 (MIM) Cl] and 10 mg of crosslinking agent polyethylene glycol diglycidyl ether (molecular weight 500) into 1 mL of purified water and uniformly mixing them. 2+ 2Cl -

[0085] In the cutting process, the large card after the screen printing process and the enzyme solution fixing process is cut into a batch of independent single-person bio-sensors on a laser cutting machine. Finally, the uniformly mixed film solution is coated on the surfaces of the working electrode, the counter electrode and the reference electrode of the single-person bio-sensor in the form of dip coating, and cured at room temperature for 24 h to prepare for subsequent tests. The film solution is prepared by adding 80 mg of poly-4-vinylpyridine (molecular weight 160000) and 20 mg of polyethylene glycol diglycidyl ether (molecular weight 500) into 5 mL of 80% ethanol solution and uniformly mixing them.

[0086] Test method:

[0087] ​A batch of biosensors as a control was placed in a series of PBS buffer (pH 7.0) containing different glucose concentrations, the test temperature was 37°C, the working voltage was set to 0.05V, for each glucose concentration of PBS buffer, the measurement was repeated 4 times, the output current of each sensor was measured by an electrochemical workstation model chi-1000c (Shanghai Chenhua Instrument Co., Ltd.), and then the coefficient of variation (CV) of the measured current value for each glucose concentration of PBS buffer was calculated. The detection results are shown in Table 3. At the same time, a standard curve graph was drawn with the glucose concentration as the abscissa and the measured output current as the ordinate, as shown in Figure 12 .

[0088] Table 3

[0089]

[0090] As can be seen from Table 3, in the biosensors as a control, since the size of the working electrode is not fixed, there is a certain difference in the size of the working electrode of different biosensors, so for each glucose concentration of PBS buffer, the test results of different biosensors are quite different, and the precision is obviously worse than that of the biosensor manufactured by the present application with a fixed working electrode size, and the CV is large (at different glucose concentrations, the CV is about 10%, and the highest value is 13.5%).

[0091] Table 4

[0092]

[0093] Since each glucose concentration of PBS buffer was measured 4 times, according to the standard curve determined in Figure 13 , the theoretical value of the glucose concentration corresponding to each current value measured in Table 3 for each glucose concentration of PBS buffer was calculated (as shown in Table 4), and then the calculated glucose concentration theoretical value was taken as the abscissa, and the true value of the glucose concentration in the PBS buffer was taken as the ordinate, to draw a correlation graph between the calculated glucose concentration theoretical value and the true value of the glucose concentration in the PBS buffer, as shown in Figure 13 . As can be seen from Figure 13 , R 2 =0.9479, R=0.9736, which is less than 0.99, which indicates that the correlation between the theoretical value and the true value of the glucose concentration is poor, and the deviation between the theoretical value and the true value is large.

[0094] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A biosensor comprising a substrate (6), a first conductive layer (2), a first insulating layer (3), a second conductive layer (4) and a second insulating layer (5), characterized in that, The first conductive layer (2) is formed on one side surface of the substrate (6); the first insulating layer (3) comprises a first upper part (31) and a first lower part (32) which are arranged at intervals and are formed on the upper and lower ends of the first conductive layer (2) respectively, and the interval between the first upper part (31) and the first lower part (32) exposes a region of the first conductive layer (2) to form a first electrode (21); the second conductive layer (4) is formed on the other side surface of the substrate (6) or the first upper part (31), and the second insulating layer (5) comprises a second upper part (51) and a second lower part (52) which are arranged at intervals and are formed on the upper and lower ends of the second conductive layer (4) respectively, and the interval between the second upper part (51) and the second lower part (52) exposes a region of the second conductive layer (4) to form a second electrode (41).

2. The biosensor of claim 1, wherein, The first insulating layer (3) does not cover the region of the far end of the first conductive layer (2) to form a first contact (22), and the second insulating layer (5) does not cover the region of the far end of the second conductive layer (4) to form a second contact (42).

3. The biosensor of claim 1, wherein, Further comprising a third conductive layer (7) and a third insulating layer (8), the third conductive layer (7) is formed on the substrate (6) or the second upper part (51), and the third insulating layer (8) comprises a third upper part (81) and a third lower part (82) which are arranged at intervals and are formed on the upper and lower ends of the third conductive layer (7) respectively, and the interval between the third upper part (81) and the third lower part (82) exposes a region of the third conductive layer (7) to form a third electrode (71).

4. The biosensor of claim 3, wherein, The third insulating layer (8) does not cover the region of the far end of the third conductive layer (7) to form a third contact (72).

5. A biosensor according to claim 3, characterized in that, The first electrode (21) is a working electrode, and the second electrode (41) and the third electrode (71) are respectively a counter electrode or a reference electrode.

6. A biosensor according to claim 5, wherein, The size of the working electrode ranges from 1.0 mm to 3.5 mm, the size of the reference electrode ranges from 0.5 mm to 1.0 mm, and the size of the counter electrode ranges from 1.0 mm to 4.0 mm.

7. A method of manufacturing a biosensor comprising screen printing, enzyme solution immobilization and cutting, characterized by, The screen printing specifically comprises the following steps: 1) Take the substrate (6), and screen print the conductive carbon ink on one side surface of the substrate (6) to form the first conductive layer (2); 2) Screen print the insulating ink on the first conductive layer (2) by using a screen plate provided with two pattern parts which are kept at a first fixed distance d1 to form the first upper part (31) and the first lower part (32) of the first insulating layer (3), the first upper part (31) of the first insulating layer (3) covers the upper end of the first conductive layer (2), the first lower part (32) covers the lower end of the first conductive layer (2), and the interval between the first upper part (31) and the first lower part (32) exposes a region of the first conductive layer (2) to form the first electrode (21); 3) Screen print the conductive carbon ink on the other side surface of the substrate (6) or the first upper part (31) of the first insulating layer (3) to form the first conductive layer (2); 4) screen printing insulating ink on the second conductive layer (4) by using a screen with two patterned sections maintaining a second fixed distance d2, to form a second upper part (51) and a second lower part (52) of the second insulating layer (5), the second upper part (51) of the second insulating layer (5) covering the upper end of the second conductive layer (4), the second lower part (52) covering the lower end of the second conductive layer (4), the interval between the second upper part (51) and the second lower part (52) leaving the area of the second conductive layer (4) exposed to form the second electrode (41); 5) stacking and arranging the conductive layer and the insulating layer in sequence on both sides of the substrate (6) to form a plurality of electrodes according to the above steps.

8. The manufacturing method according to claim 7, wherein The first electrode (21) is a working electrode, and the first fixed distance is 1.0 mm to 3.5 mm; the second electrode (41) is a counter electrode or a reference electrode, if the second electrode (41) is a reference electrode, the second fixed distance is 0.5 mm to 1.0 mm; if the second electrode (41) is a counter electrode, the second fixed distance is 1.0 mm to 4.0 mm.

9. The production method according to claim 7, wherein The first insulating layer (3) does not completely cover the distal end of the first conductive layer (2), thereby leaving an exposed area at the distal end of the first conductive layer (2) to form a first contact (22); the second insulating layer (5) does not completely cover the distal end of the second conductive layer (4), thereby leaving an exposed area at the distal end of the second conductive layer (4) to form a second contact (42); similarly, the Nth insulating layer does not completely cover the distal end of the Nth conductive layer, thereby leaving an exposed area at the distal end of the Nth conductive layer to form an Nth contact, N being an integer greater than or equal to 1.

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