Body Fluid Glucose Test Strips and Their Preparation Method

Abstract

This application provides a body fluid glucose test strip, comprising a substrate, an electrode layer, an insulating layer, a bio-enzyme layer, and a siphon structure. The electrode layer is disposed on the substrate and includes a sequentially arranged connection area, a transmission area, and a reaction area. Along the direction away from the substrate, a basic electrode layer, a basic resistance layer, and a range control layer are sequentially arranged on the electrode layer. The insulating layer is disposed on the side of the electrode layer away from the substrate from the transmission area. The bio-enzyme layer is disposed in the reaction area of ​​the electrode layer and is used to react with glucose in the body fluid to generate an electrical signal. The siphon structure is used to draw body fluid to the bio-enzyme layer through a siphon effect. The body fluid glucose test strip of this application has a relatively simple structure and can achieve accurate, non-invasive, rapid, and controllable detection of micromolar glucose concentrations in body fluids such as saliva. This application also provides a method for preparing the body fluid glucose test strip, which is easy to mass-produce.

Description

Technical Field

[0001] This application belongs to the field of medical devices, and more specifically, relates to a blood glucose test strip for body fluids and its preparation method. Background Technology

[0002] Currently, common non-invasive blood glucose monitoring technologies on the market can be categorized into near-infrared spectroscopy, microwave detection, optical rotation, and energy metabolism conservation methods. These technologies mostly measure relevant parameters of blood glucose concentration indirectly, then use complex conversion formulas to obtain the blood glucose concentration. Research data shows that salivary glucose has a significant linear correlation with blood glucose, effectively addressing the challenge of weak correlation with blood glucose levels. Furthermore, compared to other tissue fluids such as tears, urine, and sweat, saliva samples are easier to collect, providing a new technical pathway for non-invasive blood glucose detection.

[0003] However, blood glucose testing based on saliva presents significant challenges. Firstly, saliva has a complex composition, with glucose concentrations at the micromolar level, demanding extremely high precision and sensitivity from the detection system. This involves numerous interdisciplinary technologies from biochemistry, microelectronics, electrochemistry, and biomaterials, including bioreaction enzyme design, picoampere-level current detection, chemical reaction rate control, and interference filtering. Based on common knowledge of the principles, structure, and manufacturing processes of conventional finger-prick blood test strips, it is known that strip size and structure affect production efficiency, while electrode material type, circuit distribution structure, length, width, and shape influence detection accuracy and sensitivity. The bioreaction system, including the sample volume, directly relates to core electrochemical technical indicators such as detection range and accuracy. However, the aforementioned test strip technologies for accurate, non-invasive, rapid, and controllable detection of micromolar concentrations of glucose based on saliva and other bodily fluids have not yet been mastered. Summary of the Invention

[0004] The purpose of this application is to provide a body fluid glucose test strip and its preparation method, so as to solve the technical problem that the existing test strip technology for accurate, non-invasive, rapid and controllable detection of glucose at micromolar concentrations based on body fluids such as saliva has not yet been overcome.

[0005] To achieve the above objectives, the technical solution adopted in this application is: to provide a body fluid glucose test strip and its preparation method, comprising:

[0006] Substrate;

[0007] An electrode layer is disposed on the substrate. The electrode layer includes a connection area for connecting an external blood glucose meter interface, a transmission area for transmitting electrical signals, and a reaction area for generating electrical signals, which are arranged sequentially. The electrode layer includes a base electrode layer disposed on the substrate. The electrode layer in the reaction area also includes a base resistance layer and a range control layer disposed on the electrode layer of the substrate.

[0008] An insulating layer is disposed on the side of the electrode layer away from the substrate in the transmission region;

[0009] The bio-enzyme layer, located in the reaction zone of the electrode layer, is used to react with glucose in body fluids to generate electrical signals.

[0010] In one embodiment, the base electrode layer is a silver-silver chloride paste layer, the base resistor layer is a graphene-carbon paste layer, and the range control layer is a PEDOT:PSS layer.

[0011] In one embodiment, the silver content in the silver-silver chloride paste layer is 30% to 80% by mass; and / or,

[0012] The graphene-carbon paste layer contains 30% to 80% graphene by mass.

[0013] In one embodiment, the thickness of the silver-silver chloride paste layer ranges from 0.001 to 0.1 mm, the thickness of the graphene-carbon paste layer ranges from 0.001 to 0.1 mm, and the thickness of the PEDOT:PSS layer ranges from 0.001 to 0.005 mm.

[0014] In one embodiment, the electrode layer includes a working electrode, a reference electrode, a backup electrode, a signal electrode, and a counter electrode. The working electrode, the reference electrode, the backup electrode, the signal electrode, and the counter electrode all include the base electrode layer. The portions of the working electrode, the backup electrode, and the counter electrode located in the reaction region also include the base resistive layer and the range control layer. The base resistive layer is disposed on the side of the base electrode layer away from the substrate, and the range control layer is disposed on the side of the base resistive layer away from the substrate.

[0015] In one embodiment, a siphon structure is further included, which covers the bio-enzyme layer. The siphon structure includes a siphon portion for drawing body fluid into the bio-enzyme layer.

[0016] In one embodiment, the siphon structure includes:

[0017] The first channel sheet has one end located on the side of the insulating layer away from the substrate and the other end located in the reaction region of the electrode layer. The first channel sheet has an opening groove facing away from the connection area, and the bio-enzyme layer is located in the opening groove.

[0018] A first hydrophilic membrane is applied to the side of the first channel sheet away from the substrate, and the portion of the first hydrophilic membrane corresponding to the opening groove forms the siphon portion.

[0019] In one embodiment, the siphon structure includes:

[0020] The second channel sheet has one end located on the side of the insulating layer away from the substrate, and the other end located at the edge of the reaction region of the electrode layer;

[0021] The third channel sheet is disposed in the reaction region of the electrode layer and is spaced apart from the second channel sheet; the bio-enzyme layer is located between the second channel sheet and the third channel sheet.

[0022] A second hydrophilic membrane is applied to the side of the second channel sheet and the third channel sheet away from the substrate, and the siphon portion is formed on the portion of the second hydrophilic membrane corresponding to the space between the second channel sheet and the third channel sheet.

[0023] A method for preparing a body fluid glucose test strip, comprising the following steps:

[0024] Prepare the substrate;

[0025] The electrode layer is prepared on the substrate;

[0026] The insulating layer covers the transmission region of the electrode layer;

[0027] Prepare biological enzyme solutions according to the detection range and accuracy requirements;

[0028] The bio-enzyme layer is formed by coating the reaction zone of the electrode layer with the bio-enzyme solution.

[0029] In one embodiment, the step of fabricating the electrode layer on the substrate includes the following steps:

[0030] The base electrode layer is fabricated on the substrate corresponding to the positions of the working electrode, the reference electrode, the spare electrode, the signal electrode, and the counter electrode.

[0031] The basic resistive layer and the range control layer are fabricated on the basic electrode layer located in the reaction region portion of the working electrode, the standby electrode, and the counter electrode.

[0032] In one embodiment, the step of preparing the bio-enzyme solution according to the detection range and accuracy requirements is as follows:

[0033] Prepare neutral phosphate buffer solution;

[0034] Dissolve glucose oxidase and peroxidase in the neutral phosphate buffer at a mass ratio of (0.45–0.8):1 to prepare a second solution with a mass concentration of 10–60 mg / ml.

[0035] Graphene and potassium ferrocyanide in a mass ratio of (0.05–0.15):1 are dissolved in the second solution to obtain the bio-enzyme solution, wherein the total mass concentration of the graphene and potassium ferrocyanide in the bio-enzyme solution ranges from 15 to 50 mg / ml.

[0036] The beneficial effects of the body fluid glucose test strip provided in this application are as follows: Compared with the prior art, the body fluid glucose test strip of this application has a simpler test strip structure, a regular test strip shape, and convenient detection operation. Body fluids such as saliva react with the biological enzyme layer to generate an electrical signal, which is transmitted to the blood glucose meter for detection through the electrode layer. The electrode layer includes a basic electrode layer, a basic resistance layer, and a range adjustment layer, which adjusts the detection range from 1mM to several mM, achieving a detection accuracy of 10μM, which meets the requirements for measuring the glucose content of body fluids at the micromolar level (10-1000μM). By connecting to the external glucose meter interface and cooperating with the blood glucose meter to detect glucose concentration, it is possible to achieve accurate, non-invasive, rapid, and controllable detection of micromolar glucose concentration in body fluids such as saliva.

[0037] The beneficial effects of the preparation method of the body fluid glucose test strip provided in this application are as follows: Compared with the prior art, the preparation method of the body fluid glucose test strip in this application achieves accurate, rapid and controllable detection of micromolar glucose by designing the type, quantity, thickness and shape of electrode materials, the distribution structure, length, width and shape of screen printing lines, different conductive materials, a specially made biological reaction enzyme system, and a siphon structure. It also achieves large-scale low-cost production by optimizing the overall appearance, layering and size structure of the test strip. The raw materials used are easy to obtain, and the test strip is easy to achieve large-scale and batch production. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 An exploded structural diagram of the first type of body fluid glucose test strip provided in this application embodiment;

[0040] Figure 2 A schematic diagram of the substrate and electrode layer of the first type of body fluid glucose test strip provided in this application embodiment;

[0041] Figure 3 A schematic diagram of the substrate, electrode layer, and insulating layer of the first type of body fluid glucose test strip provided in this application embodiment;

[0042] Figure 4 A schematic diagram of the substrate, electrode layer, insulating layer, and biological enzyme layer of the first type of body fluid glucose test strip provided in this application embodiment;

[0043] Figure 5 This is a schematic diagram of the structure of the first body fluid glucose test strip provided in the embodiments of this application at the siphon structure;

[0044] Figure 6 This is a schematic diagram of the exploded structure of the second type of body fluid glucose test strip provided in the embodiments of this application;

[0045] Figure 7 This is a schematic diagram of the structure of the second type of body fluid glucose test strip provided in the embodiments of this application;

[0046] Figure 8 A flowchart illustrating the preparation method of the body fluid glucose test strip provided in this application embodiment;

[0047] Figure 9 A flowchart illustrating the preparation method of the electrode layer in the preparation method of the body fluid glucose test strip provided in the embodiments of this application;

[0048] Figure 10 A flowchart illustrating the preparation method of the biological enzyme solution in the preparation method of the body fluid glucose test strip provided in the embodiments of this application;

[0049] Figure 11 A linear relationship curve obtained by using a known concentration glucose verification test strip with the body fluid glucose test strip provided in the embodiments of this application;

[0050] Figure 12 The graph shows the detection effect of the body fluid glucose test strip provided in the embodiments of this application, using real saliva to verify the test strip's detection effect.

[0051] The following are the labeling elements in the figure:

[0052] 1. Substrate;

[0053] 2. Electrode layer; 21. Reference electrode; 22. Backup electrode; 221. Second front section; 222. Second rear section; 23. Signal electrode; 24. Working electrode; 241. First front section; 242. First rear section; 25. Counter electrode; 251. Third front section; 252. Third rear section; 26. Connecting area; 27. Transmission area; 28. Reaction area;

[0054] 3. Insulating layer; 4. Bio-enzyme layer;

[0055] 5. Siphon structure; 51. First channel sheet; 511. Opening groove; 52. First hydrophilic membrane; 61. Second channel sheet; 62. Second hydrophilic membrane; 63. Third channel sheet. Detailed Implementation

[0056] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0057] It should be noted that when a component is referred to as being "fixed to" or "attached to" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0058] It should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0059] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0060] In this specification, references to "one embodiment," "some embodiments," or simply "embodiment" mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. Furthermore, in one or more embodiments, specific features, structures, or characteristics may be combined in any suitable manner.

[0061] Please refer to the following: Figure 1 , Figure 2 , Figure 3 and Figure 4The following describes the body fluid glucose test strip provided in the embodiments of this application. The body fluid glucose test strip includes a substrate 1, an electrode layer 2, an insulating layer 3, and a bio-enzyme layer 4. The electrode layer 2 is disposed on the substrate 1 and includes a connection area 26, a transmission area 27, and a reaction area 28, arranged sequentially. The connection area 26 is used for connecting to an external blood glucose meter interface, the transmission area 27 is used for transmitting electrical signals, and the reaction area 28 is used for generating electrical signals. The electrode layer 2 includes a basic electrode layer disposed on the substrate 1. The electrode layer 2 also includes a basic resistance layer and a range control layer in the reaction area 28, disposed on the basic electrode layer. The insulating layer 3 is disposed on the side of the transmission area 27 of the electrode layer 2 away from the substrate 1, and the bio-enzyme layer 4 is disposed in the reaction area 28 of the electrode layer 2, used to react with glucose in the body fluid to generate an electrical signal.

[0062] The body fluid glucose test strip of this application embodiment has a relatively simple structure and regular shape, making the detection operation convenient. The body fluids such as saliva react with the biological enzyme layer 4 to generate an electrical signal, which is transmitted to the blood glucose meter for detection via the electrode layer 2. The electrode layer 2 includes a basic electrode layer, a basic resistance layer, and a range control layer, which controls the detection range from 1 mM to several mM, achieving a detection accuracy of 10 μM. This meets the requirements for measuring the glucose content of body fluids such as saliva at the micromolar level (10-1000 μM). By connecting to the external blood glucose meter interface through the connection area 26, and cooperating with the blood glucose meter to detect glucose concentration, accurate, non-invasive, rapid, and controllable detection of micromolar glucose concentration in body fluids such as saliva can be achieved.

[0063] The working process of the body fluid glucose test strip in this embodiment of the application is as follows:

[0064] First, collect saliva into a sampling tube. Then, use a pipette to draw the saliva from the sampling tube and drop it onto the reaction zone 28 of the test strip. The saliva reacts with the enzyme layer 4 to generate an electrical signal. Insert the contact area 26 of the test strip into the interface of the blood glucose meter. The transmission area 27 transmits the electrical signal generated by the reaction zone 28 to the blood glucose meter, generating electrochemical parameters and obtaining the blood glucose level.

[0065] In one embodiment, the base electrode layer is a silver-silver chloride paste layer, the base resistive layer is a graphene-carbon paste layer, and the range control layer is a PEDOT:PSS layer. Silver-silver chloride material exhibits minimal resistance change and good adhesion before and after stretching, making it suitable for screen printing. Screen-printing the silver-silver chloride paste layer to serve as the base electrode results in good conductivity and stability. The silver-silver chloride paste layer is a relatively mature and low-cost electrode printing process. Choosing silver-silver chloride as the base electrode layer facilitates processing, provides the basic circuitry, serves as a carrier for the graphene-carbon paste layer and the PEDOT:PSS layer, and saves costs.

[0066] Graphene has excellent electrical conductivity and a large specific surface area, making the surface structure of the graphene-carbon paste layer more suitable for electron migration. The graphene-carbon paste layer serves to conduct electricity and provide basic resistance, thereby effectively improving detection sensitivity. The graphene-carbon paste layer is used as a basic resistor.

[0067] PEDOT:PSS is an aqueous solution of a high-molecular-weight polymer with high conductivity. Depending on the formulation, aqueous solutions with varying conductivity can be obtained. This product is composed of two substances: PEDOT and PSS. PEDOT is a polymer of EDOT (3,4-ethylenedioxythiophene monomer), and PSS is polystyrene sulfonate. The combination of these two substances significantly improves the solubility of PEDOT. The PEDOT:PSS layer serves as a conductive layer and as a graphene dispersant, with adjustable dispersibility.

[0068] In one embodiment, the silver content in the silver-silver chloride paste layer is 30%-80% by mass, preferably 60%. This ensures extremely high stability and reversibility of the electrode layer 2.

[0069] The graphene content in the graphene-carbon paste layer is 30%-80% by mass, preferably 20%. This ensures that electrode layer 2 has good stability, reproducibility, and anti-interference ability. The surface of the graphene-modified graphene-carbon paste layer is completely covered by a graphene film. This structure increases the specific surface area of ​​the electrode (referring to the total area per unit mass of material), which is beneficial for electron transport in the graphene-carbon paste layer and improves the electrochemical activity of the graphene-carbon paste layer.

[0070] In one embodiment, the thickness of the silver-silver chloride paste layer ranges from 0.001 to 0.1 mm, preferably 0.005 mm. The thickness of the graphene-carbon paste layer ranges from 0.001 to 0.1 mm, preferably 0.02 mm. The thickness of the PEDOT:PSS layer ranges from 0.001 to 0.005 mm, preferably 0.005 mm. The silver-silver chloride paste layer serves as the base electrode, the graphene-carbon paste layer serves as the base resistor, and the PEDOT:PSS layer is used to adjust the detection range. By adjusting the thickness ranges of the silver-silver chloride paste layer, the graphene-carbon paste layer, and the PEDOT:PSS layer, the actual needs of detection with different ranges and sensitivities can be met, thereby controlling the resistance range of electrode layer 2, controlling the signal transmission of electrode layer 2, and ensuring the measurement of glucose concentration at the micromolar level.

[0071] In one embodiment, see Figure 1 , Figure 2 , Figure 3 and Figure 4The electrode layer 2 includes a working electrode 24, a reference electrode 21, a standby electrode 22, a signal electrode 23, and a counter electrode 25. The working electrode 24, the reference electrode 21, the standby electrode 22, the signal electrode 23, and the counter electrode 25 include a silver-silver chloride paste layer. The portion of the working electrode 24, the standby electrode 22, and the counter electrode 25 located in the reaction zone 28 also includes a graphene-carbon paste layer and a PEDOT:PSS layer. The graphene-carbon paste layer and the PEDOT:PSS layer are located on the side of the silver-silver chloride paste layer away from the substrate 1.

[0072] The working electrode 24, the standby electrode 22, and the counter electrode 25 located in the reaction zone 28 can be sequentially stacked with a silver-silver chloride paste layer, a graphene-carbon paste layer, and a PEDOT:PSS layer along the direction away from the substrate 1.

[0073] Alternatively, the working electrode 24, the standby electrode 22, and the counter electrode 25 located in the reaction zone 28 may be sequentially stacked with a silver-silver chloride paste layer, a PEDOT:PSS layer, and a graphene-carbon paste layer along the direction away from the substrate 1.

[0074] Alternatively, a mixed layer can be formed by mixing the graphene-carbon paste layer and the PEDOT:PSS layer. The working electrode 24, the standby electrode 22, and the counter electrode 25 located in the reaction zone 28 can be sequentially stacked with a silver-silver chloride paste layer and a mixed layer in a direction away from the substrate 1.

[0075] Electrode layer 2 adopts a 4-8 electrode system, of which 3 electrodes are used to construct a three-electrode electrochemical reaction system (working electrode 24, reference electrode 21, and counter electrode 25), and the remaining electrodes are used as signal electrode 23 or backup electrode 22. Preferably, there are 5 electrodes, of which 3 electrodes are used for electrochemical reaction and 2 electrodes are used for test strip connection signal.

[0076] The reference electrode 21 and signal electrode 23 are only provided with a basic electrode layer. The working electrode 24 includes a first front section 241 and a first rear section 242, the spare electrode 22 includes a second front section 221 and a second rear section 222, and the counter electrode 25 includes a third front section 251 and a third rear section 252. The first front section 241, the second front section 221, and the third front section 251 are located in the connection area 26 and the transmission area 27, and the first rear section 242, the second rear section 222, and the third rear section 252 are located in the reaction area 28. The first front section 241, the second front section 221, and the third front section 251 are only provided with a basic electrode layer, and the first rear section 242, the second rear section 222, and the third rear section 252 are provided with a basic electrode layer, a basic resistance layer, and a range control layer along the direction away from the substrate 1.

[0077] The reference electrode 21 serves as a reference electrode when measuring various electrode potentials. The signal electrode 23 is only used to connect the blood glucose meter signal and only has a basic electrode layer, which saves materials. The working electrode 24, the counter electrode 25, and the backup electrode 22 have a three-layer structure in the reaction zone 28, consisting of a basic electrode layer, a basic resistance layer, and a range control layer. This structure allows for better reception of the detection results in the reaction zone 28 and transmission of the reaction results of saliva and other bodily fluids with the biological enzyme layer 4 to the blood glucose meter.

[0078] In one embodiment, see Figure 1 , Figure 2 , Figure 3 and Figure 4 The working electrode 24, reference electrode 21, standby electrode 22, signal electrode 23, and counter electrode 25 are arranged along the length of the substrate 1, resulting in a compact structure and high space utilization. The signal electrode 23 is located in the contact area 26 and is used to connect the blood glucose meter signal. The reference electrode 21 and counter electrode 25 are located on opposite sides of the electrode layer 2, while the working electrode 24 and standby electrode 22 are located between them. The standby electrode 22 can be used as the working electrode 24 when necessary. The width of the working electrode 24 in the reaction area 28 and transmission area 27 is greater than its width in the contact area 26. The width of the working electrode 24 is adjustable; by controlling the width of the working electrode 24, the actual needs of detection with different ranges and sensitivities can be met. The resistance of the working electrode 24 can be controlled to adjust its signal transmission efficiency.

[0079] In one embodiment, the body fluid glucose test strip further includes a siphon structure 5, which covers the bio-enzyme layer 4. The siphon structure 5 includes a siphon portion for drawing body fluids such as saliva into the bio-enzyme layer 4 through a siphon effect. By providing the siphon structure 5, the total amount of body fluids such as saliva reacting with the bio-enzyme can be automatically controlled.

[0080] Alternatively, the siphon structure 5 can be omitted, and the total amount of saliva and other bodily fluids can be controlled by using a pipette to add saliva and other bodily fluids drop by drop.

[0081] In one embodiment, see Figure 1 and Figure 5The siphon structure 5 includes a first channel sheet 51 and a first hydrophilic membrane 52. One end of the first channel sheet 51 is located on the side of the insulating layer 3 away from the substrate 1, and the other end is located in the reaction zone 28 of the electrode layer 2. An opening groove 511 is provided on the first channel sheet 51, facing away from the contact area 26. The bio-enzyme layer 4 is located within the opening groove 511. The first hydrophilic membrane 52 covers the side of the first channel sheet 51 away from the substrate 1, and the portion of the first hydrophilic membrane 52 corresponding to the opening groove 511 forms a siphon portion. With the opening groove 511 on the first channel sheet 51 and the bio-enzyme layer 4 located within it, the first channel sheet 51 surrounds the bio-enzyme layer 4 on three sides, creating a gap. When the test strip is placed in a saliva or other bodily fluid environment, the saliva or other bodily fluid will automatically siphon into the siphon structure 5 along the direction of the gap, and fill the siphon portion under the hydrophilic action of the first hydrophilic membrane 52, achieving quantitative siphoning.

[0082] In one embodiment, see Figure 3 , Figure 6 and Figure 7 The siphon structure 5 may further include a second channel sheet 61, a third channel sheet 63, and a second hydrophilic membrane 62. One end of the second channel sheet 61 is located on the side of the insulating layer 3 away from the substrate 1, and the other end is located at the edge of the reaction zone 28 of the electrode layer 2. The third channel sheet 63 is located in the reaction zone 28 of the electrode layer 2, spaced apart from the second channel sheet 61. The bio-enzyme layer 4 is located between the second channel sheet 61 and the third channel sheet 63. The second hydrophilic membrane 62 covers the second channel sheet 61 and the third channel sheet 63, and is located on the side away from the substrate 1. The portion of the second hydrophilic membrane 62 corresponding to the area between the second channel sheet 61 and the third channel sheet 63 forms a siphon portion. The second channel sheet 61 and the third channel sheet 63 surround the bio-enzyme layer 4 on both sides, creating two gaps. After the test strip is placed in a saliva or other bodily fluid environment, the saliva or other bodily fluid will automatically siphon into the siphon structure 5 along the gap direction, and fill the siphon portion under the hydrophilic action of the second hydrophilic membrane 62, achieving quantitative siphoning.

[0083] Please see Figure 1 and Figure 8 A method for preparing a body fluid glucose test strip, comprising the following steps:

[0084] S01: Preparation of substrate 1. In some specific embodiments, substrate 1 is machined from PET material, with dimensions ranging from 0.2-0.4 mm in thickness, 5.0-8.0 mm in width, and 35.0-60.0 mm in length; preferably 0.3 mm in thickness, 6.5 mm in width, and 50.0 mm in length, to facilitate mass production. Substrate 1 serves as a support and facilitates the fabrication of other structures on it.

[0085] S02: Prepare electrode layer 2 on substrate 1. In some specific embodiments, electrode layer 2 can be printed on substrate 1 using screen printing technology. Electrode layer 2 is used to facilitate connection to a blood glucose meter and transmit the electrical signal generated by the reaction of the biological enzyme layer with glucose in bodily fluids such as saliva to the blood glucose meter.

[0086] S03: An insulating layer 3 is placed over the transmission area 27 of the electrode layer 2. In some specific embodiments, the thickness of the insulating layer 3 ranges from 0.01 to 0.1 mm; preferably 0.02 mm. The insulating layer 3 serves to insulate and protect the electrode layer 2 of the transmission area 27. It also facilitates the hand holding the test strip and inserting it into the blood glucose meter.

[0087] S04: Prepare the bio-enzyme solution according to the detection range and accuracy requirements. In some specific embodiments, the bio-enzyme solution needs to be customized, and the glucose oxidase and peroxidase method or the hexokinase and glucose-6-phosphate dehydrogenase method can be selected. This scheme uses the glucose oxidase and peroxidase method, and by customizing the bio-enzyme solution, it meets the detection accuracy requirement of 10 μM and the detection range requirement of 1 mM to several mM, realizing the detection of salivary glucose content at the micromolar level (10-1000 μM).

[0088] S05: A bio-enzyme is coated in the reaction zone 28 of electrode layer 2 to form a bio-enzyme layer 4. In some specific embodiments, a customized bio-enzyme is coated to ensure that the bio-enzyme reacts with glucose in saliva in the reaction zone 28 of electrode layer 2 to generate an electrical signal. The working electrode 24, the counter electrode 25, and the backup electrode 22 are configured with a three-layer structure in the reaction zone 28, consisting of a basic electrode layer, a basic resistance layer, and a range control layer. This allows for better reception of the detection effect in the reaction zone 28 and transmission of the reaction effect of saliva and other bodily fluids with the bio-enzyme layer 4 to the blood glucose meter.

[0089] In some embodiments, the preparation of the body fluid glucose test strip further includes step S06: preparing a siphon structure 5, wherein the volume of the siphon portion ranges from 10-100 μl, preferably 30 μl. The volume of the siphon portion determines the sample volume of body fluids such as saliva. By controlling the volume of the siphon portion, the total amount of reaction between the biological enzyme and body fluids such as saliva can be controlled. The siphon structure 5 can be selected as a unidirectional siphon (with a first hydrophilic membrane 52 and a first channel sheet 51) or a bidirectional siphon (with a second channel sheet 61, a third channel sheet 63, and a second hydrophilic membrane 62), used to draw saliva into the biological enzyme layer 4 through the siphon effect.

[0090] In one embodiment, see Figure 1 , Figure 2 , Figure 8 and Figure 9 The step of preparing electrode layer 2 on substrate 1 includes the following steps:

[0091] S201: A base electrode layer is screen-printed on the substrate 1 at the positions corresponding to the working electrode 24, reference electrode 21, spare electrode 22, signal electrode 23, and counter electrode 25. The base electrode layer is a silver-silver chloride paste layer, wherein the silver-silver chloride ratio in the silver-silver chloride paste layer ranges from 30% to 80%, preferably 60%, and the thickness of the silver-silver chloride paste layer ranges from 0.001 to 0.1 mm, preferably 0.005 mm. The silver-silver chloride paste layer is a relatively mature and low-cost electrode printing process. Choosing silver-silver chloride as the base electrode layer is convenient for processing, provides the basic circuit, provides a carrier for the graphene-carbon paste layer and the PEDOT:PSS layer, and can save costs.

[0092] S202: A basic resistive layer and a range control layer are screen-printed on the basic electrode layer located in the reaction zone 28 of the working electrode 24, the spare electrode 22, and the counter electrode 25. The basic resistive layer is a graphene-carbon paste layer, with a graphene-carbon ratio ranging from 5% to 60%, preferably 20%, and a thickness ranging from 0.001 to 0.1 mm, preferably 0.02 mm. The range control layer is a PEDOT:PSS layer, with a thickness ranging from 0.001 to 0.05 mm, preferably 0.005 mm. The graphene-carbon paste layer is used for conductivity and to provide basic resistance, while the PEDOT:PSS layer is used for conductivity and as a graphene dispersant, and can control the range.

[0093] By adjusting the thickness range of the silver-silver chloride paste layer, the graphene-carbon paste layer, and the PEDOT:PSS layer, the actual needs of detection with different ranges and sensitivities can be met, thereby controlling the resistance range of electrode layer 2, controlling the signal transmission of electrode layer 2, and ensuring the measurement of glucose concentration at the micromolar level.

[0094] In one embodiment, see Figure 1 , Figure 2 , Figure 8 and Figure 10 The steps involve preparing the bio-enzyme solution according to the detection range and accuracy requirements. The customized bio-enzyme solution preparation process is as follows:

[0095] S401: Preparation of neutral phosphate buffer. In some specific embodiments, Na₂HPO₄ solution is mixed with NaH₂PO₄ solution to obtain 1M (M is mol / L) phosphate buffer, which is then diluted 20 times with water to obtain 50mM (mM is mmol / L) diluted phosphate buffer. The pH is then adjusted to 7.0 with 1M NaH₂PO₄ solution to obtain the first solution. The Na₂HPO₄ in the Na₂HPO₄ solution can have a mass-to-volume ratio of 9.77% (the ratio range can be fine-tuned), and the NaH₂PO₄ in the NaH₂PO₄ solution can have a mass-to-volume ratio of 3.74% (the ratio range can be fine-tuned).

[0096] S402: Dissolve glucose oxidase and peroxidase in neutral phosphate buffer (first solution) to prepare a second solution with a mass concentration of 10-60 mg / ml, preferably 30 mg / ml. The mass ratio of glucose oxidase to peroxidase ranges from 45% to 80%, preferably 60%.

[0097] S403: Graphene and potassium ferrocyanide in a mass ratio of (0.05–0.15):1 are dissolved in a second solution to obtain a bio-enzyme solution. The total mass concentration of graphene and potassium ferrocyanide in the bio-enzyme solution ranges from 15 to 50 mg / ml. The preferred mass ratio of graphene to potassium ferrocyanide is 0.08:1, and the preferred total mass concentration of graphene and potassium ferrocyanide in the bio-enzyme solution is 25 mg / ml. Graphene acts as an electrical conductor; mixing it with potassium ferrocyanide improves the transmission of electron changes during chemical reactions. Potassium ferrocyanide is a reducing agent that reacts with hydrogen peroxide produced by glucose oxidase and glucose, thereby generating a change in current through changes in the oxidation state of iron ions. The current intensity corresponds to the glucose concentration.

[0098] The biological enzyme solution reacts with glucose in bodily fluids such as saliva to generate an electrical signal, which is then transmitted to the blood glucose meter through electrode layer 2 to detect the glucose content in the saliva.

[0099] The preparation method of the body fluid glucose test strip in this application embodiment achieves accurate, rapid and controllable detection of micromolar glucose by designing the type, quantity, thickness and shape of electrode materials, the distribution structure, length, width and shape of screen printing lines, different conductive materials, a specially made biological reaction enzyme system, and a siphon structure. By optimizing the overall appearance, layering and size structure of the test strip, large-scale low-cost production can be achieved. The selected raw materials are easy to obtain, and the test strip is easy to achieve large-scale and batch production.

[0100] See Figure 11 The x-axis represents glucose concentration (in mM, where mM is mmol / L), and the y-axis represents the signal value. Known glucose concentrations of 0.01 mM, 0.02 mM, 0.03 mM, 0.05 mM, 0.08 mM, and 1.0 mM were used. The linear correlation R² was as high as 0.94, Y = -8.4501420309X - 32434880648, indicating that the detection accuracy of this embodiment can reach 10 μM, and the detection range can reach 1 mM, meeting the requirements for accurate detection of micromolar glucose concentration in saliva.

[0101] See Figure 12The x-axis represents the time signal value, and the y-axis represents the current signal value. The saliva sample used was from the same period corresponding to a finger-prick blood glucose level of 8.5 mM. After eight consecutive tests, the data showed excellent consistency, verifying the stability of this embodiment.

[0102] The body fluid glucose test strip of this application embodiment can be used for glucose detection in various body fluids, such as saliva, tears, urine, and tissue fluid.

[0103] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A blood glucose test strip for bodily fluids, characterized in that, include: Substrate; An electrode layer is disposed on the substrate. The electrode layer includes a connection area for connecting an external blood glucose meter interface, a transmission area for transmitting electrical signals, and a reaction area for generating electrical signals, which are arranged sequentially. The electrode layer includes a base electrode layer disposed on the substrate. The electrode layer in the reaction area also includes a base resistance layer and a range control layer disposed on the electrode layer of the substrate. An insulating layer is disposed on the side of the electrode layer away from the substrate in the transmission region; A biological enzyme layer, located in the reaction region of the electrode layer, is used to react with glucose in body fluids to generate an electrical signal. A siphon structure is provided, which covers the bio-enzyme layer. The siphon structure includes a siphon portion for drawing body fluid into the bio-enzyme layer. The base electrode layer is a silver-silver chloride paste layer, the base resistor layer is a graphene-carbon paste layer, and the range control layer is a PEDOT:PSS layer. The silver-silver chloride paste layer contains 30% to 80% silver by mass; and / or, The graphene-carbon paste layer contains 30% to 80% graphene by mass. The thickness of the silver-silver chloride paste layer ranges from 0.001 to 0.1 mm, the thickness of the graphene-carbon paste layer ranges from 0.001 to 0.1 mm, and the thickness of the PEDOT:PSS layer ranges from 0.001 to 0.005 mm. Among them, the detection range is controlled by the range control layer to achieve a detection accuracy of 10μM, which meets the requirements for measuring the glucose content of body fluids at the micromolar level of 10-1000μM. The siphon structure includes: The first channel sheet has one end located on the side of the insulating layer away from the substrate and the other end located in the reaction region of the electrode layer. The first channel sheet has an opening groove, the opening groove is oriented away from the connection area, and the bio-enzyme layer is located in the opening groove. A first hydrophilic membrane is applied to the side of the first channel sheet away from the substrate, and the portion of the first hydrophilic membrane corresponding to the opening groove forms the siphon portion. The second channel sheet has one end located on the side of the insulating layer away from the substrate, and the other end located at the edge of the reaction region of the electrode layer; The third channel sheet is disposed in the reaction region of the electrode layer and is spaced apart from the second channel sheet; the bio-enzyme layer is located between the second channel sheet and the third channel sheet. A second hydrophilic membrane is applied to the side of the second channel sheet and the third channel sheet away from the substrate, and the siphon portion is formed on the portion of the second hydrophilic membrane corresponding to the space between the second channel sheet and the third channel sheet.

2. The body fluid glucose test strip as described in claim 1, characterized in that, The electrode layer includes a working electrode, a reference electrode, a backup electrode, a signal electrode, and a counter electrode. The working electrode, the reference electrode, the backup electrode, the signal electrode, and the counter electrode all include the base electrode layer. The portions of the working electrode, the backup electrode, and the counter electrode located in the reaction region also include the base resistance layer and the range control layer. The base resistance layer and the range control layer are disposed on the side of the base electrode layer away from the substrate.

3. A method for preparing a body fluid glucose test strip as described in claim 1 or 2, characterized in that, Includes the following steps: Prepare the substrate; The electrode layer is prepared on the substrate; The insulating layer covers the transmission region of the electrode layer; Prepare biological enzyme solutions according to the detection range and accuracy requirements; The bio-enzyme layer is formed by coating the reaction zone of the electrode layer with the bio-enzyme solution.

4. The method for preparing the body fluid glucose test strip as described in claim 3, characterized in that, The steps involve preparing a biological enzyme solution according to the required detection range and accuracy. The preparation process of the biological enzyme solution is as follows: Prepare neutral phosphate buffer solution; Dissolve glucose oxidase and peroxidase in the neutral phosphate buffer at a mass ratio of (0.45–0.8):1 to prepare a second solution with a mass concentration of 10–60 mg / ml. Graphene and potassium ferrocyanide in a mass ratio of (0.05–0.15):1 are dissolved in the second solution to obtain the bio-enzyme solution, wherein the total mass concentration of the graphene and potassium ferrocyanide in the bio-enzyme solution ranges from 15 to 50 mg / ml.

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