A nanowire-nanotube hybrid sensor and a preparation method and application thereof

Abstract

The application discloses a nanowire-nanotube hybrid sensor and a preparation method and application thereof. The nanowire-nanotube hybrid sensor has two regions of a nanochannel and a nanowire electrode by using a conical double-channel microcapillary. A substrate solution is precisely released to a specific site of a single cell through the nanochannel. After a to-be-measured glycosidase recognizes the substrate molecules, the glycosidic bond is cut to release active substances of the electrode. The nanowire electrode is used for real-time quantitative detection of the active substances, so that quantitative detection of glycosidase substances at a single / subcellular level is realized. The application constructs an efficient and universal detection strategy of non-electrochemical active glycosidase by using the nanowire-nanotube hybrid sensor and an electrical signal conversion method of the reaction of specific glycosidase and a substrate, widens the detection range of electrochemical target objects, and provides a promising method for high-time-space-resolution quantitative monitoring of hydrolytic enzymes at a single / subcellular level.

Description

Technical Field

[0001] This invention relates to the technical fields of chemistry, biomedicine and the manufacture or processing of nanostructures, and particularly to a nanowire-nanotube hybrid sensor and its preparation method and application. Background Technology

[0002] Lysosomes are important organelles in eukaryotic cells, containing various hydrolytic enzymes specifically designed to break down biological macromolecules such as proteins, nucleic acids, and polysaccharides. Immune cell lysosomes can release these hydrolytic enzymes extracellularly via exocytosis to perform various immune functions, including pathogen killing, antigen presentation, and target cell killing. Studies have shown that abnormal lysosomal secretion leads to the ineffective clearance of pathogens and cellular waste, resulting in dysregulation of cell signaling and triggering chronic inflammation, storage diseases, and autoimmune diseases. Glycosidases, as important enzymes in lysosomes that hydrolyze glycosidic bonds, participate in the degradation of pathogenic microbial glycans and play a crucial role in disrupting and dissolving the integrity of pathogens. Therefore, quantitative monitoring and precise regulation of immune cell lysosomal glycosidases in terms of time, space, and release volume are of great significance for understanding immune function and the development of diseases, and can contribute to providing new strategies for disease prevention and treatment. However, existing glycosidase monitoring methods, such as fluorescence, immunoelectron microscopy, and enzyme-linked immunosorbent assay (ELISA), can often only obtain information on glycosidase activity in population cells or qualitative information on glycosidase at the single / subcellular level. Moreover, these techniques usually require cell fixation, making it difficult to perform high spatiotemporal resolution, high sensitivity, and high specificity quantitative monitoring of specific glycosidases in different lysosomal secretion forms under conditions of maintaining high cell viability.

[0003] Therefore, there is an urgent need to develop sensors with high spatiotemporal resolution, high sensitivity, and high specificity for highly selective real-time quantitative monitoring of glycosidases secreted by lysosomes at the single / subcellular level under living cell conditions. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a nanowire-nanotube hybrid sensor, its fabrication method, and its application.

[0005] In a first aspect, a nanowire-nanotube hybrid sensor is provided, comprising: The first microtube has one end sealed by liquid metal injection and the other end provided with a conductive nanowire; one end of the conductive nanowire is connected to the liquid metal and sealed, and the other end extends out of the first microtube. The second microtube is a through-tube that connects to the first microtube to form a parallel dual-channel micro-nanotube. The first microtube and the second microtube are independent working tubes.

[0006] In one possible implementation, the conductive nanowire comprises a nanowire and a conductive layer covering the nanowire; the conductive layer is carbon.

[0007] In one possible implementation, both the first and second microtubes are tapered tubes.

[0008] In a second aspect, a bio-enzyme-modified nanowire-nanotube hybrid sensor is provided, comprising the nanowire-nanotube hybrid sensor described in the first aspect; the conductive nanowire comprises a nanowire and a conductive layer coated on the nanowire; the conductive layer is platinum particles; and the conductive nanowire is coated with a bio-enzyme-modified layer.

[0009] In one possible implementation, the bio-enzyme-modified layer is coated onto the conductive nanowire via enzyme cross-linking, comprising a bio-enzyme, a cross-linking agent, and an enzyme-stabilizing protein.

[0010] In one possible implementation, the bioenzyme includes at least one of glucose oxidase and galactose oxidase.

[0011] Thirdly, a method for fabricating the nanowire-nanotube hybrid sensor described in the first aspect is provided, comprising the following steps: First and second micro-nanotubes are drawn to form parallel dual-channel micro-nanotubes; A conductive layer is coated onto nanowires to form conductive nanowires; Liquid metal was injected into one end of the first micro / nanotube for sealing; One end of the conductive nanowire is connected to the liquid metal, and the other end extends out into the first micro-nano tube opening; Gas is introduced into the second micro-nanotube, and the portion of the conductive nanowire located inside the first micro-nanotube is sealed.

[0012] Fourthly, a method for preparing the bio-enzyme-modified nanowire-nanotube hybrid sensor described in the second aspect is provided, comprising the following steps: First and second micro-nanotubes are drawn to form parallel dual-channel micro-nanotubes; A conductive layer is coated onto nanowires to form conductive nanowires; Bioenzymes were immobilized on a conductive layer using an enzyme cross-linking method to obtain bioenzyme-modified conductive nanowires. Liquid metal was injected into one end of the first micro / nanotube for sealing; One end of a bio-enzyme-modified conductive nanowire is connected to liquid metal, and the other end extends out into a first micro-nanotube opening. Gas was introduced into the second micro-nanotube, and the portion of the bio-enzyme-modified conductive nanowire located inside the first micro-nanotube was sealed.

[0013] Fifthly, the application of the nanowire-nanotube hybrid sensor described in the first aspect in the quantitative detection of enzyme activity during the reaction of substrates and glycosidases to generate electrochemically active small molecules is provided, and the application methods include: A glycosidase-specific substrate solution with electrochemically active molecules was released using a second-micron tube. The electrical signals of electroactive molecules released by the reaction of glycosidase with the specific substrate are monitored in real time using a first-micron tube. The corresponding electrical quantity value is obtained by calculating based on the electrical signal; Plot a standard curve of enzyme activity versus charge value to quantitatively calculate glycosidase activity.

[0014] In one possible implementation, the glycosidase-specific substrate solution comprises: a glycoside molecule formed by linking a glycosidase recognition group and an electrochemically active unit via a glycosidic bond; the electrochemically active unit comprises at least one of 4-aminophenol and dopamine; and the glycosidase recognition group is a specific recognition group of the glycosidase.

[0015] Sixthly, the application of the bio-enzyme-modified nanowire-nanotube hybrid sensor described in the second aspect in detecting lysosomal glycosidase release at the single or subcellular level is provided, and the application method includes the following steps: Release of glycosidase substrate solution using a second-micron tube; The electrochemical signal of hydrogen peroxide converted from glycosidases released by single or subcellular cells after reacting with substrates and bio-enzyme-modified layers was monitored in real time using platinum particles in a first-micron tube.

[0016] In one possible implementation, the glycosidase substrate solution is a glycosidase-specific substrate solution.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a nanowire-nanotube hybrid sensor and its fabrication method. A hybrid sensor combining nanotube and nanowire electrodes is constructed using a pre-stretched tapered dual-channel microtube as the main body. This sensor is combined with an enzyme substrate molecule that integrates enzyme recognition and electrochemical activity units. The target glycosidase cleaves the substrate to release electrochemically active small molecules. The nanowire-nanotube hybrid sensor allows for precise injection of the enzyme substrate and detection of the electrochemically active small molecules converted by the target enzyme.

[0018] This invention provides an efficient and universal signal conversion strategy for non-electrochemically active lysosomal glycosidases, expands the range of substances that can be detected electrochemically, develops a new electrochemical measurement system for the exocytotic release of lysosomal glycosidases, and realizes high spatiotemporal resolution quantitative detection of lysosomal glycosidases at the single / subcellular level under living cell conditions. It has broad application prospects in the preparation of nanosensors and the quantitative monitoring of non-electrochemically active substances at the single / subcellular level. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the 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 based on these drawings without creative effort.

[0020] Figure 1 This is a flowchart of the fabrication method of a carbon conductive nanowire-nanotube hybrid sensor; Figure 2 These are bright-field micrographs and scanning electron microscope images of a carbon conductive nanowire-nanotube hybrid sensor. Figure 3 This is a flowchart of the fabrication method of a platinum conductive nanowire-nanotube hybrid sensor modified with biological enzymes. Figure 4 This is a scanning electron microscope image of a platinum conductive nanowire-nanotube hybrid sensor modified with bio-enzymes. Figure 5 These are cyclic voltammograms of a carbon conductive nanowire-nanotube hybrid sensor detecting a mixture of glycosidase and substrate containing electroactive groups. Figure A shows the cyclic voltammogram of 4-aminophenyl-β-D-glucopyranoside and β-glucopyranoside after the reaction, and Figure B shows the cyclic voltammogram of 4-aminophenyl-β-D-galactopyranoside and β-galactopyranoside after the reaction. Figure 6 These are standard curves of the charge and enzyme activity of β-glucosidase and β-galactosidase detected by a carbon conductive nanowire-nanotube hybrid sensor. Figure A is the standard curve of β-glucosidase, and Figure B is the standard curve of β-galactosidase. Figure 7 Figure A shows a schematic diagram of the release of glycosidase simulated by a single micron-sized capillary and the detection of glycosidase using a carbon conductive nanowire-nanotube hybrid sensor, along with the ampere signal results. Figure B is a schematic diagram of the detection and the ampere signal detection results. Figure 8 This is a bright-field micrograph of a carbon conductive nanowire-nanotube hybrid sensor detecting the release of glycosidases at the frustrated phagocytic opening of macrophages. Figure 9 This is an amperometric signal graph and enzyme activity statistics graph of β-glucosidase and β-galactosidase released from the phagocytic port of macrophages using a carbon conductive nanowire-nanotube hybrid sensor. In graph A, the amperometric detection results of glycosidase are shown, and in graph B, the results are based on... Figure 6 The calculated enzyme activity statistics graph; Figure 10 This is a schematic diagram of a platinum conductive nanowire-nanotube hybrid sensor modified with bio-enzymes for detecting glycosidases. Figure 11 The graphs show the amperometric signal results and charge statistics of β-glucosidase and β-galactosidase released from the phagocytic port of macrophages by a bio-enzyme-modified platinum conductive nanowire-nanotube hybrid sensor. In the graph, A is the amperometric detection result of glycosidase, and B is the charge statistics. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. 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.

[0023] Example 1 A method for fabricating a carbon conductive nanowire-nanotube hybrid sensor, as follows: Figure 1 As shown, it includes the following steps: (1) Preparation of cone-shaped nanopore body: A conical nanopore body was drawn from a dual-channel borosilicate glass capillary (outer diameter 1.5 mm, length 10 cm, model BT-150-10, Sutter Instruments, USA) using a P-2000 drawing instrument (P2000, Sutter Instruments, USA). The drawing parameters HEAT=550, FIL=4, VEL=28, DEL=250, and PUL=0 were adjusted to achieve an aperture of 0.5-5 μm for the nanopore body.

[0024] (2) Preparation of carbon-coated conductive nanowires: A. Purification of silicon carbide nanowires Weigh 1-10 mg of silicon carbide nanowires (Nanjing XFNANO Materials Tech Co., Ltd, China) and ultrasonically disperse them in 1-10 mL of anhydrous ethanol solution. Centrifuge at 1500 rpm for 3 min to remove short, fine nanowires and impurities from the supernatant, and then ultrasonically disperse the remaining nanowires evenly. Repeat the above operation twice, centrifuging at 1000 rpm for 2 min and then at 500 rpm for 2 min. After centrifugation purification, collect the bottom silicon carbide nanowires and dry them in an oven at 80 °C.

[0025] B. Silicon carbide nanowires coated with a carbon layer 0.1–5 g of purified silicon carbide nanowires were weighed and spread evenly in a quartz boat. The quartz boat was placed in a tube furnace and chemically vapor-deposited with butane for 20–60 min in an oxygen-free environment at 600–900 °C, thereby coating the surface of the silicon carbide nanowires with a dense carbon layer. Subsequently, the carbon-coated nanowires were collected and ultrasonically dispersed in anhydrous ethanol solution.

[0026] (3) Assemble carbon conductive nanowire-nanotube hybrid sensor Conductive nanowires (one of carbon-coated silicon carbide nanowires and platinum-coated silicon carbide nanowires) uniformly dispersed in solvent are dropped onto the center of a glass slide. The solvent is dried by heating at 90-120°C. Then, the back of the glass slide is cut along the center line with a glass cutter, and the glass slide is pried open from the front to expose the single conductive nanowires at its edges.

[0027] Liquid metal (gallium indium tin zinc alloy) 0.5-2 cm long is injected into one end of a conical dual-channel glass capillary. Centrifugation is used to push the liquid metal to a position 5-20 μm from the tip of the capillary. Under a 40x objective lens, a single conductive nanowire exposed at the edge of a glass slide is inserted into the main channel of the capillary containing the liquid metal using a micromanipulation platform, extending the conductive nanowire 1-10 μm beyond the opening as needed. An inert gas is introduced into the ends of the dual tubes while the capillary containing the conductive nanowire is immersed in molten paraffin solution to seal and insulate the electrode side.

[0028] It should be noted that liquid metal has a certain viscosity and requires centrifugal force to reach a certain distance from the glass tube opening, rather than flowing directly to it. The empty space at the tube opening, where no liquid metal is filled, is sealed with paraffin wax for insulation. After sealing, the liquid metal will stabilize at the position reached after centrifugation and will not flow. In subsequent testing, a conductive copper wire is inserted into the liquid metal to achieve conductivity.

[0029] Example 2 A method for fabricating a bio-enzyme-modified platinum conductive nanowire-nanotube hybrid sensor is as follows: Figure 3 As shown, it includes the following steps: (1) Based on Case 1, a column of ultrapure water was injected into the non-liquid metal side of the conical double-channel glass capillary using a micro-injection gun with a Microloader (Eppendorf, Germany) to prevent the modification solution from entering the nanochannel due to capillary action during the modification of the enzyme.

[0030] (2) Bundle 4-10 double-channel glass capillaries with platinum conductive nanowires obtained in step (1) into a bundle, align the conical tips and fix the tips facing downwards. Add 0.1% polyethyleneimine solution to the conical tips, ensuring that the solution forms a suspended droplet due to tension at the capillary tips, and then dry the solution by irradiating with an infrared lamp for 30-60 min.

[0031] (3) Continue to add enzyme crosslinking solution (100 U / mL enzyme aqueous solution + 5 mg / mL polyethylene glycol diglycidyl aqueous solution + 2 mg / mL bovine serum albumin aqueous solution) to the tip of the bound electrode and ensure that it forms droplets. Place the bound capillary in a refrigerated container at 4°C for 6-12 h to promote enzyme crosslinking to the electrode surface.

[0032] (4) Inert gas is introduced into the tail end of the modified enzyme dual-channel glass capillary to remove the aqueous solution in step (1). The inside of the nanotube and the electrode are cleaned with ultrapure water and then inert gas is introduced to remove the solution. The above steps are repeated 3 times to obtain a nanowire-nanotube hybrid sensor with a single-sided modified bioenzyme electrode. The sensor is stored at 4°C to maintain enzyme activity.

[0033] Example 3 A carbon conductive nanowire-nanotube hybrid sensor was used to detect electrochemical molecules released after glycosidases react with substrates containing electroactive groups, in order to assess their performance.

[0034] During the test, the liquid metal end of the first micron tube of the carbon conductive nanowire-nanotube hybrid sensor is connected to a copper wire as the working electrode, and Ag / AgCl is the reference electrode. Both are immersed in the test solution, and the detection is performed by applying voltage through an electrochemical workstation.

[0035] Test 1: 1 mM of 4-aminophenyl-β-D-glucopyranoside and 4-aminophenyl-β-D-galactopyranoside were mixed with their corresponding β-glucosidase and β-galactosidase, and reacted at 25 °C for 1 min. Cyclic voltammetry was used to detect the ability of the nanowire-nanotube hybrid sensor to measure the 4-aminophenol molecules released by glycosidase cleavage. The detection principle is as follows: Due to the weak electrochemical activity of the substrate itself, no obvious oxidation signal can be observed only in the substrate solution. Figure 5(Dashed line). When the substrate and its corresponding glycosidase are mixed and reacted, the glycosidase recognizes and cleaves the glycosidic bond of p-aminophenol on the substrate, thereby releasing the highly electroactive molecule p-aminophenol. Furthermore, carbon conductive nanowires can detect an oxidation signal similar to that of p-aminophenol. Figure 5 (Colored curves). The above results demonstrate that the carbon conductive nanowire-nanotube hybrid sensor has a sensitive detection capability for 4-aminophenol produced by the reaction of glycosidase and substrate.

[0036] Test 2: 10 mM of 4-aminophenyl-β-D-glucopyranoside and 4-aminophenyl-β-D-galactopyranoside were mixed with β-glucosidase and β-galactosidase of corresponding different enzyme activities and reacted at 25 °C. A carbon conductive nanowire-nanotube hybrid sensor was used to apply a +600 mV potential (vs Ag / AgCl) to monitor the amperometric signal generated in real time. Subsequently, the amperometric signal was integrated over 1 min of reaction to obtain the corresponding charge value, and a standard curve of enzyme activity versus charge was further plotted, as shown below. Figure 6 As shown in the figure. This enzyme activity-charge standard curve is used to quantitatively calculate glycosidase activity in cell assays.

[0037] Test 3: The release of glycosidases from cells is simulated using single cone-shaped nanotubes filled with glycosidases. For example... Figure 7 As shown in Figure A, the nanotubes of the nanowire-nanotube hybrid sensor are loaded with substrates containing electroactive groups, while the individual nanotubes are loaded with the corresponding glycosidases. Using a micromanipulator, the nanowire-nanotube hybrid sensor and the individual nanotubes are moved to a distance of 1 μm between their tips. The solutions of both are then released via a microinjection apparatus (microinjection parameters: Pi = 10⁻¹⁰⁰ hPa, t = 3 s, Pc = 5 hPa). The nanowire-nanotube hybrid sensor detects the generated ampere signal in real time at a potential of +600 mV (vs Ag / AgCl). Figure 7 As shown in B, the nanowire-nanotube hybrid sensor has a sensitive detection capability for simulated glycosidase release.

[0038] Example 4 The application of a carbon conductive nanowire-nanotube hybrid sensor for quantitative monitoring of glycosidase release at the phagocytic orifice during macrophage phagocytosis of glass fibers includes the following steps: (1) A carbon conductive nanowire-nanotube hybrid sensor was constructed using the method of Example 1; (2) Using a micro-injection gun with a microloader (Eppendorf, Germany), the substrate solution of the enzyme to be tested (at least one of 4-aminophenyl-β-D-glucopyranoside and 4-aminophenyl-β-D-galactopyranoside) is loaded into the nanotube of the nanowire-nanotube hybrid sensor.

[0039] (3) Glass nanofibers were incubated on RAW 264.7 macrophages seeded in small culture dishes to induce them to form a frustrated phagocytic model. The small culture dishes were then placed on the platform of an inverted microscope.

[0040] (4) All electrochemical monitoring was performed in an inverted fluorescence microscope equipped with a 40× objective lens, a micromanipulator, a microinjector, and a grounded Faraday cage with a patch clamp. A self-made Ag / AgCl electrode was used as the reference electrode in a dual-electrode system throughout the experiment. The holder fixing the nanowire-nanotube hybrid sensor was connected to the patch clamp amplifier and then connected to the microinjector via a flexible tube to form a sealed system. Figure 8 As shown, the carbon conductive nanowire-nanotube hybrid sensor was positioned at the phagocytic opening of a macrophage that was engulfing glass fibers with the aid of a micromanipulator.

[0041] (5) The detection potential of the patch clamp was set to 600 mV and the detection sampling frequency was 1 kHz during the electrochemical monitoring process.

[0042] (6) Apply air pressure through a microinjector to precisely inject the substrate solution into the macrophage phagocytic port. The microinjector parameters are: Pi=10-100 hPa, t=3s, Pc=5 hPa. The patch clamp records the ampere signal during the experiment in real time.

[0043] (7) For example Figure 9 As shown in Figure A, this carbon conductive nanowire-nanotube hybrid sensor can detect the ampere signals generated by β-glucosidase and β-galactosidase. By integrating the ampere signals and using the enzyme activity-charge standard curve equation obtained in Example 3, quantitative information about enzyme activity can be calculated. Figure 9 As shown in B. This method can also be used to quantify the activities of a series of glycosidases, such as α-glucosidase and α-mannosidase.

[0044] Example 5 A bio-enzyme-modified platinum conductive nanowire-nanotube hybrid sensor is used to monitor the release of glycosidases at the phagocytic opening during the frustrated phagocytosis of glass fibers by macrophages.

[0045] like Figure 10As shown, this embodiment uses the bio-enzyme-modified platinum conductive nanowire-nanotube hybrid sensor prepared in Example 2. The nanowire electrode of this bio-enzyme-modified platinum conductive nanowire-nanotube hybrid sensor consists of silicon carbide nanowires coated with platinum particles and modified glucose oxidase. The second microtube releases a substrate without electroactive groups. This substrate reacts with glycosidase secreted by lysosomes to generate glucose, which is further converted by glucose oxidase to H2O2. H2O2 is oxidized on the surface of the platinum particles. During the oxidation process, electron transfer occurs, enabling the detection of an electrical signal, thereby achieving electrochemical detection of glycosidase released by lysosomes secreted by living cells.

[0046] Following the same operational steps as in Example 4, after the bio-enzyme-modified nanowire-nanotube hybrid sensor is positioned at the macrophage phagocytic port, it releases octyl-β-D-glucopyranoside and lactose, respectively. Ampere signals generated by β-glucosidase and β-galactosidase can be detected. Figure 11 As shown in A; integrating its amperometric signal can yield information on the electrical quantities produced by β-glucosidase and β-galactosidase, such as Figure 11 As shown in B, the hybrid sensor modified with bio-enzymes involves two enzymatic reactions in the glycosidase detection process, resulting in low detection efficiency. It can only obtain information on the electrical charge generated by glycosidase conversion, making it difficult to accurately quantify enzyme activity.

[0047] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanowire-nanotube hybrid sensor, characterized in that, include: The first micrometer tube has liquid metal injected into one end to form a cap, and conductive nanowires are placed at the other end. One end of the conductive nanowire is connected to the liquid metal and sealed, while the other end extends out into a first micron tube; The second microtube is a through-tube that connects to the first microtube to form a parallel dual-channel micro-nanotube. The first microtube and the second microtube are independent working tubes.

2. The nanowire-nanotube hybrid sensor according to claim 1, characterized in that, The conductive nanowire comprises a nanowire and a conductive layer covering the nanowire; the conductive layer is carbon.

3. A bio-enzyme-modified nanowire-nanotube hybrid sensor, characterized in that, The invention includes the nanowire-nanotube hybrid sensor of claim 2; the conductive nanowire comprises a nanowire and a conductive layer coated on the nanowire; the conductive layer is platinum particles; and the conductive nanowire is coated with a bio-enzyme modified layer.

4. The bio-enzyme-modified nanowire-nanotube hybrid sensor according to claim 3, characterized in that, The bio-enzyme modified layer is coated onto the conductive nanowires via enzyme cross-linking and includes a bio-enzyme, a cross-linking reagent, and an enzyme-stabilizing protein.

5. The bio-enzyme-modified nanowire-nanotube hybrid sensor according to claim 3, characterized in that, The bioenzyme includes at least one of glucose oxidase and galactose oxidase.

6. The method for fabricating the nanowire-nanotube hybrid sensor as described in claim 2, characterized in that, Includes the following steps: First and second micro-nanotubes are drawn to form parallel dual-channel micro-nanotubes; A conductive layer is coated onto nanowires to form conductive nanowires; Liquid metal was injected into one end of the first micro / nanotube for sealing; One end of the conductive nanowire is connected to the liquid metal, and the other end extends out into the first micro-nano tube opening; Gas is introduced into the second micro-nanotube, and the portion of the conductive nanowire located inside the first micro-nanotube is sealed.

7. The method for preparing the bio-enzyme-modified nanowire-nanotube hybrid sensor according to any one of claims 3-5, characterized in that, Includes the following steps: First and second micro-nanotubes are drawn to form parallel dual-channel micro-nanotubes; A conductive layer is coated onto nanowires to form conductive nanowires; Bioenzymes were immobilized on a conductive layer using an enzyme cross-linking method to obtain bioenzyme-modified conductive nanowires. Liquid metal was injected into one end of the first micro / nanotube for sealing; One end of a bio-enzyme-modified conductive nanowire is connected to liquid metal, and the other end extends out into a first micro-nanotube opening. Gas was introduced into the second micro-nanotube, and the portion of the bio-enzyme-modified conductive nanowire located inside the first micro-nanotube was sealed.

8. The application of the nanowire-nanotube hybrid sensor of claim 2 in the quantitative detection of enzyme activity during the reaction of substrate and glycosidase to generate electrochemically active small molecules, characterized in that, Application methods include: A glycosidase-specific substrate solution with electrochemically active molecules was released using a second-micron tube. The electrical signals of electroactive molecules released by the reaction of glycosidase with the specific substrate are monitored in real time using a first-micron tube. The corresponding electrical quantity value is obtained by calculating based on the electrical signal; Plot a standard curve of enzyme activity versus charge value to quantitatively calculate glycosidase activity.

9. The application according to claim 8, characterized in that, The glycosidase-specific substrate solution comprises: a glycoside molecule formed by linking a glycosidase recognition group and an electrochemically active unit via a glycosidic bond; the electrochemically active unit comprises at least one of 4-aminophenol and dopamine; and the glycosidase recognition group is a specific recognition group of the glycosidase.

10. The application of the bio-enzyme-modified nanowire-nanotube hybrid sensor according to any one of claims 3-5 in detecting lysosomal glycosidase release at the single or subcellular level, characterized in that, The application method includes the following steps: Release of glycosidase substrate solution using a second-micron tube; The electrochemical signal of hydrogen peroxide converted from glycosidases released by single or subcellular cells after reacting with substrates and bio-enzyme-modified layers was monitored in real time using platinum particles in a first-micron tube.

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