An electrochemical biosensor, its preparation method and application

Abstract

This application relates to the field of biosensor technology, specifically to an electrochemical biosensor, its fabrication method, and its application. The electrochemical biosensor includes a substrate layer, a conductor layer, and an insulating capping layer. The conductor layer includes a first working electrode, a counter electrode, and a second working electrode arranged sequentially at intervals. The first and second working electrodes share a counter electrode, and the center-to-center distance between the first and second working electrodes and the counter electrode is 1 mm to 3 mm. The insulating capping layer has a through-hole extending perpendicular to a first side surface, at least partially exposing the first working electrode, the counter electrode, and the second working electrode to form a reaction chamber. The counter electrode is located in the center of the reaction chamber. This application employs a structure where the working electrodes share a counter electrode to achieve dual-index detection. By setting the center-to-center distance between the counter electrode and the working electrode to 1 mm to 3 mm and placing the counter electrode in the center of the reaction chamber, the sensor achieves a balance between miniaturization and improved detection performance.

Description

Technical Field

[0001] This application relates to the field of biosensor technology, specifically to an electrochemical biosensor, its preparation method, and its application. Background Technology

[0002] Electrochemical biosensor test strips have been widely used for the point-of-care testing of biomarkers such as blood glucose, uric acid, and ketone bodies. With the increasing demand for health management among patients with chronic diseases such as diabetes and hypertension, multifunctional test strips capable of simultaneously detecting multiple indicators have become an important development direction. These test strips can obtain at least two test results with a single blood sample and sample addition, greatly improving testing efficiency and reducing patient discomfort.

[0003] Existing multi-index test strips mostly employ multiple independent electrode systems, meaning each detection index corresponds to an independent three-electrode system: a working electrode, a counter electrode, and a reference electrode. Detecting N indexes requires 3N electrodes, resulting in large test strip areas, high blood consumption, and high manufacturing costs, hindering product miniaturization. Furthermore, flawed electrode arrangement designs make them prone to electrochemical crosstalk or short circuits due to air bubbles during sample siphoning, reducing detection accuracy. In other words, the market lacks an electrochemical biosensor that can effectively control test strip area, reduce sample requirements, and ensure good consistency across multiple channels while maintaining the detection accuracy of multiple working electrodes independently detecting various biological indicators.

[0004] Based on this, how to optimize the electrode structure design of multi-parameter sensors to support multiple detection indicators while ensuring accurate simultaneous detection of multiple indicators and minimizing the test strip area as much as possible. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides an electrochemical biosensor, its preparation method, and its application.

[0006] This application provides an electrochemical biosensor, including a substrate layer, a conductor layer, and an insulating capping layer; the substrate layer has a first side and a second side disposed opposite to each other; the conductor layer is disposed on the first side of the substrate layer, and the conductor layer includes a first working electrode, a counter electrode, and a second working electrode disposed sequentially at intervals, the first working electrode and the second working electrode sharing the counter electrode, and the center distance between the first working electrode, the second working electrode and the counter electrode is 1mm-3mm; the insulating capping layer is disposed on the side of the conductor layer away from the substrate layer, and the insulating capping layer has a hole penetrating in a direction perpendicular to the first side, the hole at least partially exposing the first working electrode, the counter electrode and the second working electrode to form a reaction chamber, the counter electrode being located in the middle of the reaction chamber.

[0007] Furthermore, the thickness of the counter electrode is 10 μm to 20 μm, preferably 15 μm; the area of ​​the counter electrode is 1.5 mm² to 3.0 mm², preferably 2.0 mm². Positioning the counter electrode in the center of the reaction chamber ensures a uniform distribution of electric field lines, placing the two working electrodes in a symmetrical electrochemical environment and reducing signal deviation caused by positional differences.

[0008] Furthermore, the conductor layer also includes an impedance electrode, which is spaced apart from the first working electrode and located at the end of the first working electrode away from the counter electrode; or, the impedance electrode is spaced apart from the second working electrode and located at the end of the second working electrode away from the counter electrode; the impedance electrode and the counter electrode are connected to form an integrated common electrode, which is used as the counter electrode during electrochemical detection and as the impedance electrode during impedance detection.

[0009] Furthermore, the common electrode is in a non-enclosed, circular shape, and the first or second working electrode is at least partially disposed within the circular area.

[0010] Furthermore, the common electrode includes a first electrode element and a second electrode element spaced apart, and a third electrode element connected between the first electrode element and the second electrode element. The first electrode element is disposed between the first working electrode and the second working electrode. The second electrode element is disposed at the end of the first working electrode or the second working electrode away from the first electrode element. The third electrode element is disposed perpendicular to the first electrode element and the second electrode element. The center distance between any two adjacent electrode components in the first working electrode, the second working electrode, the first electrode element, and the second electrode element is 1mm-3mm.

[0011] Furthermore, the width of the counter electrode is 0.4-0.8 mm; the width of the impedance electrode is 0.2-0.4 mm; the width of the first working electrode is 0.2-0.8 mm; and the width of the second working electrode is 0.2-1.2 mm.

[0012] Furthermore, the impedance electrode includes a first impedance electrode and a second impedance electrode. The first impedance electrode is located at the inlet end of the reaction chamber and is arranged in pairs with the second impedance electrode. The second impedance electrode is disposed between the counter electrode and the first impedance electrode.

[0013] The distance between the first impedance electrode and the second impedance electrode is 0.2 to 0.5 mm; preferably 0.3 mm.

[0014] The thickness of the first impedance electrode and the second impedance electrode is 10-20 μm; preferably 15 μm.

[0015] The area of ​​the first impedance electrode and the second impedance electrode is 0.3 to 0.6 mm²; preferably 0.4 mm².

[0016] This application further designs the spacing, thickness, and area of ​​the impedance electrodes. On the one hand, it integrates three major functions: sample filling detection, sample quality assessment, and electrochemical detection auxiliary correction. On the other hand, it works in coordination with the counter electrode system, significantly improving the intelligence and measurement reliability of the sensor without adding additional lead channels. Furthermore, it makes the structure as compact as possible, reduces the test strip area, and further achieves an extremely compact layout, reducing the test strip area and blood consumption.

[0017] The first and second impedance electrodes form an impedance detection circuit used for sample introduction detection, background impedance measurement, and post-reaction impedance measurement. The two impedance electrodes typically use the same material and printing process to ensure detection symmetry. The second impedance electrode also functions as a "grounding" node: when a detection voltage is applied, this electrode is grounded to provide a reference potential for the entire measurement system.

[0018] Furthermore, the length from the injection port to the injection electrode is 2-5 mm, preferably 3 mm, to ensure that the sample quickly fills the cavity under capillary action, while minimizing the blood volume (approximately 1.0-1.5 μL).

[0019] The electrochemical biosensor of this application has a length of 30-35 mm and a width of 7-7.5 mm, which meets the requirements for miniaturization.

[0020] Furthermore, the material of each lead in the electrode lead area is conductive silver paste; the materials of the sample injection electrode, the first working electrode, the common counter electrode, the second working electrode, and the impedance electrode are all carbon paste.

[0021] Furthermore, it also includes a hydrophilic cover sheet, which is attached to the side of the insulating cover layer away from the conductor layer. The hydrophilic cover sheet is grooved to fit with the reaction chamber to form a capillary reaction chamber. The hydrophilic cover sheet is connected to the insulating cover layer by a double-sided adhesive layer.

[0022] The reaction chamber is connected to the outside via the inlet. Liquid samples are introduced into the reaction chamber through capillary action, simultaneously covering all electrodes. The relative positions of the electrodes within the reaction chamber are arranged as follows: the impedance electrodes are positioned close to the inlet, the sample-supplementing detection electrode is located at the rear of the injection channel, and the counter electrode is close to the two working electrodes, ensuring that the sample contacts each electrode area sequentially after entering and ultimately covers all working areas simultaneously.

[0023] In this application, the first and second working electrodes are circuitically independent, connected to different electrode leads and measurement channels respectively. The counter electrode serves as a common terminal, simultaneously connected to both measurement channels or switched via an analog switch. The first and second working electrodes are circuitically independent, connected to different electrode leads and measurement channels respectively. The two working electrodes have their own independent signal acquisition channels, without interference, but they share the same counter electrode as a common terminal for the electrochemical reaction. Specifically, the first working electrode is connected to the instrument's first measurement channel via a first electrode lead, and the second working electrode is connected to the instrument's second measurement channel via a second electrode lead. The counter electrode is simultaneously connected to the common reference terminal of both measurement channels. During detection, the instrument ensures that different potentials can be applied to the two channels simultaneously and current signals can be independently acquired through timing control of the analog switch, while the counter electrode always serves as a common terminal grounded or connected to a reference potential. This design ensures circuit isolation while reducing the space occupied by a counter electrode.

[0024] Furthermore, the first working electrode is used to detect blood glucose, and the second working electrode is used to detect blood ketones.

[0025] Furthermore, the center-to-center distance between the first and second working electrodes has a significant impact on the mutual interference of dual-path detection. Too small a distance may increase capacitive coupling, while too large a distance may lead to differences in sample diffusion. The center-to-center distance between the first and second working electrodes is 2mm to 6mm; preferably 3mm to 5mm; preferred values ​​include, but are not limited to, 2mm, 3mm, 3.5mm, 4mm, 5mm, and 6mm; more preferred values ​​are 3mm, 3.5mm, and 4mm; even more preferred is 3.5mm.

[0026] Furthermore, since the linear range of blood ketone concentration testing is relatively low (0–8 mmol / L), in order to enhance the signal, the area of ​​the second working electrode is larger than that of the first working electrode, and the area ratio of the first working electrode to the second working electrode is 1:1 to 1:4, preferably 1:2; this ensures that the two signal intensities are comparable at typical concentrations, which facilitates subsequent circuit processing.

[0027] Considering the wide linear range of blood glucose concentration testing (0–33.3 mmol / L), the area of ​​the first working electrode is designed to be 0.6 mm²–1.0 mm², preferably 0.8 mm². The area of ​​the second working electrode is 0.8 mm²–2.0 mm², preferably 1.6 mm².

[0028] Furthermore, the thickness of the first working electrode and the second working electrode is 10μm to 20μm.

[0029] Furthermore, the substrate layer is one or more of polyethylene terephthalate (PET), polycarbonate (PC), polyimide (PI), polyvinyl chloride (PVC), and fiberglass board (FR-4); the thickness of the substrate layer is 0.2 to 0.5 mm.

[0030] Preferably, the base layer is made of polyethylene terephthalate (PET) material, and the thickness of the base layer is 0.35 mm.

[0031] The selection of the substrate material and the design of the thickness parameters in this application enable its mechanical flexibility and rigidity to be adjusted to suit different application scenarios.

[0032] Furthermore, the conductor layer includes independent electrode leads, each connected to a subsequent electrode, with a lead width of 0.3–0.6 mm, preferably 0.4 mm, to ensure low resistance and printability.

[0033] Furthermore, the conductor layer also includes a sample filling detection electrode located at the end of the sample inlet of the reaction chamber. This electrode works in conjunction with the counter electrode to perform the sample filling detection process: when the sample is detected to be introduced, a voltage is applied to the electrode and the change in current or impedance is monitored to determine whether the sample has completely filled the reaction chamber.

[0034] The sample injection satisfies that the thickness of the detection electrode is 10-20 μm, preferably 15 μm; The sample injection satisfies that the area of ​​the detection electrode is 0.4 to 1.0 mm², preferably 0.6 mm².

[0035] The sample introduction in this application meets the design requirements of the detection electrode thickness and area, enabling it to generate sufficient detection signals without occupying too much cavity area or affecting the electric field distribution of the main detection area, thus achieving a balance between signal strength and occupied area.

[0036] Furthermore, the insulating covering layer is a UV-curable insulating ink; the insulating covering layer covers the area except for the electrode area and the lead end contact; the thickness of the insulating covering layer is 12-14 μm.

[0037] The insulating coating of this application has the advantages of rapid curing, no ink diffusion, and chemical resistance.

[0038] On the other hand, this application provides a method for preparing an electrochemical biosensor, comprising the following steps: Preparation of conductor layer: The conductor layer is formed on the substrate layer by screen printing technology. The printing and drying temperature is 100-120℃, preferably 110-115℃; the drying time is 8-15 minutes, preferably 10 minutes. Forming a reaction chamber: An insulating covering layer is provided on the conductor layer and the electrode area is exposed through openings to form a reaction chamber; Bio-modification of working electrodes: Different biometric elements are respectively covered on the first working electrode and the second working electrode using screen printing technology.

[0039] Furthermore, the first working electrode is used to detect blood glucose, and the second working electrode is used to detect blood ketones; a solution containing glucose dehydrogenase is applied to the first working electrode for drying at a temperature of 60-70°C for 15-20 minutes; a solution containing β-hydroxybutyrate dehydrogenase is applied to the second working electrode for drying at a temperature of 45-55°C for 30-50 minutes.

[0040] Thirdly, this application provides an application of an electrochemical biosensor that can be used to simultaneously detect the content of glucose and β-hydroxybutyric acid in a sample.

[0041] The beneficial effects of this application are as follows: This application's electrochemical biosensor employs a shared counter electrode structure for the working electrode to achieve dual-index detection. The center-to-center spacing of the counter electrode and the working electrode is designed to be 1mm-3mm, with the counter electrode positioned in the center of the reaction chamber. This multi-factor synergy achieves a balance between miniaturization and improved detection performance. The sensor's structural design significantly reduces the test strip area, decreases sample volume, and ensures highly consistent reaction conditions for both working electrodes, thus improving the accuracy of dual-index detection. The counter electrode is located at the geometric and electric field center of the chamber, and the equal distances from both working electrodes to the counter electrode result in symmetrical electric field intensity distribution, symmetrical reactant diffusion paths, comparable solution resistance, and similar double-layer charging currents. This further minimizes the differences in sensitivity, linear range, and response current values ​​between the two working electrodes. The spacing design of the two working electrodes effectively avoids crosstalk and short-circuit risks, improving reliability. Attached Figure Description

[0042] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the structure of the first electrochemical biosensor in this application; Figure 2 for Figure 1 Schematic diagram of the structure of the intermediate reaction chamber; Figure 3 This is a schematic diagram of the structure of the second type of electrochemical biosensor in this application; Figure 4 This is a schematic diagram of a common electrode structure in this application; Figure 5This is a schematic diagram of another common electrode structure in this application; Figure 6 This is a schematic diagram of the structure of the third type of electrochemical biosensor in this application; Figure 7 This is a schematic diagram of the structure of the fourth type of electrochemical biosensor in this application; Figure 8 This is a schematic diagram of the capillary reaction chamber in this application; Figure 9 The current response curve for glucose content; Figure 10 The current response curve is shown for the β-hydroxybutyric acid content.

[0043] in: 1. Substrate layer, 2. Conductor layer, 21. First working electrode, 22. Counter electrode, 23. Second working electrode, 3. Insulating cover layer, 4. Reaction chamber, 41. Capillary reaction chamber, 5. Impedance electrode, 51. First impedance electrode, 52. Second impedance electrode, 6. Common electrode, 61. First electrode element, 62. Second electrode element, 63. Third electrode element, 7. Sample injection and detection electrode, 8. Biomodified layer, 9. Hydrophilic cover plate. Detailed Implementation

[0044] Unless otherwise specified, all reagents and instruments used in the following examples are commercially available products.

[0045] This embodiment provides an electrochemical biosensor, such as Figure 1 , Figure 2 As shown, the system includes a base layer 1, a conductor layer 2, and an insulating cover layer 3. The base layer 1 has a first side and a second side disposed opposite to each other. The conductor layer 2 is disposed on the first side of the base layer 1 and includes a first working electrode 21, a counter electrode 22, and a second working electrode 23 disposed sequentially at intervals. The first working electrode 21 and the second working electrode 23 share the counter electrode 22. The center distance between the first working electrode 21, the second working electrode 23, and the counter electrode 22 is 1mm-3mm. The insulating cover layer 3 is disposed on the side of the conductor layer 2 away from the base layer 1 and has a hole that penetrates in a direction perpendicular to the first side. The hole at least partially exposes the first working electrode 21, the counter electrode 22, and the second working electrode 23 to form a reaction chamber 4. The counter electrode 22 is located in the middle of the reaction chamber 4.

[0046] Understandable, such as Figure 1 , Figure 2 As shown, the first working electrode 21, the counter electrode 22, and the second working electrode 23 can be arranged laterally at intervals along the sample flow direction; or they can be arranged as follows: Figure 3 The samples are arranged vertically at intervals parallel to the direction of sample flow.

[0047] Understandably, the thickness of the counter electrode 22 is 10μm to 20μm, and its area is 1.5mm² to 3.0mm². By limiting the thickness and area of ​​the counter electrode, the optimal match between the charge transfer capability, surface reaction uniformity, and solution resistance of the counter electrode is achieved without expanding the reaction chamber, thereby optimizing detection sensitivity, response speed, and measurement repeatability. Simultaneously, this, combined with the centrally positioned counter electrode and the 1-3mm center-to-center spacing, ensures a high degree of symmetry between the two working electrodes in terms of electric field distribution, solution resistance, and reactant diffusion conditions.

[0048] Based on the previous embodiment, another electrochemical biosensor is provided, in which an impedance electrode 5 is added to the conductor layer 2. The impedance electrode 5 is spaced apart from the first working electrode 21 and disposed at the end of the first working electrode 21 away from the counter electrode 22; or, the impedance electrode 5 is spaced apart from the second working electrode 23 and disposed at the end of the second working electrode 23 away from the counter electrode 22. The impedance electrode 5 and the counter electrode 22 are connected to form an integrated common electrode 6, which is used as the counter electrode 22 during electrochemical detection and as the impedance electrode 5 during impedance detection. Figure 4 As shown, the impedance electrode 5 is disposed at the end of the second working electrode 23 furthest from the counter electrode 22; as Figure 5 As shown, the impedance electrode 5 is disposed at the end of the first working electrode 21 away from the counter electrode 22.

[0049] This application integrates the counter electrode and impedance electrode into a shared electrode, achieving dual-function reuse of electrochemical detection and impedance detection without increasing the total number of electrodes or the test strip area. The two detection modes switch quickly and without interference, significantly improving the sensor's multi-functional integration and sample information acquisition capabilities. The counter electrode achieves engineering-optimal values ​​of "sufficiently large" in terms of area, thickness, and spatial layout.

[0050] Understandably, the common electrode 6 is in a non-enclosed, ring-shaped configuration, with the first working electrode 21 or the second working electrode 23 between the counter electrode 22 and the impedance electrode 5 located at least partially within the ring-shaped region. This non-enclosed, ring-shaped structure further expands the effective area, allowing the counter electrode's charge transfer capacity, electric field coverage, and reactant supply capacity to surpass the requirements of the working electrode without increasing the reaction chamber or increasing or minimizing the sample volume. This eliminates the possibility of the counter electrode acting as a performance bottleneck, achieving high sensitivity, high stability, and high consistency in both electrochemical and impedance detection modes.

[0051] Understandably, the non-enclosed ring shape can be C-shaped, L-shaped, V-shaped, arc-shaped, rectangular with a notch, serpentine, etc. The non-enclosed ring layout of the shared electrode causes the alternating electric field lines to converge from all sides towards the working electrode, resulting in a more concentrated electric field intensity distribution; during impedance detection, the impedance change amplitude is larger at the same analyte concentration, thus improving sensitivity; the non-enclosed design can effectively avoid short circuits or crosstalk.

[0052] Understandably, the impedance electrode 5 includes a first impedance electrode 51 and a second impedance electrode 52, such as... Figure 6 As shown, the first impedance electrode 51 and the second impedance electrode 52 are sequentially arranged upstream of the second working electrode 23 along the sample flow direction; the second impedance electrode 52 is connected to the counter electrode 22 to form a common electrode 6.

[0053] In another embodiment, the structure of the common electrode 6 is further described, as follows: Figure 4 As shown, the common electrode 6 includes a first electrode element 61, a second electrode element 62, and a third electrode element 63 connected between the first electrode element 61 and the second electrode element 62, arranged at intervals. The first electrode element 61 is arranged between the first working electrode 21 and the second working electrode 23, the second electrode element 62 is located at the end of the second working electrode 23 away from the first electrode element 61, and the third electrode element 63 is arranged perpendicularly to the first electrode element 61 and the second electrode element 62. The second working electrode 23 is arranged perpendicularly to the third electrode element 63. The center-to-center distance between adjacent electrode components of the first working electrode 21, the first electrode element 61, the second working electrode 23, and the second electrode element 62 is 1mm-3mm. The specific vertical-parallel composite structure of the common electrode optimizes the electric field distribution, improves impedance detection resolution, and makes the structure more compact and suitable for small-area test strips.

[0054] In another embodiment, such as Figure 6 As shown, the conductor layer 2 also includes a sample introduction satisfaction detection electrode 7, located at the end of the sample inlet of the reaction chamber 4. This electrode cooperates with the counter electrode 22 for the sample introduction satisfaction detection process: when sample introduction is detected, a voltage is applied to this electrode and the current or impedance change is monitored to determine whether the sample completely fills the reaction chamber 4. Figure 3 As shown, along the sample flow direction, the sample injection satisfies the requirement that the detection electrode 7 is positioned in front of the first working electrode 21.

[0055] In another embodiment, such as Figure 7 As shown, the electrochemical biosensor also includes a biomodification layer 8, which is disposed on the side of the insulating cover layer 3 away from the conductor layer 2. Different bio-recognition elements are modified on the working electrode according to the detection requirements.

[0056] Biometric elements can be selected from any two of the following categories: (1) Enzymes: glucose oxidase (GOD), glucose dehydrogenase (GDH), hexokinase (HK), β-hydroxybutyrate dehydrogenase (β-HBDH), uricase, cholesterol oxidase (ChOx), cholesterol esterase (ChE), glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO), lactate oxidase (LOx), lactate dehydrogenase (LDH), pyruvate oxidase (PyOx), creatininase, creatinase, sarcosine oxidase (SOX), urease, glutamate dehydrogenase (GLDH), alcohol dehydrogenase (ADH), alcohol oxidase (AOx), choline oxidase, lysine oxidase, etc. (2) Cofactor-dependent enzyme system: requires coenzyme NADH / NAD + NADPH / NADP + Enzyme systems such as PQQ, for example, require NAD. + β-hydroxybutyrate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, etc.; (3) Ion-selective recognition element: used to detect electrolytes (K+). + Na + Cl - Ca² + Mg² + Li + Ionophores such as valinomycin (K) + Selectivity), crown ether compounds (Na + Selective), tri(dodecyl)methylammonium chloride (Cl) - Selective), ETH 129 (Ca²) + (selective) etc.; (4) Small molecule recognition element: a specific enzyme or molecularly imprinted polymer used to detect small molecules such as uric acid, creatinine, and urea; (5) Antibody / antigen recognition system: Antibodies (such as cTnI, NT-proBNP, PSA, CRP, etc.) or antigens used to detect specific protein markers.

[0057] Different combinations of biometric elements include, but are not limited to: Enzyme-enzyme combinations (e.g., glucose dehydrogenase + β-hydroxybutyrate dehydrogenase; glucose oxidase + uricase; cholesterol oxidase + glycerol kinase; lactate oxidase + pyruvate oxidase, etc.). Enzyme-ion carrier combination (e.g., glucose oxidase + valamicin, to achieve simultaneous measurement of blood glucose and blood potassium); Ion carrier-ion carrier combination (e.g., valamicin + ETH 129, to achieve simultaneous measurement of blood potassium and blood calcium); Enzyme-antibody combination (e.g., glucose oxidase + anti-cTnI antibody, to achieve simultaneous measurement of blood glucose and cardiac troponin); Any combination of any two different identification elements.

[0058] In another embodiment, such as Figure 7 , Figure 8 As shown, the electrochemical biosensor also includes a hydrophilic cover plate 9, which is attached to the side of the insulating cover layer 3 away from the conductor layer 2. The hydrophilic cover plate 9 is grooved to fit with the reaction chamber 4 to form a capillary reaction chamber 41. The hydrophilic cover plate 9 is connected to the insulating cover layer 3 by a double-sided adhesive layer.

[0059] Next, taking an electrochemical biosensor that simultaneously detects blood glucose and blood ketones as an example, we will conduct comparative experiments on key parameters related to the structure of the biosensor. To demonstrate the objectivity of the experimental data, electrochemical biosensors with the same structure (such as...) will be used. Figure 1 The sensor shown has its counter electrode located between the first working electrode and the second working electrode.

[0060] Firstly, the study investigated the effect of the center-to-center distance between the first and second working electrodes on the detection performance. The area of ​​the first working electrode was fixed at 0.8 mm², the area of ​​the second working electrode at 1.6 mm², and the area of ​​the counter electrode at 2.0 mm², while only the center-to-center distance between the first and second working electrodes was changed.

[0061] Test method: Venous whole blood containing glucose concentration of 10 mmol / L and β-hydroxybutyrate concentration of 2.0 mmol / L was tested. Each group was tested 10 times and the average value was taken. The following indicators were recorded: blood glucose response current (GLU), blood ketone response current (KET), blood glucose CV%, and blood ketone CV%. The test results are shown in Table 1 below.

[0062] Table 1. Influence of the center-to-center distance between the first and second working electrodes on detection performance.

[0063] The data in the table above shows that the center-to-center distance between the two working electrodes has a relatively small impact on the response current but a significant impact on precision. When the electrode distance is within the range of 3.0–4.0 mm, the response currents for blood glucose and blood ketones are stable, with CV values ​​all below 3.0%, and the CV value is lowest at a distance of 3.5 mm. When the distance increases to more than 5.0 mm, the response current decreases, and the CV value increases significantly, indicating that excessively large distances can lead to uneven sample diffusion and signal attenuation. Although performance is acceptable with a distance less than 2.0 mm, excessively small distances may increase capacitive coupling between the electrodes.

[0064] Secondly, the influence of the area ratio of the first and second working electrodes on the detection sensitivity was studied. The center-to-center distance between the first and second working electrodes was fixed at 3.5 mm, the area of ​​the counter electrode was 2.0 mm², and the area of ​​the first working electrode was kept constant at 0.8 mm². Only the area of ​​the second working electrode was changed to form different area ratios.

[0065] Test method: Venous whole blood containing glucose concentration of 10 mmol / L and β-hydroxybutyrate concentration of 2.0 mmol / L was tested. Each group was tested 10 times and the average value was taken. The test results are shown in Table 2 below.

[0066] Table 2. Influence of the area ratio of the first and second working electrodes on detection performance.

[0067] The data in the table above show that as the area of ​​the second working electrode increases, the blood ketone response current increases linearly, while the blood glucose response current remains relatively stable. The blood ketone CV value is lowest at an area ratio of 1:2, and is less than 3.6% at both 1:1.5 and 1:3. When the area ratio is less than 1:1, the blood ketone signal is too weak, with a CV value as high as 4.0%; when the area ratio is greater than 1:4, the signal is too strong, the linear range narrows, and the CV value rebounds.

[0068] Thirdly, the study investigates the combined effects of the center-to-center distance and area ratio of the first and second working electrodes. The electrode area is 2.0 mm², while the area of ​​the first working electrode remains constant at 0.8 mm². The area of ​​the second working electrode is changed to form different area ratios.

[0069] Experimental method: The following four types of samples were tested to comprehensively evaluate the performance of each group. Each group was tested 10 times and the average value was taken. The test results are shown in Table 3 below.

[0070] Sample S1 (normal concentration): glucose 10.0 mmol / L, β-hydroxybutyrate 2.0 mmol / L, hematocrit 42%; Sample S2 (hypoketosis): Glucose 5.0 mmol / L, β-hydroxybutyrate 0.2 mmol / L, hematocrit 42%; Sample S3 (hyperglycemic interference): glucose 30.0 mmol / L, β-hydroxybutyrate 0.5 mmol / L, hematocrit 42%; Sample S4 (upper limit of linear ketone levels): glucose 5.0 mmol / L, β-hydroxybutyrate 6.0 mmol / L, hematocrit 42%.

[0071] In Table 3, the interference deviation of sample S3 refers to the difference between the sensor's current response to the target substance (such as blood ketone) and the theoretically expected current in the presence of high concentrations of interfering substances (such as high blood sugar).

[0072] Calculation formula: Interference deviation = I 实测 I 理论 Specific calculation steps: First, establish a calibration curve for the target analyte in a standard sample free of interfering substances: I = k × C + b, where k is the sensitivity and b is the intercept. For a sample containing interfering substances, the concentration of the target analyte is known (e.g., blood ketone 0.5 mmol / L). Calculate the theoretical current based on the calibration curve: Itheoretical = k × 0.5 + b. Then, measure the actual current of the sample to obtain the measured current Imeasured. Subtracting the two yields the interference bias.

[0073] For Group 1, the center distance between the first and second working electrodes is 3.5 mm, and the area ratio is 1:2. The calibration curve is I = 1.1710 × C + 0.0403, R 2 =0.9921. Under sample S3, I 理论 =1.1710×0.5+0.0403=0.62580μA; I 实测 =0.64760μA, with an interference bias of +0.022μA.

[0074] For Group 2, the center distance between the first and second working electrodes is 3.0 mm, and the area ratio is 1:2. The calibration curve is I = 1.1761 × C + 0.0251, R 2 =0.9918. Under sample S3, I 理论 =1.1761×0.5+0.0251=0.61315μA; I 实测 =0.64040μA, with an interference bias of +0.027μA.

[0075] For Group 3, the center distance between the first and second working electrodes is 3.5 mm, and the area ratio is 1:1.5. The calibration curve is I = 0.8804 × C + 0.0182, R 2 =0.9919. In sample S3, I 理论 =0.8804×0.5+0.0182=0.47858μA; I 实测 =0.45840μA, with an interference bias of +0.020μA.

[0076] In Table 3, the relative current deviation (%) in sample S4 refers to the percentage deviation of the measured current from the theoretical current, which is used to evaluate the linearity of the sensor at high concentrations.

[0077]

[0078] Group 1: The center distance between the first and second working electrodes is 3.5 mm, and the area ratio is 1:2. The calibration curve is I = 1.1710 × C + 0.0403, R 2 =0.9921. In sample S4, I理论 =1.1710×6+0.0403=7.06630μA; I 实测 =6.7291μA, with a relative current deviation of -4.77%.

[0079] Group 2: The center distance between the first and second working electrodes is 3.0 mm, and the area ratio is 1:2. The calibration curve is I = 1.1761 × C + 0.0251, R 2 =0.9918. In sample S4, I 理论 =1.1761×6+0.0251=7.08170μA; I 实测 =6.65870μA, with a relative current deviation of -5.97%.

[0080] Group 3: The center distance between the first and second working electrodes is 3.5 mm, and the area ratio is 1:1.5. The calibration curve is I = 0.8804 × C + 0.0182, R 2 =0.9919. In sample S4, I 理论 =0.8804×6+0.0182=5.30060μA; I 实测 =4.95845μA, with a relative current deviation of -6.45%.

[0081] Table 3. Comprehensive Influence of Center Distance and Area Ratio of the First and Second Working Electrodes on Detection Performance

[0082] All three sensor groups performed well in samples with normal concentrations. In low ketone samples (0.2 mmol / L), the ketone current signal of group 1 was 0.28 μA with a CV value of 3.1%, significantly better than groups 2 and 3. This indicates that a center-to-center distance of 3.5 mm and an area ratio of 1:2 between the first and second working electrodes resulted in a stronger signal and better repeatability at low concentrations. Under hyperglycemic interference, the ketone current deviations of all three groups were small, and their anti-interference capabilities were comparable, meeting the expected requirements. In high ketone samples (6.0 mmol / L), groups 1 and 2 showed good linearity, with relatively smaller current deviations than group 3.

[0083] In summary, Group 1 is slightly better than the other two groups in terms of low-concentration signal intensity, detection precision, and linear range. All three groups can meet the requirements in terms of overall performance, but Group 1 has the greater advantage.

[0084] Fourthly, the effects of electrode thickness and electrode area on detection performance were studied. The center-to-center distance between the first and second working electrodes was fixed at 3.5 mm, and the areas of the first and second working electrodes remained unchanged at 0.8 mm² and 1.6 mm².

[0085] Test method: Venous whole blood containing glucose concentration of 10 mmol / L and β-hydroxybutyrate concentration of 2.0 mmol / L was tested. Each group was tested 10 times and the average value was taken. The test results are shown in Table 4 below.

[0086] Table 4. Effects of electrode thickness and area on detection performance

[0087] When the electrode thickness is 15 μm and the area is 2.0 mm², the coefficient of variation (CV) of blood glucose is the lowest (1.1%) and the CV of blood ketone is the lowest (1.5%), indicating that the detection precision is optimal under this condition. Although the response current is not the highest, the coefficient of variation is a key indicator for measuring the reliability of the sensor, indicating that the electron transmission efficiency is the best and the electric field distribution is the most uniform under this combination, which are the optimal parameters.

[0088] When the thickness is 10 μm and the area is 3.0 mm², both the response current and CV value are within acceptable ranges. Performance decreases slightly when the thickness is 20 μm and the area is 1.5 mm², but remains acceptable. Outside the preferred range: excessively thin thickness (5 μm) and excessively large area (3.5 mm²) lead to increased resistance, lower current, and higher CV value; excessively thick thickness (25 μm) and excessively small area (1.0 mm²) result in decreased current and increased CV value due to electrode material redundancy and edge effects. Therefore, the acceptable range for the counter electrode thickness is 10–20 μm, preferably 15 μm; the acceptable range for the counter electrode area is 1.5–3.0 mm², preferably 2.0 mm².

[0089] Fifthly, the study investigated the effect of using different materials for the counter electrode and working electrode on the detection performance. The center-to-center distance between the first and second working electrodes was fixed at 3.5 mm, the area of ​​the first working electrode was 0.8 mm², the area of ​​the second working electrode was 1.6 mm², and the area of ​​the counter electrode was 2.0 mm².

[0090] Test method: Venous whole blood containing 10 mmol / L glucose and 2.0 mmol / L β-hydroxybutyrate was tested. Each group was tested 10 times, and the average value was taken. The sensor was subjected to accelerated treatment at 75℃ for three days. The test results are shown in Table 5 below.

[0091] The formula for calculating the rate of change of current in the table is as follows:

[0092] Where I 新鲜 For the unaged response current, I 老化后 The response current after accelerated aging at 75℃ for 3 days.

[0093] Table 5. Influence of Electrode Material on Detection Performance

[0094] Analysis of the data in the table above shows that all three material combinations can achieve dual-parameter detection. For material B (silver paste counter electrode), the stability decreases rapidly due to the easy oxidation of silver paste during storage. Material C (gold paste working electrode) has a stronger signal and a lower CV value, but gold paste is more expensive and suitable for high-end products. Material A (carbon paste) achieves the best balance between performance and cost, and has excellent stability, making it the best choice. Alternative materials (silver paste, gold paste) are also acceptable in specific application scenarios.

[0095] In another embodiment, a method for preparing an electrochemical biosensor is provided, comprising the following steps: Preparation of conductor layer: The conductor layer is formed on the substrate layer by screen printing technology. After printing, it is dried and cured. The drying temperature is 100-120℃ and the drying time is 8-15 minutes. Forming a reaction chamber: An insulating covering layer is provided on the conductor layer and the electrode area is exposed through openings to form a reaction chamber; Biomodification of working electrodes: Different bio-recognition elements are respectively covered on the first working electrode and the second working electrode.

[0096] Understandably, the electrode area of ​​the reaction chamber can be treated with a hydrophilic coating, or the area around the reaction chamber can be treated with a hydrophobic coating; both can be done using conventional methods.

[0097] Understandably, the biomodification of the working electrode can be performed using methods such as electrochemical deposition, inkjet printing, dotting, screen printing, or microcontact printing.

[0098] By establishing standardized manufacturing processes, the same production line can flexibly switch to produce sensors with different combinations of performance indicators. Among them, the screen printing and drying process for the conductor layer can fully solidify the conductive paste, forming a low-resistance, high-adhesion electrode. Under this process, the electrode edges are clear and there is no diffusion halo.

[0099] Next, the drying temperature and drying time in the preparation of the conductor layer were tested as follows: PET film with a thickness of 0.35 mm was cut into 10 cm * 10 cm sheets, 10 mL of conductive carbon paste was poured onto one end of the screen, and the conductive carbon paste was scraped onto the screen in one go with a scraper. The screen was lifted and the printed substrate was taken out. The printed substrate was placed on the platform and leveled at room temperature for 5 minutes before the drying experiment was carried out. The experimental conditions and experimental results are shown in Table 6 below. Among them, the test items are: (1) Electrode thickness was measured by a thickness gauge. Five measurement points (center and four corners) were selected in the electrode area and the average value was taken. (2) Resistivity was measured by a four-probe tester. Five positions were measured for each sample and the average value was taken. (3) Adhesion test: According to ISO 2409:2020 and ASTM D3359 standards, a 6×6 grid was drawn on the electrode surface using a cross-cutting knife (1 mm spacing). After being pasted with tape, it was quickly peeled off at a 180° angle. The area of ​​peeling was observed under a magnifying glass and rated according to 0-5. Level 0 indicates no detachment, and level 5 indicates severe detachment.

[0100] Table 6. Effect of drying conditions on conductor layer properties

[0101] The data in the table above shows that if the temperature is too low, the solvent evaporates slowly, the resin cures incompletely, and the slurry does not shrink sufficiently, resulting in a larger electrode thickness. Conversely, if the temperature is too high, the solvent evaporates rapidly, the substrate shrinks, and the resin undergoes excessive cross-linking, resulting in a smaller electrode thickness. Too low a temperature (90℃) or too short a time (8 min) leads to insufficient solvent evaporation, resulting in a porous electrode with high resistivity and poor adhesion. Too high a temperature (125℃) or too long a time (30 min) easily causes excessive cross-linking or degradation of the carbon paste resin, making the electrode brittle and reducing adhesion. A temperature of 110℃ and a time of 10 min yields a dense and uniform electrode film with the lowest resistivity, achieving optimal adhesion.

[0102] Another embodiment provides a method for preparing a biosensor that can simultaneously detect blood glucose and blood ketones. The only difference from the preparation method in the previous embodiment is the biomodification step of the working electrode. Specifically, after covering the first working electrode with a reaction solution containing glucose dehydrogenase, a drying step is performed at a temperature of 60-70°C for 15-20 minutes.

[0103] After covering the second working electrode with the reaction solution containing β-hydroxybutyrate dehydrogenase, a drying step is performed at a temperature of 45-55℃ for 30-50 minutes.

[0104] The bio-modification process matches the thermal stability of different biorecognition elements, improves enzyme activity retention and thus enhances detection sensitivity; the drying process reduces enzyme diffusion, allowing the enzyme solution to form a film rapidly in the electrode area without diffusing into the surrounding insulating area, and prevents cross-contamination between different working electrodes, thus defining the modification area.

[0105] Understandably, solutions containing glucose dehydrogenase and solutions containing β-hydroxybutyrate dehydrogenase can use conventional reaction solutions.

[0106] To demonstrate the experimental results, the solutions used in this application, specifically those containing glucose dehydrogenase, primarily comprise the following components by mass percentage: 2.1% adenine flavin nucleoside glucose dehydrogenase (FAD-GDH), 9.3% hexaammonium trichloride ruthenium chloride, 0.8% polyethylene glycol octylphenyl ether, and 4.2% maltitol. The solutions containing β-hydroxybutyrate dehydrogenase primarily comprise the following components by mass percentage: 0.7% β-hydroxybutyrate dehydrogenase, 0.4% coenzyme NAD, and 0.4% hexaammonium trichloride ruthenium chloride.

[0107] Next, the effects of the different drying conditions mentioned above on the performance of the enzyme-modified electrode were studied, with the drying temperature being 110℃ and the drying time being 10 minutes during the preparation of the conductor layer.

[0108] Firstly, the drying temperature of the second working electrode (WE2, carbon electrode, with a β-hydroxybutyrate dehydrogenase solution application volume of 0.2 μL / mm², where mm² refers to the electrode area) was fixed at 50°C and the drying time was 40 minutes, while only the drying temperature and time of the first working electrode (WE1, carbon electrode, with a glucose dehydrogenase solution application volume of 0.2 μL / mm²) were changed.

[0109] Test method: Venous whole blood with a glucose concentration of 10 mmol / L was tested. Each group was tested 10 times and the average value was taken. The experimental results are shown in Table 7 below.

[0110] Table 7. Effect of drying conditions of the first working electrode on detection performance

[0111] Based on the experimental results of drying conditions for the first working electrode (WE1, glucose dehydrogenase), the highest blood glucose response current (3.62 μA) and the lowest coefficient of variation (2.3%) were observed at a drying temperature of 65℃ and a drying time of 15 minutes, indicating uniform and dense enzyme membrane drying, which represents the optimal process parameters. Under the conditions of 60℃ for 15–20 minutes and 65℃ for 20 minutes, the response current was 3.12–3.58 μA, with a CV value of 2.5%–3.7%, indicating uniform enzyme membrane drying and acceptable performance. At 70℃ for 15 minutes... At the initial drying time, the response current was 3.5 μA and the CV was 2.8%, indicating that the enzyme membrane remained uniformly dry, which was within the acceptable range. At 70℃ for 20 minutes, slight cracking occurred, with a current of 3.4 μA and a CV of 3.5%, showing a slight decrease in performance but still usable. Beyond the acceptable range, at 50℃ for 25 minutes, the enzyme membrane remained moist and not completely dry, with a current of only 3.05 μA and a CV as high as 4.8%. At 80℃ for 10 minutes, the enzyme membrane cracked severely and peeled off, with the current dropping to 2.85 μA and the CV reaching 6.2%, both of which were unacceptable. Therefore, the acceptable drying temperature range for WE1 is 60–70℃, and the acceptable drying time range is 15–20 minutes, with the optimal range being 65℃ for 15 minutes.

[0112] Secondly, the drying temperature of the first working electrode (WE1, carbon electrode, glucose dehydrogenase solution dispensing volume of 0.2 μL / mm²) was fixed at 65℃ and the drying time was 15 minutes. Only the drying temperature and time of the second working electrode (WE2, carbon electrode, β-hydroxybutyrate dehydrogenase solution dispensing volume of 0.2 μL / mm²) were changed. The test was repeated 10 times for each group and the average value was taken. The experimental results are shown in Table 8 below.

[0113] Table 8. Effect of drying conditions on detection performance of the second working electrode

[0114] Based on the experimental results of drying conditions for the second working electrode (WE2, β-hydroxybutyrate dehydrogenase), the highest blood ketone response current (0.45 μA) and the lowest coefficient of variation (3.1%) were observed at a drying temperature of 50℃ and a time of 40 minutes, indicating uniform and dense enzyme membrane drying, representing the optimal process parameters. Within the range of 45–55℃ and 30–50 minutes, the response current was 0.40–0.44 μA, with a CV value of 3.3%–4.2%, indicating uniform enzyme membrane drying and acceptable performance. Outside of these acceptable ranges, at 40℃ and 50 minutes, the enzyme membrane remained moist and not completely dried, resulting in a current of only 0.32 μA and a CV as high as 6.5%; at 60℃ and 30 minutes, the enzyme membrane cracked and peeled off, with a current of 0.35 μA and a CV of 5.2%, both unacceptable. Therefore, the acceptable drying temperature range for WE2 is 45–55℃, and the acceptable drying time range is 30–50 minutes, with the optimal range being 50℃ and 40 minutes.

[0115] Next, the current response curves of the electrochemical biosensor under different concentration combinations were verified (covering blood glucose 0-800 mg / dL, blood ketones 0-9 mmol / L, and intra-batch precision). To demonstrate the experimental results, the preparation method of the electrochemical biosensor is as follows: Preparation of conductor layer: The conductor layer is formed on the substrate layer by screen printing technology. The electrode material in the conductor layer is conductive carbon paste. After printing, it is dried and cured at 110℃ for 10 minutes. The center distance between the first working electrode and the second working electrode is 3.5mm. The area of ​​the counter electrode is 2mm², the area of ​​the first working electrode is 0.8mm², and the area of ​​the second working electrode is 1.6mm².

[0116] Forming a reaction chamber: An insulating covering layer is provided on the conductor layer and the electrode area is exposed through openings to form a reaction chamber; Biomodification of the working electrode: A solution containing glucose dehydrogenase was applied to the first working electrode for drying at a concentration of 0.25 μL / mm², a drying temperature of 65 °C, and a drying time of 17 minutes; a solution containing β-hydroxybutyrate dehydrogenase was applied to the second working electrode for drying at a concentration of 0.25 μL / mm², a drying temperature of 50 °C, and a drying time of 40 minutes.

[0117] Reaction chamber assembly: A capillary reaction chamber is formed by bonding a double-sided adhesive layer and a hydrophilic cover.

[0118] The obtained electrochemical biosensor was tested, and its structure is as follows: Figure 7 As shown, the test was conducted at room temperature using a multi-channel electrochemical detector. The key steps are as follows: Venous blood samples containing different concentrations of glucose and β-hydroxybutyric acid (hematocrit 42%) were added. Each concentration combination was tested 10 times for intra-batch CV calculation. Each time, 1.0 μL of the test solution was aspirated and the test solution was automatically drawn into the sensor inlet using capillary action.

[0119] Sensor parameter settings: first working electrode voltage 0.3V, second working electrode voltage 0.25V, detection time 8S.

[0120] Figure 9 , Figure 10 The current response curves for different concentration combinations are shown below. The first working electrode current showed good linearity with glucose concentration in the range of 0-800 mg / dL, with R²=0.9981; the second working electrode current showed good linearity with β-hydroxybutyrate concentration in the range of 0-9 mmol / L, with R²=0.9921; intra-assay CV%: blood glucose 1.8%-3.2%, blood ketones 2.5%-4.1%.

[0121] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0122] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An electrochemical biosensor, characterized in that, include: A base layer having a first side and a second side disposed opposite to each other; A conductor layer is disposed on the first side surface. The conductor layer includes a first working electrode, a counter electrode, and a second working electrode arranged sequentially at intervals. The first working electrode and the second working electrode share the counter electrode. The center-to-center distance between the first working electrode, the second working electrode, and the counter electrode is 1mm-3mm. An insulating cover layer is disposed on the side of the conductor layer away from the substrate layer. The insulating cover layer has a hole that extends through the first side in a direction perpendicular to the first side. The hole at least partially exposes the first working electrode, the counter electrode, and the second working electrode to form a reaction chamber. The counter electrode is located in the middle of the reaction chamber.

2. The electrochemical biosensor according to claim 1, characterized in that, The thickness of the counter electrode is 10μm-20μm; the area of ​​the counter electrode is 1.5mm²-3.0mm².

3. The electrochemical biosensor according to claim 1, characterized in that, The conductor layer further includes an impedance electrode, which is spaced apart from the first working electrode and located at the end of the first working electrode away from the counter electrode; or, The impedance electrode is spaced apart from the second working electrode and is located at the end of the second working electrode away from the counter electrode; The impedance electrode is connected to the counter electrode to form an integrated common electrode. The common electrode is used as the counter electrode during electrochemical detection and as the impedance electrode during impedance detection.

4. An electrochemical biosensor according to claim 3, characterized in that, The common electrode is in a non-enclosed, circular shape, and the first working electrode or the second working electrode is at least partially disposed within the circular area.

5. An electrochemical biosensor according to claim 4, characterized in that, The common electrode includes a first electrode element and a second electrode element arranged at intervals, and a third electrode element connected between the first electrode element and the second electrode element. The first electrode element is disposed between the first working electrode and the second working electrode. The second electrode element is disposed at the end of the first working electrode or the second working electrode away from the first electrode element. The third electrode element is arranged perpendicular to the first electrode element and the second electrode element. The center distance between any two adjacent electrode components in the first working electrode, the second working electrode, the first electrode element, and the second electrode element is 1mm-3mm.

6. An electrochemical biosensor according to claim 1, characterized in that, The first working electrode is used to detect blood glucose, and the second working electrode is used to detect blood ketones.

7. An electrochemical biosensor according to claim 6, characterized in that, The area ratio of the first working electrode to the second working electrode is 1:1 to 1:4; the area of ​​the first working electrode is 0.6 mm²-1.0 mm², and the area of ​​the second working electrode is 0.8 mm²-2.0 mm².

8. The method for preparing the electrochemical biosensor according to any one of claims 1-7, characterized in that, Includes the following steps: Preparation of conductor layer: The conductor layer is formed on the substrate layer by screen printing technology. The printing and drying temperature is 100-120℃ and the drying time is 8-15 minutes. Forming a reaction chamber: An insulating covering layer is provided on the conductor layer and the electrode area is exposed through openings to form a reaction chamber; Bio-modification of working electrodes: Different biometric elements are respectively covered on the first working electrode and the second working electrode using screen printing technology.

9. The method for preparing an electrochemical biosensor according to claim 8, characterized in that, After covering the first working electrode with the reaction solution containing glucose dehydrogenase, a drying step is performed at a temperature of 60-70℃ for 15-20 minutes. After covering the second working electrode with the reaction solution containing β-hydroxybutyrate dehydrogenase, a drying step is performed at a temperature of 45-55℃ for 30-50 minutes.

10. The application of an electrochemical biosensor as described in any one of claims 1-7 or an electrochemical biosensor prepared according to any one of claims 8-9, characterized in that: Used for simultaneous detection of glucose and β-hydroxybutyric acid content in samples.

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