Biosensor for eliminating interference of electroactive interferent

Abstract

The invention relates to a biosensor which comprises a first working electrode, a second working electrode and a reference electrode, the voltage difference value between the first working electrode and the reference electrode is a first voltage, the voltage difference value between the second working electrode and the reference electrode is a second voltage, and the first voltage is higher than the second voltage.

Description

Technical Field

[0001] This application relates to the field of biosensors, and more specifically to a biosensor capable of eliminating interference from electroactive interfering substances. Background Technology

[0002] A biosensor is an analytical device that tightly integrates biological materials, bio-derived materials, or biomimetic materials with physicochemical sensors or sensing microsystems based on optics, electrochemistry, temperature, piezoelectricity, magnetism, or micromechanics. It is typically used to rapidly detect the concentration of specific chemical substances in test solutions or human tissue fluids, such as glucose, lactic acid, urea, uric acid, cholesterol, and a range of amino acid compounds.

[0003] Taking implantable current sensors as an example, they primarily utilize the electrochemical oxidation-reduction properties of the detected substance. Typically, under a certain voltage, they detect the products of enzyme-catalyzed reactions (such as hydrogen peroxide) to indirectly determine the concentration of the detected substance. The working electrode usually includes a sensing layer in direct contact with the electrode conductive layer. The detected substance, on the surface of the electrode sensing layer, undergoes an enzyme-catalyzed oxidation-reduction reaction, generating a corresponding current. When the detected substance reaches the surface of the sensing layer and is consumed, there is a certain linear relationship between the detected current value and the concentration of the chemical substance. However, this linear relationship can be affected by interference from other biomolecules in the environment. Generally, based on their mechanism of action, molecules can be divided into electrochemically active molecules and non-electrochemically active molecules.

[0004] Electrochemically active molecules are those that undergo oxidation reactions when a high oxidation potential is applied to the working electrode, generating interfering currents and affecting the accuracy of the detection of the analyte. Non-electrochemically active interfering molecules do not possess the above characteristics; they generally have similar structures or properties to the substrate, causing the molecular recognition element in the sensor to mistakenly identify them as the substrate, thus affecting measurement accuracy.

[0005] Currently, most commercially available bioanalytical sensors employ molecular recognition elements. Because the analytical enzymes used (such as glucose oxidase and lactase) have strong specificity and do not respond to structures other than the analyte, interference from non-electrochemically active biomolecules can be eliminated. To eliminate interference from electrochemically active molecules, the most common methods are as follows: First, using a selective membrane on the sensor. These membranes typically filter and regulate the diffusion flux of molecules and ions participating in the reaction based on size, charge, and polarity, effectively limiting the entry of interfering molecules. Second, lowering the oxidation potential of the working electrode to prevent the oxidation of interfering substances on the electrode and the generation of interfering currents. Third, applying the same voltage to the two working electrodes in a four-electrode system using a potentiostat to achieve interference subtraction.

[0006] However, the first and second methods mentioned above both affect the response signal of the sensor's main reaction to some extent, limiting their practicality. The third method uses dual working electrodes, but practical experience has shown that when the same voltage is applied to both electrodes, due to competing reactions, the electron mediator on the electrode without the added analytical enzyme reacts with electroactive interfering substances, generating an additional current signal. This leads to a partial deviation in the response current deduction, thus failing to completely eliminate the influence of electroactive interfering substances. Examples of electroactive interfering substances include ascorbic acid, uric acid, and hydroxyurea. Therefore, a new method needs to be developed that can eliminate the interference of electroactive interfering substances without affecting the main reaction of the detected substance, thereby improving the accuracy of the test results. Summary of the Invention

[0007] This application is made in view of the problems existing in the prior art, and aims to provide a biosensor that can eliminate the influence of electroactive interfering substances.

[0008] To achieve the above objectives, a first aspect of this application provides a biosensor comprising a first working electrode, a second working electrode, and a reference electrode, wherein the voltage difference between the first working electrode and the reference electrode is a first voltage, the voltage difference between the second working electrode and the reference electrode is a second voltage, and the first voltage is higher than the second voltage.

[0009] According to the design of this application, by using a sensor with dual working electrodes, different voltages are applied to the two working electrodes, and the current signals of the two working electrodes are subtracted by an algorithm to finally obtain a current signal that only the detected substance responds to, thus eliminating the interference of other electroactive interfering substances and making the detection results more accurate.

[0010] In some preferred embodiments, the first voltage is at least 0.03V higher than the second voltage. This reduces the impact of electroactive interferences.

[0011] In some preferred embodiments, the biosensor further includes a substrate, with the first working electrode and the second working electrode located on opposite sides of the substrate. This prevents the two electrodes from interfering with each other during operation, resulting in more accurate analysis results.

[0012] In some preferred embodiments, the biosensor further includes a polymer film layer that at least covers the surfaces of the first and second working electrodes. This improves the selectivity and biocompatibility of the biosensor for the detected substance.

[0013] In some preferred embodiments, the first working electrode is an enzyme electrode containing an immobilized enzyme layer, while the second working electrode does not contain an immobilized enzyme layer. Therefore, by offsetting the working current on the second working electrode (which does not contain an immobilized enzyme layer), current interference caused by electroactive interfering substances can be counteracted, improving the accuracy of the results.

[0014] In some preferred embodiments, the immobilized enzyme layer may contain one or more selected from glucose oxidase, glucose dehydrogenase, hydroxybutyrate dehydrogenase, myocardial flavin, coenzyme, and lactase. Thus, by selecting suitable enzymes for the immobilized enzyme layer based on the high specificity of the enzymes, the electrode's ability to highly identify the analyte is improved, thereby increasing the accuracy of the results.

[0015] In some preferred embodiments, the first voltage can be 0.04–0.2V, and the second voltage can be 0–0.02V. Therefore, using different voltages on the first and second working electrodes can improve analytical accuracy.

[0016] In some preferred embodiments, the first voltage can be 0.04–0.05V, and the second voltage can be 0–0.005V. Therefore, using different voltages on the first and second working electrodes can improve analytical accuracy.

[0017] In some preferred embodiments, the polymer film layer includes a vinylpyridine polymer, a crosslinking agent, and a modifier, wherein the vinylpyridine polymer has a mass fraction of 80% to 99.9% in the polymer film layer. This results in a more stable electrode structure.

[0018] In some preferred embodiments, the biosensor further includes a counter electrode, with the first working electrode and the counter electrode located on one side of the substrate, and the second working electrode and the reference electrode located on the other side of the substrate. This allows for more accurate current measurements and improves analytical accuracy.

[0019] A second aspect of this application provides an analyte monitoring method, comprising: implanting a biosensor obtained according to a first aspect of this application into the body of a subject; applying a first voltage to a first working electrode to obtain a first current signal associated with the first working electrode; applying a second voltage to a second working electrode, wherein the first voltage is higher than the second voltage, to obtain a second current signal associated with the second working electrode; calculating the difference between the first current signal and the second current signal; and obtaining the concentration of the analyte based on the obtained difference.

[0020] According to the method of this application, the current signal measured by the first working electrode (WE1) containing the fixed enzyme layer is the superposition of the current of the detected substance and the electroactive interfering substance, while the current signal measured by the second working electrode (WE2) without the fixed enzyme layer is only the current of the electroactive interfering substance. In order to reduce the influence of the electroactive interfering substance on the response of the detected substance, different voltages are used on WE1 and WE2. The different current values ​​obtained on different electrodes are subtracted by an algorithm to obtain the response current of the pure detected substance, which better eliminates the interference of the electroactive interfering substance and obtains more accurate analysis results.

[0021] A third aspect of this application provides an analyte monitoring system, including a processing device, for obtaining the concentration of the analyte using the analyte monitoring method of the second aspect of this application.

[0022] According to the monitoring system of this application, by utilizing a dual working electrode system, the substance to be tested can be stably and accurately detected in the presence of electroactive interfering substances. This avoids the shortcomings of existing technologies in effectively eliminating impurity interference, making the electrodes safe, non-toxic, stable in performance, and reusable. It also effectively eliminates the interference of coexisting inorganic and organic substances on the measurement, thereby improving the accuracy of the test results. Attached Figure Description

[0023] Figure 1a This is a front structural schematic diagram of a biosensor according to an embodiment of this application.

[0024] Figure 1b This is a schematic diagram of the back structure of a biosensor according to an embodiment of this application.

[0025] Figure 2 A partial schematic diagram of the electrode structure of a biosensor according to an embodiment of this application is shown.

[0026] Figure 3 This is a schematic diagram of the structure of the biosensor according to this application.

[0027] Figures 4a-4c The diagram shows the current signal changes in Examples 1 to 3.

[0028] Figures 5a-5c The diagram shows the changes in current signals for comparative examples 1 to 3.

[0029] Explanation of reference numerals in the attached figures

[0030] 1 Substrate; 2 Conductor layer; 3 Separating membrane; 4a First working electrode; 4b Second working electrode; 4c Counter electrode; 4d Reference electrode; 5 Micro workstation; 6 Polymer film layer. Detailed Implementation

[0031] The above-described objectives, other objectives, advantages, and features of this application will become apparent from the following description of embodiments given with reference to the accompanying drawings. However, the scope of this application is not limited to the embodiments described herein and can be implemented in various different forms. Embodiments are provided to make the description of this application more thorough and to fully convey the scope of this application to those skilled in the art.

[0032] In the following description of the embodiments, terms such as “comprising,” “including,” and “having” should be interpreted as indicating the presence of the features, numbers, steps, components, elements, or parts described herein, or combinations thereof, and do not exclude the presence of one or more other features, numbers, steps, components, elements, parts, or combinations thereof, or the possibility of adding the same features, numbers, steps, components, elements, parts, or combinations thereof.

[0033] All figures, values, and / or expressions used in this specification to represent the amounts of components, reaction conditions, polymer compositions, and blends are approximate values, reflecting the various uncertainties that arise when these values ​​are obtained from inherently different things, and therefore should be understood to be modified by the term "about" unless otherwise stated. Unless specifically stated or obvious from the context, the term "about" as used herein should be understood to mean within the normal tolerances in the field, such as within 2 standard deviations of the average. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Furthermore, it should be understood that if a numerical range is disclosed in the specification, unless otherwise stated, the range includes all consecutive values ​​from the minimum to the maximum value of the range. Additionally, if the range refers to integers, unless otherwise stated, the range includes all integers from the minimum to the maximum integer of the range.

[0034] The present application will now be described in further detail with reference to the accompanying drawings and specific embodiments. In the drawings, the same components or components with the same function are denoted by the same symbols, and repeated descriptions of them are omitted.

[0035] Current bioanalytical sensors typically employ a potentiostat to apply the same voltage to two working electrodes in a four-electrode system to subtract interference. However, due to the interaction between electroactive interfering substances and electron mediators on the electrode surface under higher voltage, some deviation occurs when the response current is superimposed, failing to accurately subtract the influence of electroactive interfering substances. Therefore, a new method is needed to eliminate the interference of electroactive interfering substances without affecting the main reaction of the analyte, thereby improving the accuracy of test results.

[0036] To achieve the above objectives, a first aspect of this application provides a biosensor. The electrode structure of the biosensor according to embodiments of this application is as follows: Figure 1a and 1bAs shown, this is the core component of the biosensor involved in this application. The biosensor can be used to acquire physiological parameter information of the host. It can be applied to the host, who can acquire their own physiological parameter information through a monitoring device applied to themselves. Typically, the biosensor can be fitted to partially implantable body surface sites such as the host's arm, back, abdomen, waist, or leg to monitor physiological parameters. For example, it can be implanted subcutaneously to acquire sensing signals (e.g., electrical signals). When the biosensor is applied to the host, it can come into contact with the host's tissue fluid or blood to measure the analyte levels in the tissue fluid or blood.

[0037] The biosensor involved in this application includes a first working electrode, a second working electrode, and a reference electrode. The voltage difference between the first working electrode and the reference electrode is a first voltage, and the voltage difference between the second working electrode and the reference electrode is a second voltage. The first voltage is higher than the second voltage.

[0038] The biosensor structure designed according to this application is as follows: Figure 1a , Figure 1b and Figure 2 As shown, by using a sensor with dual working electrodes, different voltages are applied to the two working electrodes, with the voltage applied to the first working electrode being higher than that to the second working electrode. Corresponding current signals are obtained on the two working electrodes respectively, and the two current signals are subtracted by an algorithm to finally obtain a current signal that only the detected substance responds to, thus eliminating the interference of other electroactive interfering substances and making the detection results more accurate.

[0039] In this application, the first voltage and the second voltage refer to the voltage difference between the working electrode and the reference electrode 4d, i.e., the voltage of the reference electrode 4d is assumed to be 0V. The current is mainly generated by the circuit composed of the working electrode and the counter electrode 4c, and therefore depends on the voltage difference between the working electrode and the counter electrode 4c.

[0040] In some preferred embodiments, the first voltage is at least 0.03V higher than the second voltage. The relatively low voltage at the second electrode 4b reduces the reaction between electroactive interfering substances and the electron mediator on the second electrode surface, thereby reducing interference current caused by the electroactive interfering substances and improving the accuracy of the detection results. The inventors unexpectedly discovered through experimentation that the interference signal elimination effect is optimal when the difference between the first and second voltages is at least 0.03V.

[0041] In some embodiments, the biosensor further includes a substrate, the substrate being positioned as follows: Figure 2As shown. Substrate 1 can be a flexible substrate. The flexible substrate can be made generally of at least one of polyethylene (PE), polypropylene (PP), polyimide (PI), polystyrene (PS), polyethylene terephthalate (PET), and polyethylene terephthalate (PEN). In other embodiments, the flexible substrate can also be made generally of metal foil, ultrathin glass, a single-layer inorganic film, a multilayer organic film, or a multilayer inorganic film. In some embodiments, substrate 1 can also be a non-flexible substrate. Non-flexible substrates can generally include materials with relatively low conductivity, such as ceramics, alumina, or silicon dioxide.

[0042] In some embodiments, the first working electrode and the second working electrode are located on opposite sides of the substrate. Figure 2 This figure illustrates an exemplary structure of the electrodes of a biosensor according to an embodiment of this application. As can be seen from the figure, two working electrodes 4a and 4b are located on opposite sides of the substrate 1. When the sensor is working, the electrodes on both sides perform tests independently, minimizing mutual interference and resulting in more accurate analysis results. Furthermore, distributing the electrodes on both sides of the substrate 1 saves space, reducing the substrate length.

[0043] In some embodiments, the biosensor further includes a polymer membrane layer, such as Figure 2As shown. The polymer membrane layer 6 includes a semi-permeable membrane layer and a biocompatible membrane layer. The position and structure of the semi-permeable membrane layer and the biocompatible membrane layer are not specifically limited. The selection of the semi-permeable membrane layer can improve the selective permeability of the sensor to the detected substance, thereby improving the accuracy of the detection results. Specifically, it can include a diffusion control layer to control the permeability of the detected substance and an anti-interference layer to reduce the entry of other interfering substances into the electrode surface. The diffusion control layer can reduce the amount of detected substance diffused to the working electrode surface, ensuring sufficient substances participating in the reaction of the bioenzyme layer. The concentration of the detected substance becomes the main limiting factor for the electrode current, thus ensuring that the current magnitude accurately reflects the concentration of the detected substance and significantly increasing the linear range of the biosensor. The biocompatible membrane layer can improve the biocompatibility of the biosensor. The biocompatible membrane can be made of plant materials. Plant materials can be sodium alginate, tragacanth gum, pectin, gum arabic, xanthan gum, guar gum, agar, etc., or natural material derivatives including starch derivatives, cellulose derivatives, etc. The biocompatible membrane can also be made of artificial synthetic materials. Synthetic materials can be polyolefins: povidone, polyvinyl alcohol, polyisobutylene pressure-sensitive adhesive, ethylene-vinyl acetate copolymer, etc.; polyacrylic acids: acrylic resin, carboxyethylene-sucrose, carboxyethylene-pentaerythritol copolymer, polyacrylate pressure-sensitive adhesive, etc.; polyoxyethylene: polyoxyethylene fatty acid ester, polyoxyethylene-polyoxypropylene copolymer, etc.; polyesters: polylactic acid, polyglycolic acid-lactide, polydinonyl sebacate, polycyanoalkylamino ester, polyether polyurethane, etc. This reduces the human body's immune response to the biosensor, extending its lifespan. The semi-permeable membrane can also be biocompatible. This avoids the need for biocompatible membranes, reducing manufacturing costs.

[0044] In some preferred embodiments, the polymer film layer covers at least the surfaces of the first and second working electrodes. To reduce the amount of raw materials used, the polymer film layer 6 may partially cover the surface of the biosensor. Since the electrochemical reactions detected by the biosensor occur primarily on the surfaces of the first and second working electrodes 4a and 4b, to reduce the influence of interfering substances, the polymer film layer 6 should cover at least the surfaces of the first and second working electrodes 4a and 4b.

[0045] In some embodiments, the first working electrode is an enzyme electrode containing an immobilized enzyme layer, while the second working electrode does not contain an immobilized enzyme layer. For example... Figure 2The first working electrode 4a contains an immobilized enzyme layer. The enzyme catalyzes an oxidation-reduction reaction in the analyte (e.g., glucose, ketones, or lactic acid), forming a circuit with the reference electrode 4d and the counter electrode 4c to generate a current signal indicating the concentration of the analyte. The second working electrode 4b does not contain the corresponding enzyme and does not react with the analyte. It forms a circuit with the reference electrode 4d and the counter electrode 4c, generating a current signal solely from electroactive interfering substances. By subtracting the two current signals, interference currents caused by electroactive interfering substances can be canceled, improving the accuracy of the results.

[0046] In some embodiments, the immobilized enzyme layer may contain one or more selected from glucose oxidase, glucose dehydrogenase, hydroxybutyrate dehydrogenase, myocardial flavin, coenzyme, and lactase. It is known that biological enzymes are highly specific; a specific enzyme can only react with a specific substrate and is unaffected by other substances. Since the testing environment of biosensors is generally complex, many interfering substances can generate a certain current signal on the electrode. Therefore, selecting a suitable enzyme in the immobilized enzyme layer ensures that only the target analyte reacts on the electrode surface, thereby generating a current signal. Using biological enzymes improves the high recognition performance of the working electrode for the analyte, thus improving the accuracy of the results. It is known that substances commonly analyzed include glucose, urea, uric acid, ketone bodies, creatinine, and lactic acid. Ketone bodies (which can be simply referred to as ketones) are a collective term for acetoacetic acid, β-hydroxybutyrate, and acetone, intermediate products of fatty acid oxidation and decomposition in the liver. In this application, the content of ketone bodies can be monitored by detecting any one of acetoacetic acid, β-hydroxybutyrate, and acetone. Therefore, the highly specific enzymes corresponding to these substances being tested are glucose oxidase, glucose dehydrogenase, hydroxybutyrate dehydrogenase, myocardial flavin, coenzyme, lactase, etc.

[0047] In some preferred embodiments, the content (mass fraction) of glucosase in the immobilized enzyme layer can be 40% to 70%. For example, the content (mass fraction) of glucosase can be 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%. This facilitates the reaction of glucose.

[0048] In some preferred embodiments, the content of hydroxybutyrate dehydrogenase in the immobilized enzyme layer can be 10-20%. For example, the content of hydroxybutyrate dehydrogenase can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. This promotes the redox reaction of ketones. In some preferred embodiments, the content of myocardial flavin in the immobilized enzyme layer can be 5-20%. For example, the content of myocardial flavin can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. This allows for the transfer of electrons generated by the β-BHB reaction and improves the electron transfer efficiency in the working electrode. In some preferred embodiments, the content of coenzyme in the immobilized enzyme layer can be 10-30%. For example, the coenzyme content can be 10%, 12%, 14%, 15%, 16%, 18%, 19%, 20%, 21%, 22%, 25%, 26%, 27%, 28%, 29%, or 30%. This promotes the oxidation of hydroxybutyrate dehydrogenase, thereby transferring electrons generated in the β-BHB reaction. Hydroxybutyrate dehydrogenase, myocardial flavin enzyme, and coenzymes are often used together to analyze ketone concentrations. Coenzymes include nicotinamide adenine dinucleotide, etc.

[0049] In some embodiments, the first working electrode 4a and the second working electrode 4b may further include an electron mediator. The electron mediator may be a redox polymer. In some preferred embodiments, the redox polymer may be a metal redox polymer, which can participate in redox reactions through covalent bonds, coordination bonds, or ionic bonds. In some preferred embodiments, the metal redox polymer may be selected from at least one of poly(vinylferrocene), quaternized poly(4-vinylpyridine) of ferrocyanide, quaternized poly(1-vinylimidazolium) of ferrocyanide, quaternized poly(4-vinylpyridine) of ferrocyanide, quaternized poly(1-vinylimidazolium) of osmium 2,2'-bispyridine complex coordinated to poly(1-vinylimidazolium), osmium 2,2'-bispyridine complex coordinated to poly(4-vinylpyridine), cobalt 2,2'-bispyridine complex coordinated to poly(1-vinylimidazolium), or cobalt 2,2'-bispyridine complex coordinated to poly(4-vinylpyridine). In some preferred embodiments, preferably, the metal polymer can be an osmium 2,2'-bispyridine complex coordinated to poly(4-vinylpyridine).

[0050] The primary reason for the additional interference current generated by the electroactive interfering substance is the additional side reaction between the exposed electron mediator not covered by the enzyme layer and the electroactive interfering substance on the working electrode without an immobilized enzyme layer, and it is not significantly related to the choice of enzyme type in the immobilized enzyme layer. Therefore, the biosensor involved in this application is applicable to four-electrode systems containing different immobilized enzyme layers.

[0051] In some embodiments, the first voltage can be 0.04–0.2V, and the second voltage can be 0–0.02V. Under a voltage condition of 0.04–0.2V, due to the catalytic effect of the immobilized enzyme layer, the detected substance undergoes a redox reaction on the electron mediator surface of the first working electrode 4a, resulting in the first working current I. WE1 The initial voltage should not be too high to prevent side reactions on the surface of the first working electrode 4a. By applying a higher voltage to the first working electrode 4a than to the second working electrode 4b, the analyte is ensured to react fully on the surface of the first working electrode 4a, resulting in a stronger current signal. Applying a lower voltage to the second working electrode 4b reduces the redox reaction capability of electroactive interfering substances, thereby weakening the occurrence of other side reactions between electroactive interfering substances and electron mediators on the surface of the second working electrode 4b, which does not contain a fixed enzyme layer, under higher voltage conditions, thus improving analytical accuracy.

[0052] In some preferred embodiments, the first voltage can be 0.04–0.05V, and the second voltage can be 0–0.005V. Furthermore, to avoid side reactions such as water electrolysis at the first working electrode 4a, the first voltage should not be too high, preferably not exceeding 0.05V. The difference between the first and second voltages should be at least 0.03V.

[0053] In some embodiments, the polymer membrane layer includes a vinylpyridine polymer, a crosslinking agent, and a modifier, wherein the vinylpyridine polymer comprises 80% to 99.9% by mass in the polymer membrane layer. It is known that polymer membrane layers can be configured to be permeable to the detectable substance and cover an immobilized enzyme layer; therefore, it is necessary to restrict the permeation of the detectable substance. Vinylpyridine polymers have good selective permeability and good biocompatibility, making them a preferred choice. Furthermore, the permeability coefficient of the polymer membrane layer can be adjusted by selecting the degree of polymerization, the degree of crosslinking, and regulating the modifier, thereby increasing the stability of the polymer membrane layer. Simultaneously, the amount of the detectable substance can be controlled within the linear range of the biosensor, improving the sensitivity and accuracy of the biosensor.

[0054] In some preferred embodiments, the thickness ratio of the polymer membrane layers can be adjusted to give the polymer membrane layers different permeability coefficients. In this case, by adjusting the permeability coefficient of the polymer membrane layer, the amount of analyte that permeates can be controlled, making the current generated by the working electrode more accurate, thereby making the biosensor more sensitive.

[0055] In some preferred embodiments, the crosslinking agent may be polyethylene glycol diglycidyl ether. The modifier may be a polydimethylsiloxane modifier. This improves the stability of the polymer film, thereby enhancing the performance of the biosensor.

[0056] In some preferred embodiments, the biosensor further includes a counter electrode, with the first working electrode and the counter electrode located on one side of the substrate, and the second working electrode and the reference electrode located on the other side of the substrate.

[0057] The reference electrode 4d can form a known and fixed potential difference with the tissue fluid or blood. By measuring the potential difference between the first working electrode 4a or the second working electrode 4b and the tissue fluid or blood through the potential difference formed between the reference electrode 4d and the first working electrode 4a or the second working electrode 4b, the voltage generated by the first working electrode 4a and the second working electrode 4b can be accurately determined. Therefore, the voltage generated by the first working electrode 4a or the second working electrode 4b can be obtained more accurately, and the electronic system can automatically adjust and maintain the voltage at the working electrode according to a preset voltage value, so that the measured current signal can more accurately reflect the concentration level of the analyte in the tissue fluid or blood.

[0058] Counter electrode 4c helps regulate the electrochemical reaction rate, provides additional electron transfer pathways, increases electrode recombination, maintains ion balance in the electrolyte, and improves the accuracy of experimental data. Counter electrode 4c can be made of at least one of platinum, silver, silver chloride, palladium, titanium, iridium, gold, glassy carbon, or graphite.

[0059] Typically, a first working electrode 4a, a second working electrode 4b, a counter electrode 4c, and a reference electrode 4d are disposed on the substrate 1 of the implanted portion of the biosensor. The first working electrode 4a and the counter electrode 4c are located on one side of the substrate, and the second working electrode 4b and the reference electrode 4d are located on the other side of the substrate (see [reference]). Figure 2In this case, placing the four electrodes in pairs on both sides of the substrate optimizes the voltage drop of the sensor electrodes, reduces errors, and further reduces the interference of the electric field on the sensor system. Meanwhile, since wearing comfort is related to the implantation depth, a longer substrate 1 results in a deeper implantation, which may increase wearing discomfort. Therefore, placing the four electrodes in pairs on both sides of the substrate improves substrate utilization. Setting up four electrodes within a limited substrate space reduces the substrate length, thereby reducing sensor implantation pain and improving wearing comfort.

[0060] While the biosensors disclosed herein may include a reference electrode and a counter electrode, it is not necessary to receive, reference, utilize, or otherwise process signals from the reference electrode to determine concentration values. That is, since the first and second working electrodes are individually referenced to the reference electrode, corrections for the first and second current signals are canceled when the signal difference is determined. In other words, the first and second working electrodes are internally referenced to each other. Therefore, the biosensors described herein overcome the drift problems associated with conventional biosensors by eliminating the need for signal correction using a reference electrode.

[0061] In the biosensor of this application, all electrodes are formed on a conductor layer 2, and the conductor layer 2 is isolated from each other, at least in the portion implanted under the skin. The conductor layer 2 may be made of at least one selected from gold, glassy carbon, graphite, silver, silver chloride, palladium, titanium, and iridium. In this case, the conductor layer 2 has good conductivity and can suppress electrochemical reactions, thereby improving the stability of the conductor layer 2.

[0062] A second aspect of this application provides an analyte monitoring method, comprising: implanting a biosensor obtained according to a first aspect of this application into the body of a subject; applying a first voltage to a first working electrode to obtain a first current signal associated with the first working electrode; applying a second voltage to a second working electrode, wherein the first voltage is higher than the second voltage, to obtain a second current signal associated with the second working electrode; calculating the difference between the first current signal and the second current signal; and obtaining the concentration of the analyte based on the obtained difference.

[0063] According to the method of this application, the current signal measured by the first working electrode 4a containing the immobilized enzyme layer is the superposition of the currents of the analyte and the electroactive interfering agent to generate a first current signal, while the current signal measured by the second working electrode 4b without the immobilized enzyme layer is only the current of the electroactive interfering agent to generate a second current signal. This allows the difference between the first and second current signals to be correlated with the concentration of the analyte. Furthermore, to reduce the influence of interfering agents on the response of the analyte, different voltages are applied to the first and second working electrodes 4a and 4b, resulting in corresponding current signals at different electrodes. The second current signal is subtracted from the first current signal using an algorithm, and the difference in signal magnitudes can be correlated with the concentration of the analyte. The signal difference can be calculated manually or automatically using a suitable processor. The results obtained through this method better eliminate the interference from electroactive interfering agents, leading to more accurate analytical results.

[0064] In some embodiments, the method of this application may include accessing a lookup table containing multiple concentration values ​​of the detected substance and corresponding differences between a first current signal and a second current signal to calculate the concentration of the detected substance. In other embodiments, the method of this application may include accessing a calibration curve comparing the concentration values ​​of the detected substance to the corresponding differences between the first current signal and the second current signal to calculate the concentration of the detected substance.

[0065] According to some implementations, the first current signal and the second current signal may each contain a peak volt-ampere voltage. This peak volt-ampere voltage can be determined using cyclic voltammetry, differential pulse voltammetry, pulse-wave voltammetry, square wave voltammetry, etc. Depending on the type of voltammetric scan performed, a suitable position on the observed curve can be determined to identify the peak volt-ampere voltage for each substance. Those skilled in the art will be able to make this determination based on the type of voltammetric scan to be performed.

[0066] According to various embodiments, the first current signal and the second current signal can be measured simultaneously or not simultaneously. Measurements that are not simultaneous may include, for example, performing a volt-ampere scan of each working electrode separately, without applying a voltage to the other working electrode. According to some embodiments, measurements in this manner can be performed using a single channel. In other embodiments, the first current signal and the second current signal can be simultaneously monitored at each working electrode for simultaneous measurement.

[0067] A third aspect of this application provides an analyte monitoring system, including a processing device, for obtaining the concentration of the analyte using the analyte monitoring method of the second aspect of this application.

[0068] In some preferred embodiments, the biosensor of this application may include a processor (or processing device) that communicates with the first and second working electrodes. The processor is located in the micro workstation 5, such as... Figure 3 As shown. The processor can be configured to receive a first current signal from the first working electrode 4a and a second current signal from the second working electrode 4b. The processor can be further configured to calculate the difference between the first current signal and the second current signal, and correlate the difference between the signals to the concentration of the detected substance.

[0069] In some embodiments, the processor can be configured to access a lookup table containing the concentration values ​​of the detected substance and the corresponding differences between the first and second current signals to calculate the concentration of the detected substance. This lookup table can be filled before measuring an unknown sample by measuring multiple samples with known concentrations of the detected substance, measuring the first and second current signals, and determining the differences between them. The processor can, for example, determine which difference in the lookup table is closest to the difference measured for a known sample and then report the concentration of the detected substance accordingly. In other embodiments, the processor can interpolate between the differences in the lookup table to determine the measured concentration value of the detected substance. Interpolation can assume a linear change in the concentration of the detected substance between the reported differences.

[0070] In other embodiments, the processor can be configured to access a calibration curve of the corresponding difference between the detected substance concentration value and the first and second current signals to calculate the detected substance concentration. Like a lookup table, the calibration curve can be determined before measuring unknown samples by measuring the first and second current signals with multiple samples having known detected substance concentrations, determining the difference between them, and curve fitting the detected substance concentration and the difference to determine a calibration function. Factory calibration at the batch level is possible by determining the signal difference as a function of the detected substance concentration at the factory and assigning a lookup table or calibration curve to the biosensor. Since reference electrode calibration for each signal is not required, the signal difference within a given range of detected substance concentrations should be consistent across different sensors for a given selection of substance in the first and second active portions.

[0071] The biosensor provided in this application will be described in detail below with reference to embodiments and comparative examples.

[0072] Example

[0073] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product manual. Instruments whose manufacturers are not specified are conventional products that can be obtained commercially.

[0074] Experimental reagents:

[0075] All reagents used in this application are commercially available, standard reagents.

[0076] Preparation of phosphate buffer: Add appropriate amounts of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride to deionized water and dissolve them completely. The concentration of phosphate ions is 0.03M. Adjust the pH value to the required range using sodium hydroxide. The phosphate buffer used in this experiment has a pH value of 7–8.

[0077] Test solution preparation:

[0078] Dissolve glucose thoroughly in a phosphate buffer solution with a pH of 7-8 to prepare test solution 1 with a glucose concentration of 5 mmol / L.

[0079] Weigh out an appropriate amount of the interfering substance according to the predetermined concentration, and dissolve it completely in test solution 1 to prepare a test solution with a glucose concentration of 5 mmol / L and a specific concentration of the interfering substance. The specific types and concentrations of the interfering substances are listed in Table 1.

[0080] Table 1

[0081]

[0082] The biosensor according to this application comprises at least a four-electrode system consisting of an electrode containing a vinylpyridine polymer membrane-electron mediator-specific enzyme (first working electrode 4a, abbreviated as WE1), an electrode containing a vinylpyridine polymer membrane-electron mediator (second working electrode 4b, abbreviated as WE2), a counter electrode CE, and a reference electrode RE. All four electrodes are located under the same membrane and connected in a four-electrode system. The two working electrodes WE1 and WE2 share a set of CE and RE. WE1 and CE are located on one side of the substrate, while WE2 and RE are located on the other side. It is connected to an electrochemical workstation for testing.

[0083] An electrochemical workstation includes at least a voltage source, a current measurement module, and a signal output module. The voltage source applies voltage to the two working electrodes WE1 and WE2 respectively. The current measurement module measures the current generated by the two working electrodes WE1 and WE2 and outputs it to the signal output module. The signal output module outputs the two measured currents to the host computer, where MATLAB software performs a subtraction calculation between the two currents.

[0084] Performance testing:

[0085] Test methods

[0086] The biosensor according to this application is first placed in a test solution containing the analyte free of electroactive interferences. A voltage is then applied for testing, with V11 on WE1 and V21 on WE2. After the current signal stabilizes, the current signal on WE1 is obtained as I. WE11 The current signal on WE2 is I. WE21 The difference between the two current signals is ΔI1, which represents the current value of the analyte without electroactive interference. The biosensor is transferred to test solutions containing different concentrations of electroactive interference, and voltage is applied for testing. The voltage on WE1 is V12, and the voltage on WE2 is V22. After the current signal stabilizes, the current signal on WE1 is obtained as I. WE12 The current signal on WE2 is I. WE22 The difference between the two current signals is ΔI2, which represents the current value of the analyte under the presence of an electroactive interfering substance.

[0087] Algorithm for current signal difference: ΔI1=I WE11 -I WE21 ΔI2=I WE12 -I WE22 ,

[0088] The influence of electroactive interference on the detection results is determined by comparing the differences between the current signals ΔI1 and ΔI2. Specifically, when the difference between ΔI1 and ΔI2 is sufficiently small, for example, ΔI1 / (ΔI1-ΔI2) is less than or equal to ±6%, it indicates that the current value of the detector with the electroactive interference added is very close to the current value of the detector without the electroactive interference. In this case, it can be considered that the current signal on WE2 can subtract the current signal of the electroactive interference on WE1 when voltages V1 and V2 are applied respectively. When the difference between ΔI1 and ΔI2 is sufficiently large, for example, ΔI1 / (ΔI1-ΔI2) is greater than ±6%, it indicates that the current value of the detector with the electroactive interference added differs significantly from the current value of the detector without the electroactive interference. In this case, it can be considered that the current signal on WE2 cannot subtract the current signal of the electroactive interference on WE1 when voltages V1 and V2 are applied respectively.

[0089] Example 1

[0090] The biosensor according to this application was first placed in test solution 1 containing glucose as the analyte. A voltage was then applied for testing, with voltage V1 on WE1 at 0.05V and voltage V2 on WE2 at 0.005V. After the current signal stabilized, the sensor was transferred to test solution 2 containing ascorbic acid as an electroactive interfering substance, and the current signal was tested again. The obtained current signal results are shown in Table 3, and the current signal graph is shown below. Figure 4a As shown.

[0091] Examples 2-3

[0092] The tests were conducted using essentially the same procedure as in Example 1, except for the addition of different electroactive interfering substances, the types of which are shown in Table 2. The current signal results obtained in Examples 2 and 3 are shown in Table 3, and the current signal graphs are shown in Figures 4b and 4c.

[0093] Examples 4-6

[0094] The test was conducted using essentially the same procedure as in Example 1, except that the voltage applied to WE2 was 0V and the added electroactive interfering substances are shown in Table 2.

[0095] The current signal results obtained in Examples 4 to 6 are shown in Table 3.

[0096] Table 2

[0097]

[0098] Table 3

[0099]

[0100] Table 3 shows the current changes before and after adding electroactive interfering substances in Examples 1-6. The difference between ΔI1 and ΔI2, and the percentage of this difference to ΔI1, can be used to determine whether interference needs to be subtracted. Interference can be subtracted when the interference deviation is less than or equal to ±6%. From the calculation results in Table 3, it can be seen that when the voltage applied to WE1 is 0.05V and the voltage applied to WE2 is less than the voltage applied to WE1, the interference caused by electroactive interfering substances such as ascorbic acid, uric acid, hydroxyurea, levodopa, acetaminophen, and salicylic acid on the current of glucose being measured can be subtracted. Therefore, the interference caused by these electroactive interfering substances can be considered negligible under the method according to this application.

[0101] like Figures 4a-4cAs shown in Examples 1-3, a voltage lower than that of WE1 was applied to WE2 using a micro workstation, and current signal graphs of solutions containing different electroactive interfering substances were measured. The electroactive interfering substances were ascorbic acid, uric acid, and hydroxyurea, respectively. It can be seen from the graphs that the current changes in WE1 and WE2 before and after the addition of the electroactive interfering substances are basically consistent, that is, the values ​​of ΔI1 and ΔI2 are basically the same. It can be considered that the interference of these electroactive interfering substances on the test results is negligible under the method according to this application.

[0102] Examples 7-9

[0103] The tests were conducted using essentially the same procedure as in Example 1, except that the voltage applied to WE2 was 0.02V, and the electroactive interfering substances added are shown in Table 2. The current signal results obtained in Examples 7–9 are shown in Table 4.

[0104] Table 4

[0105]

[0106]

[0107] Table 4 shows the current changes before and after adding electroactive interfering substances in Examples 7-9. The difference between ΔI1 and ΔI2, and the percentage of this difference to ΔI1, can be used to determine whether interference needs to be subtracted. Interference deviation of ±6% or less is considered acceptable for subtraction. The calculation results in Table 4 show that when the voltage applied to WE1 is 0.05V and the voltage applied to WE2 is 0.02V, the interference caused by electroactive interfering substances such as ascorbic acid, uric acid, and hydroxyurea on the current of glucose is subtracted. Therefore, the interference caused by these electroactive interfering substances can be considered negligible under the method of this application.

[0108] Comparative Examples 1-3

[0109] In Comparative Examples 1-3, tests were conducted using essentially the same procedures as in Example 1. The difference was that the voltage applied to WE2 was the same as the voltage applied to WE1, which was 0.05V; the added electroactive interfering substances are shown in Table 5. The current signal results obtained from Comparative Examples 1-3 are shown in Table 6, and the current signal graphs are shown in Figures 5a-5c.

[0110] Table 5

[0111]

[0112] Table 6

[0113]

[0114] Table 6 shows the current changes before and after adding electroactive interfering substances in Comparative Examples 1-3. The difference between ΔI1 and ΔI2, and the corresponding percentage of this difference to ΔI1, can be used to determine whether interference needs to be subtracted. Interference can be subtracted when the interference deviation is less than or equal to ±6%. The calculation results in Table 6 show that when both WE1 and WE2 are 0.05V, the interference caused by electroactive interfering substances such as ascorbic acid, uric acid, and hydroxyurea on the current measured when the detected substance is glucose cannot be subtracted.

[0115] like Figures 5a-5c As shown in the figures, in Comparative Examples 1-3, the same voltage was applied to WE1 and WE2 using a micro workstation, and the current signals of solutions containing different electroactive interfering substances were measured. It can be seen from the figures that the changes in current on WE1 and WE2 before and after the addition of the electroactive interfering substances are inconsistent, with WE2 showing a more significant current response than WE1, resulting in a larger change in the difference between the corresponding ΔI1 and ΔI2. Therefore, it can be concluded that when the same voltage is applied to WE1 and WE2, ascorbic acid, uric acid, and hydroxyurea all cause reverse interference, leading to excessive current subtraction and thus failing to achieve the purpose of interference subtraction.

[0116] It is known that electroactive interfering substances react with electron mediator portions coated on the surfaces of a first working electrode and a second working electrode. The first working electrode is coated with a mixture of enzyme and electron mediator, while the second working electrode is coated with a solution of the electron mediator. Under higher voltages, because the second working electrode is not coated with enzyme, the exposed electron mediator portion not occupied by the enzyme undergoes an interference side reaction with an intensity different from that of the first working electrode. This results in inconsistent interference current intensities between the first and second working electrodes, making it impossible to subtract interference using the second working electrode. Therefore, it can be considered that in this type of biosensor, the type of immobilized enzyme layer on the working electrode surface does not significantly affect the current change. Therefore, the biosensor according to this application includes a four-electrode biosensor containing various immobilized enzyme layers.

[0117] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A biosensor comprising a first working electrode, a second working electrode, and a reference electrode, wherein the voltage difference between the first working electrode and the reference electrode is a first voltage, the voltage difference between the second working electrode and the reference electrode is a second voltage, and the first voltage is higher than the second voltage.

2. The biosensor of claim 1, wherein, The first voltage is at least 0.03V higher than the second voltage.

3. The biosensor of claim 1, wherein, Includes a substrate, with the first working electrode and the second working electrode located on opposite sides of the substrate.

4. The biosensor of claim 1, wherein, It also includes a polymer film layer, which at least covers the surfaces of the first working electrode and the second working electrode.

5. The biosensor of claim 1, wherein, The first working electrode is an enzyme electrode containing an immobilized enzyme layer, while the second working electrode does not contain an immobilized enzyme layer.

6. The biosensor of claim 5, wherein, The immobilized enzyme layer contains one or more selected from glucose oxidase, glucose dehydrogenase, hydroxybutyrate dehydrogenase, myocardial flavin, coenzyme, and lactase.

7. The biosensor of claim 1, wherein, The first voltage is 0.04 to 0.2V, and the second voltage is 0 to 0.02V.

8. The biosensor of claim 7, wherein, The first voltage is 0.04 to 0.05V, and the second voltage is 0 to 0.005V.

9. The biosensor according to claim 1, wherein, The polymer film layer comprises a vinylpyridine polymer, a crosslinking agent, and a modifier, wherein the vinylpyridine polymer has a mass fraction of 80% to 99.9% in the polymer film layer.

10. The biosensor according to claim 1, wherein, It also includes a counter electrode, wherein the first working electrode and the counter electrode are located on one side of the substrate, and the second working electrode and the reference electrode are located on the other side of the substrate.

11. An analyte monitoring method, wherein, The analyte monitoring method includes: The biosensor according to any one of claims 1 to 10 is implanted into the body of the subject being tested; A first voltage is applied to the first working electrode to obtain a first current signal associated with the first working electrode; A second voltage is applied to the second working electrode, wherein the first voltage is higher than the second voltage, thereby obtaining a second current signal associated with the second working electrode; Calculate the difference between the first current signal and the second current signal; The concentration of the analyte is obtained based on the difference.

12. An analyte monitoring system, comprising: A processing apparatus configured to perform the analyte monitoring method of claim 11 to obtain the concentration of the analyte.

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