Enzyme preparation composition for dehydrogenase cascade reaction and application thereof

Abstract

The invention provides an enzyme preparation combination for dehydrogenase cascade reaction and application thereof, and relates to the technical field of cascade reaction, the enzyme preparation combination is formed by integrating a double-enzyme cascade catalytic reaction system of dehydrogenase and coenzyme thereof on a nanostructure of a Schottky junction, and is used for an electrode interface of a biosensor. The technical problems that in the prior art, dehydrogenase is low in catalytic efficiency, high in working voltage, large in byproduct generation amount and large in enzyme dosage are solved, the catalytic efficiency of dehydrogenase and coenzyme thereof is improved, and equal-magnitude current signals are generated under the condition that the enzyme dosage is smaller; therefore, the production cost of test strips based on dehydrogenase electrochemical detection of blood sugar, lactic acid and the like is reduced.

Description

Technical Field

[0001] This invention relates to the technical field of cascade reactions, and in particular to an enzyme preparation combination for a dehydrogenase cascade reaction and its application. Background Technology

[0002] In modern blood glucose monitoring, glucose dehydrogenase-based test strips have become one of the mainstream technologies due to their superior anti-interference performance and accuracy. The reaction zone of the test strip is immobilized with highly efficient glucose dehydrogenase and its coenzyme. When a blood sample is added, the glucose in the blood undergoes a specific catalytic reaction with the enzyme. The glucose dehydrogenase precisely catalyzes the oxidation of glucose, while the coenzyme is converted from its oxidized state to its reduced state. The reduced coenzyme then reacts with a pre-designed chemical mediator in the reaction zone. After being reduced, the mediator diffuses to the electrode surface and is oxidized under an applied low voltage, thereby generating a weak current. The magnitude of this current is directly proportional to the glucose concentration in the blood.

[0003] Since glucose dehydrogenase is more expensive than traditional oxidases, there is an urgent need to improve the catalytic efficiency of glucose dehydrogenase and reduce the amount of enzyme used per unit of test strip, thereby enhancing the competitiveness of blood glucose test strip products.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide an enzyme preparation combination for a dehydrogenase cascade reaction, which can solve the technical problems of low catalytic efficiency, high operating voltage, many by-products, and large enzyme dosage in the existing technology of dehydrogenases. It improves the catalytic efficiency of dehydrogenases and their coenzymes, and achieves the generation of an equivalent current signal with less enzyme usage, thereby reducing the production cost of test strips for electrochemical detection of blood glucose, lactic acid, etc. based on dehydrogenases.

[0006] The second objective of this invention is to provide an application of an enzyme preparation combination for a dehydrogenase cascade reaction, which is beneficial to reducing the production cost of test strips for blood glucose, lactic acid, etc. By improving the directional transfer of enzyme catalytic reactions, it is beneficial to reduce the working potential and the generation of by-products, and also to improve the selectivity of test strip detection.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: Firstly, an enzyme preparation assembly for a dehydrogenase cascade reaction is constructed by integrating a dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme onto a Schottky junction nanostructure, and is used as an electrode interface for a biosensor.

[0008] Furthermore, the dehydrogenase and its coenzyme are glucose dehydrogenase and its coenzyme, or lactate dehydrogenase and its coenzyme.

[0009] Furthermore, in the cascaded catalytic reaction system, the electric field direction is such that electrons flow from the coenzyme to the electrode, thereby reducing the operating voltage and decreasing the generation of byproducts in the reaction.

[0010] Furthermore, the Schottky junction is composed of a combination of noble metal materials and semiconductor materials; The precious metal materials include gold, platinum, and palladium; The semiconductor materials include titanium dioxide, cadmium oxide, zinc oxide, and zinc sulfide.

[0011] Furthermore, the preparation method of the Schottky junction material includes at least one of in-situ growth and loading methods.

[0012] Furthermore, the in-situ growth method includes the following steps: In the synthesis of semiconductor nanocrystals, a metal precursor is added to carry out a one-step reaction, simultaneously generating a semiconductor interface and a metal structure, thus obtaining a Schottky junction material.

[0013] Furthermore, the load method includes the following steps: Metals are loaded onto semiconductor nanomaterials using an impregnation reduction method to obtain Schottky junction materials.

[0014] Furthermore, the dehydrogenase is legally immobilized on the surface of the Schottky junction via charge adsorption or covalent bonding.

[0015] Secondly, the use of any of the enzyme preparations described above in the preparation of blood glucose monitoring products or lactate monitoring products.

[0016] Furthermore, the blood glucose monitoring product includes blood glucose test strips; The lactate monitoring products include lactate test strips.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The enzyme preparation combination for the dehydrogenase cascade reaction provided by this invention uses the electric field at the Schottky junction interface as a directionalist for the enzymatic reaction. This can control the directionality of the dual-enzyme cascade catalytic reaction of the dehydrogenase and its coenzyme, which is beneficial to reduce the generation of byproducts (such as hydrogen peroxide) in the cascade reaction and increase the generation rate of the final product. At the same time, by utilizing the local electric field in the micro-nano material system to reduce the overall potential in the reaction chamber, the selectivity of detection can be further improved.

[0018] The application of the enzyme preparation combination for the dehydrogenase cascade reaction provided by this invention is beneficial to reducing the production cost of test strips for blood glucose, lactic acid, etc. By improving the directional transfer of enzyme catalytic reactions, it is beneficial to reduce the working potential and the generation of by-products, and also to improve the selectivity of test strip detection. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a comparison graph of the response currents obtained from Example 1 and Comparative Example 1 of the present invention. Figure 2 This is a comparison graph of the response currents obtained from Example 2 and Comparative Example 2 of the present invention. Figure 3 The concentration of hydrogen peroxide byproducts obtained in Example 4 of this invention and the tests of Comparative Example 1; Figure 4 This is a comparison of the detection data of Example 1 and Comparative Example 1 obtained from Test Example 4 of the present invention. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] In the catalytic reaction system of dehydrogenases and their coenzymes, the reduced coenzyme may transfer electrons to the target mediator or be oxidized by oxygen or other impurities in the solution, leading to electron leakage and the formation of byproducts such as hydrogen peroxide, which interferes with normal measurements. In contrast, in the single-enzyme catalytic system of oxidases, such as glucose oxidase, one of the products is hydrogen peroxide itself, so there is no need to reduce the hydrogen peroxide byproduct in the dual-enzyme cascade reaction. In addition, the dehydrogenase catalytic system operates at a relatively high voltage, and the high potential can cause some active chemical substances to directly generate electrochemical signals at the electrode, forming false signals. To solve the above problems, the technical solution of this invention is proposed.

[0023] According to a first aspect of the present invention, an enzyme preparation assembly for a dehydrogenase cascade reaction is provided, which is composed of a dual-enzyme cascade catalytic reaction system of a dehydrogenase and its coenzyme integrated on a Schottky junction nanostructure, for use as an electrode interface of a biosensor.

[0024] This invention uses the electric field at the Schottky junction interface as a directionalist for enzymatic reactions, which can control the directionality of the dual-enzyme cascade catalytic reaction of dehydrogenase and its coenzyme. This helps to reduce the generation of byproducts such as hydrogen peroxide and increase the generation rate of the final product. At the same time, by using the local electric field in the micro-nano material system to reduce the overall potential in the reaction chamber, the selectivity of detection can be further improved.

[0025] In this invention, the cascade catalytic reaction system can be various dehydrogenases and coenzymes, such as glucose dehydrogenase and its coenzyme, or lactate dehydrogenase and its coenzyme. The electric field direction must be able to promote the flow of electrons from the coenzyme to the electrode along the path from the driving substrate to the coenzyme and then to the electrode, thereby reducing the operating voltage and reducing the generation of byproducts in the reaction.

[0026] It should be noted that in traditional dehydrogenase detection systems, both the enzyme and coenzyme move freely in the solution, resulting in random reaction paths. However, under the drive of an electric field, the traditional random, diffusion-controlled cascade reaction can be transformed into a highly ordered, spatially oriented, and electrically controllable process, thereby improving the overall catalytic efficiency.

[0027] Under the influence of an electric field, the overall rate of a cascade catalytic reaction is no longer determined solely by substrate concentration and enzyme activity, but can be directly controlled by adjusting the external bias voltage, thereby altering the energy barrier of reaction intermediates near the enzyme's active site. This directional control process requires a very strong electric field, typically reaching 10⁻⁶. 7 Directional control can only be achieved with a voltage of V / m or higher. Such a directional electric field cannot be arranged in traditional electrodes. Only by combining enzymes with micro / nano structure materials can such electric field control be achieved.

[0028] It should be noted that typical micro / nanostructure materials are either conductive or insulating, which results in a uniform electric field distribution on the surface of the structure, making it impossible to achieve the required electric field strength under operable conditions. An effective electric field must be concentrated in the interface region where the reaction occurs (i.e., the micro / nanoscale space where the enzyme and coenzyme reside, rather than being diffused throughout the overall solution). The inherent characteristic of Schottky junctions is that they generate an extremely strong and localized space charge region electric field at the interface, perfectly meeting this requirement. At the same time, nano-Schottky materials can greatly increase the effective interface area and create a large number of local electric field hotspots, ensuring that enzyme molecules within the reaction region are enveloped in an effective electric field.

[0029] In a preferred embodiment, the Schottky junction is composed of a combination of noble metal materials and semiconductor materials; wherein the noble metal materials include, but are not limited to, gold, platinum and palladium; and the semiconductor materials include, but are not limited to, titanium dioxide, cadmium oxide, zinc oxide and zinc sulfide.

[0030] In this invention, the Schottky junction material can be prepared by in-situ growth or loading method. The in-situ growth method involves adding a metal precursor during the synthesis of semiconductor nanocrystals to react and generate a semiconductor interface and a metal structure simultaneously in one step, thereby obtaining the Schottky junction material. The loading method involves first preparing semiconductor nanomaterials and then loading a metal onto the semiconductor material by impregnation reduction, thereby obtaining the Schottky junction material.

[0031] In a preferred embodiment, the dehydrogenase is legally immobilized on the Schottky junction by surface charge adsorption or covalent bonding.

[0032] Without affecting enzyme activity, the enzyme is immobilized on a Schottky junction, with the enzyme close to the Schottky junction interface, thus providing electric field-driven catalysis conditions.

[0033] According to a second aspect of the present invention, there is provided the use of the enzyme combination described in any one of the above claims in the preparation of a blood glucose monitoring product or a lactate monitoring product.

[0034] In this invention, blood glucose monitoring products include, but are not limited to, blood glucose test strips; lactate monitoring products include, but are not limited to, lactate test strips.

[0035] The application of the enzyme preparation combination for the dehydrogenase cascade reaction provided by this invention is beneficial to reducing the production cost of test strips for blood glucose, lactic acid, etc. By improving the directional transfer of enzyme catalytic reactions, it is beneficial to reduce the working potential and the generation of by-products, and also to improve the selectivity of test strip detection.

[0036] In summary, this invention can actively and precisely regulate the dehydrogenase cascade reaction through external bias, and use the built-in electric field to achieve directional driving and rate control of the electron transport path, thereby improving the catalytic efficiency of dehydrogenase and coenzyme. It achieves the technical effect of generating an equivalent current signal with less enzyme usage, which not only helps to reduce the production cost of test strips, but also helps to improve the selectivity of test strip detection.

[0037] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0038] Example 1 This embodiment provides an enzyme preparation combination for a dehydrogenase cascade reaction, which is composed of a dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme integrated on a Schottky junction nanostructure, and is used as an electrode interface for a biosensor. The Schottky junction uses a Pt / TiO2 nanocomposite material.

[0039] The method for preparing the enzyme preparation combination for the dehydrogenase cascade reaction in this embodiment includes the following steps: Tetrabutyl titanate was used as a titanium precursor and reacted with chloroplatinic acid in a hydrothermal reactor at 180°C for 12 hours to form a Pt / TiO2 nanowire structure. Pt nanoparticles were uniformly distributed on the TiO2 surface, resulting in a Pt / TiO2 nanocomposite material. Glucose dehydrogenase (GDH, from Aspergillus, with an activity of 250 U / mg) and its coenzyme NAD were used. + (Nicotinamide adenine dinucleotide), Pt / TiO2 nanocomposite materials were dispersed in phosphate-buffered saline (PBS, pH 7.4), and GDH (enzyme dosage 0.1 mg / mL) and NAD were added. + (1mM) was fixed at 4°C for 12 hours by electrostatic adsorption to complete enzyme immobilization. The enzyme was brought close to the Schottky junction interface to obtain an enzyme preparation combination for the dehydrogenase cascade reaction.

[0040] Example 2 This embodiment provides an enzyme preparation combination for a dehydrogenase cascade reaction, which is composed of a dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme integrated on a Schottky junction nanostructure, and is used as an electrode interface for a biosensor. The Schottky junction uses Au / TiO2 nanocomposite material.

[0041] The method for preparing the enzyme preparation combination for the dehydrogenase cascade reaction in this embodiment includes the following steps: TiO2 nanoparticles were first synthesized by sol-gel method, then impregnated in chloroauric acid solution, and reduced with sodium borohydride to load Au nanoparticles onto the TiO2 surface, thus obtaining Au / TiO2 nanocomposite material. Lactate dehydrogenase (LDH, derived from bovine heart, with an activity of 200 U / mg) and its coenzyme NAD were used. + Au / TiO2 nanocomposite materials were dispersed in PBS buffer, and LDH (enzyme dosage 0.1 mg / mL) and NAD were added. + (1mM), the enzyme was immobilized at 4°C for 12 hours by covalent bonding (using glutaraldehyde as a cross-linking agent) to complete the enzyme immobilization. The enzyme was brought close to the Schottky junction interface to obtain the enzyme preparation combination for the dehydrogenase cascade reaction.

[0042] Example 3 This embodiment provides an enzyme preparation combination for a dehydrogenase cascade reaction, which is composed of a dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme integrated on a Schottky junction nanostructure, and is used as an electrode interface for a biosensor. The Schottky junction uses an Au / ZnO nanorod array.

[0043] The method for preparing the enzyme preparation combination for the dehydrogenase cascade reaction in this embodiment includes the following steps: First, ZnO nanorods are vertically grown on an electrode substrate, and then Au nanoparticles are sputtered to obtain an Au / ZnO nanorod array. Glucose dehydrogenase (GDH, from Acinetobacter calcitriol, with an activity of 300 U / mg) and its coenzyme PQQ (pyrroloquinoline quinone) were immobilized on Au / ZnO nanorod array electrodes by physical adsorption. The enzyme was immobilized near the Schottky junction interface, resulting in an enzyme preparation combination for the dehydrogenase cascade reaction.

[0044] Comparative Example 1 The only difference between this comparative example and Example 1 is that the dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme was not integrated on the Schottky junction, that is, no nano-Schottky junction material was modified. GDH and NAD + Mixed in PBS buffer, the enzyme was directly immobilized on the surface of a conventional carbon electrode by physical adsorption and allowed to stand at 4°C for 12 hours, during which the enzyme was naturally adsorbed. Everything else is the same as in Example 1.

[0045] Comparative Example 2 The only difference between this comparative example and Example 2 is that the dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme was not integrated on the Schottky junction, that is, no nano-Schottky junction material was modified. LDH and NAD + Mixed in PBS buffer, the enzyme was directly immobilized on the surface of a conventional carbon electrode by physical adsorption and allowed to stand at 4°C for 12 hours, during which the enzyme was naturally adsorbed. Everything else is the same as in Example 2.

[0046] Comparative Example 3 The only difference between this comparative example and Example 3 is that GDH and PQQ are directly fixed on the planar gold electrode, instead of being fixed on the Au / ZnO nanorod array electrode. Everything else is the same as in Example 3.

[0047] Comparative Example 4 The only difference between this comparative example and Example 1 is that the dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme is replaced with the single-enzyme catalytic reaction system of glucose oxidase, that is, the single-enzyme catalytic reaction system of glucose oxidase is integrated on the Schottky junction nanostructure. Everything else is the same as in Example 1.

[0048] Compared with Example 1, the single enzyme catalytic reaction system in this comparative example is not affected by the Schottky junction. The electric field formed by the Schottky junction does not play a regulatory role in single enzyme catalysis, and the measured current is completely affected by the instrument's operating voltage.

[0049] Experimental Example 1 The target product of Example 1 was coated onto the surface of a carbon electrode and dried to form a working electrode. In a standard three-electrode system (working electrode and reference electrode were Ag / AgCl, counter electrode was Pt), a voltage of 0.3V was applied, and 5mM glucose solution was added. The steady-state current generated by the working electrode was measured. The steady-state current of Comparative Example 1 was measured in the same manner. The results are shown in […]. Figure 1 .

[0050] Depend on Figure 1 As can be seen, compared with the current value of 26.1 μA generated by Comparative Example 1 as the working electrode, the current value of Example 1 of the present invention, after introducing Pt / TiO2 Schottky junction material as the working electrode, can reach 43.5 μA, with an efficiency improvement of 66.7%. This indicates that the built-in electric field of the Schottky junction interface effectively drives the transfer of electrons from the coenzyme to the electrode, accelerates the cascade reaction rate, and thus generates a higher current under the same enzyme dosage.

[0051] Experimental Example 2 The target product of Example 2 was coated onto the surface of a carbon electrode and dried to form a working electrode. In a standard three-electrode system (working electrode and reference electrode were Ag / AgCl, counter electrode was Pt), a low voltage of 0.25V was applied, and 5mM lactic acid solution was added. The steady-state current generated by the working electrode was measured. The steady-state current of Comparative Example 2 was measured in the same manner. The results are shown in […]. Figure 2 ; Depend on Figure 2 As can be seen, compared with the current value of 29.6 μA generated by Comparative Example 2 as the working electrode, the current value of Example 2 of the present invention, after introducing Au / TiO2 Schottky junction material as the working electrode, can reach 101.8 μA, and the efficiency is improved by 240%. This proves that under a low working voltage of 0.25V, the effect of introducing Schottky structure on current improvement is more significant.

[0052] Experimental Example 3 In PBS buffer (pH 7.4) containing 1 mM glucose, the current-voltage (IV) curves of Examples 3 and Comparative Examples 3-4 were measured using an electrochemical workstation. The operating voltage was scanned from 0.0 V to +0.5 V (relative to the Ag / AgCl reference electrode) at a scan rate of 10 mV / s. The steady-state current values ​​at different characteristic voltages were recorded, and the results are shown in Tables 1 and 2.

[0053] As can be seen from the data in Table 1, in the low voltage region (+0.2V and below), the current signal generated by Example 3 of the present invention is much greater than that of the conventional planar electrode of Comparative Example 3. For example, at +0.2V, the current generated by the Schottky junction in Example 3 of the present invention is 12.1μA, which is 4.3 times the current generated by Comparative Example 3 (2.8μA). This proves that the built-in electric field of the Schottky junction provides a directional driving force for the transfer of electrons from the enzyme active center to the electrode, so that the reaction can proceed efficiently when the external voltage is very low.

[0054] A horizontal comparison of the operating currents at +0.4V and +0.2V operating states shows that in Example 3, the current decreases by 45% when +0.4V drops to +0.2V, but the actual operating current is still above 10μA. In Comparative Example 3, the current decreases by 85% when +0.4V drops to +0.2V. In practical testing, an operating current of at least 10μA is typically required to ensure signal clarity and system stability. Example 3 achieves a current of 12.1μA at +0.2V, meeting the current response threshold of common sensors. Comparative Example 3, however, only achieves 2.8μA at +0.2V, far below the effective detection level, requiring the voltage to be increased to approximately +0.35V or higher to approach the 10μA threshold. This demonstrates that the present invention not only provides higher current output at low voltages but also significantly reduces the actual operating voltage requirement, which is beneficial for constructing low-power, high-sensitivity sensing systems.

[0055] In Comparative Example 4, since the catalytic process of glucose monoenzyme does not involve a cascade reaction, it is not affected by the Schottky junction. Its measured current is entirely influenced by the instrument's operating voltage. The electric field formed by the Schottky junction does not play a regulatory role in monoenzyme catalysis. As shown in Table 2, when the operating voltage decreases, the measured current of glucose oxidase drops sharply, failing to achieve the low-voltage cascade catalytic effect of the example. Therefore, compared to the monoenzyme catalytic reaction system, the present invention integrates the dual-enzyme cascade catalytic reaction system onto the nanostructure of the Schottky junction, which can effectively reduce the operating voltage.

[0056] The current generated by Example 3 is consistently much higher than that of Comparative Example 4. For example, at +0.2V, Example 3 generates a current of 12.1μA, while Comparative Example 4 generates only 1.4μA, with the former being approximately 8.6 times the latter. This indicates that Comparative Example 4 has a weaker current response at low voltages, and it is difficult for Comparative Example 4 to provide sufficient electrical signal output without applying a higher voltage.

[0057] In practical testing, 10μA is often set as the effective current threshold. In Example 3, the current at +0.2V is 12.1μA, which exceeds this threshold. In Comparative Example 4, even at +0.3V, the current is only 3.2μA, far below the threshold. The voltage must be increased to close to +0.4V to achieve a similar output level. This indicates that using a Schottky junction electrode structure can significantly reduce the operating voltage requirement of the sensor. However, the Schottky junction electrode structure is difficult to function effectively in single-enzyme systems such as glucose oxidase.

[0058] Table 1. Electrode Current-Voltage Scan Comparison

[0059] Table 2 Comparison of Electrode Current-Voltage Scans

[0060] Test Example 4 The amount of hydrogen peroxide byproduct produced after the reaction of Example 1 and Comparative Example 1 with an equal amount of 5mM glucose was tested, and the test results are as follows: Figure 3 As shown.

[0061] from Figure 3 As can be seen, the hydrogen peroxide concentration produced in Example 1 was 13.5 μM, while that produced in Comparative Example 1 was as high as 53.1 μM. The hydrogen peroxide concentration produced in Example 1 was only 25.4% of that produced in Comparative Example 1. This shows that the built-in electric field of the Schottky structure can significantly improve the directionality of the dual-enzyme cascade catalytic reaction, significantly reduce the amount of hydrogen peroxide generated, and help reduce the interference of byproducts on electrode measurements, thereby improving detection accuracy.

[0062] Example 1 and Comparative Example 1 were tested using calibration simulation solutions of different concentrations (1 mM, 2 mM, 4 mM, 8 mM, 12 mM). The comparison of the obtained test data is as follows: Figure 4 As shown, Example 1 has higher detection accuracy with an average measurement deviation of 3.3%, while the test concentration of Comparative Example 1 is generally higher than the actual concentration, and the overall curve is significantly biased upward. This is because the interference of hydrogen peroxide causes the reading to be too high, resulting in poor detection accuracy with an average measurement deviation of 11.7%. This further verifies that the Schottky structure has a positive effect on improving detection selectivity and accuracy.

[0063] In summary, this invention uses the electric field at the Schottky junction interface as a rate regulator for enzyme-catalyzed reactions, which can control multi-enzyme cascade reactions, accelerate the electron transfer rate catalyzed by enzymes, and improve the generation rate of the final product. At the same time, by utilizing the local electric field in the micro / nano material system to reduce the overall potential in the reaction chamber, the selectivity of detection can be further improved.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An enzyme preparation combination for a dehydrogenase cascade reaction, characterized in that, It is composed of a dual-enzyme cascade catalytic reaction system of dehydrogenase and its coenzyme integrated on a Schottky junction nanostructure, and is used as an electrode interface for biosensors.

2. The enzyme preparation combination according to claim 1, characterized in that, The dehydrogenase and its coenzyme are glucose dehydrogenase and its coenzyme, or lactate dehydrogenase and its coenzyme.

3. The enzyme preparation combination according to claim 2, characterized in that, In the cascaded catalytic reaction system, the electric field direction is such that electrons flow from the coenzyme to the electrode, thereby reducing the operating voltage and the generation of byproducts in the reaction.

4. The enzyme preparation combination according to any one of claims 1-3, characterized in that, The Schottky junction is made of a combination of noble metal materials and semiconductor materials; The precious metal materials include gold, platinum, and palladium; The semiconductor materials include titanium dioxide, cadmium oxide, zinc oxide, and zinc sulfide.

5. The enzyme preparation combination according to claim 4, characterized in that, The preparation method of the Schottky junction material includes at least one of in-situ growth method and loading method.

6. The enzyme preparation combination according to claim 5, characterized in that, The in-situ growth method includes the following steps: In the synthesis of semiconductor nanocrystals, a metal precursor is added to carry out a one-step reaction, simultaneously generating a semiconductor interface and a metal structure, thus obtaining a Schottky junction material.

7. The enzyme preparation combination according to claim 5, characterized in that, The load method includes the following steps: Metals are loaded onto semiconductor nanomaterials using an impregnation reduction method to obtain Schottky junction materials.

8. The enzyme preparation combination according to claim 4, characterized in that, The dehydrogenase is legally immobilized on the surface of the Schottky junction through charge adsorption or covalent bonding.

9. The use of the enzyme preparation combination according to any one of claims 1-8 in the preparation of a blood glucose monitoring product or a lactate monitoring product.

10. The application according to claim 9, characterized in that, The blood glucose monitoring products include blood glucose test strips. The lactate monitoring products include lactate test strips.

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