Graphene nano-wall electrochemical sensor with photo-thermal enhanced enzymatic activity and preparation and application of graphene nano-wall electrochemical sensor

Abstract

The invention relates to the technical field of biosensing, in particular to a graphene nano-wall electrochemical sensor with photo-thermal enhanced enzymatic activity as well as preparation and application of the graphene nano-wall electrochemical sensor. The electrochemical sensor comprises a reference electrode, a counter electrode and a working electrode, the electrochemical sensor is connected with an electrochemical workstation during use, the electrochemical sensor further comprises an insulating substrate, a graphene nano-wall, oxidase, a polymer film and a sample adding groove, the working electrode and the counter electrode are arranged on the upper surface of the insulating substrate, the graphene nano-wall is arranged on the upper surface of the working electrode, and the oxidase is arranged on the upper surface of the polymer film. The heat exchanger is used for receiving light energy and converting the light energy into heat energy; oxidase is connected with the graphene nano wall through a connecting molecule to serve as a recognition probe. The polymer film is arranged on the outer surfaces of the graphene nano wall and the oxidase and serves as a protective layer; the sample adding groove is formed in the upper surface of the insulating substrate, surrounds the working electrode and the counter electrode and is used for sample adding of an object to be detected. The electrochemical sensor has the advantages of high sensitivity, high specificity and portability.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, in particular to a graphene nano-wall electrochemical sensor for photothermal enhancement of enzyme activity and the preparation and application thereof. Background Art

[0002] Electrochemical sensing technology is an analytical method based on electrochemical principles, detecting the presence and concentration changes of substances by monitoring current, voltage, impedance, or charge. Enzyme-based electrochemical sensors are widely used to detect disease markers such as blood glucose, uric acid, and lactate. Due to their high specificity, label-free nature, and rapid response, these sensors hold great potential for periodic clinical testing of related diseases.

[0003] However, current enzyme-based electrochemical sensors have difficulty detecting trace amounts of biomarkers due to their low sensitivity. Biomarkers for some diseases are usually present at low concentrations, so the lack of device sensitivity can limit the detection of some diseases. Nanomaterials can improve the sensitivity of electrochemical sensors due to their efficient charge transfer and catalytic capabilities. Previous researchers have also tried to use nanomaterials such as graphene nanowalls, carbon nanotubes, graphene, and molybdenum sulfide as working electrode materials for electrochemical sensors, but the electrical properties of these nanomaterials have limited effect on improving device sensitivity, and their detection limits are usually in the micromolar range. Therefore, improving the catalytic activity of enzymes has become a better choice for improving device sensitivity. Summary of the Invention

[0004] To address the insufficient sensitivity of traditional enzyme-based electrochemical sensors, the present invention provides a graphene nanowall electrochemical sensor with photothermal enhancement of enzyme activity, as well as its preparation and application. This photothermal enhancement of enzyme activity graphene nanowall electrochemical sensor can sensitively and efficiently detect all small-molecule disease markers catalyzed by oxidases. Compared with currently used traditional detection methods, it offers higher sensitivity, better specificity, and stronger anti-interference capabilities, promising promising applications.

[0005] Existing nanomaterials have very strong light absorption properties, such as graphene nanowalls, bismuth selenide, and molybdenum sulfide nanosheets. These materials can convert light energy into heat, thereby raising the temperature of the enzyme's environment and enabling it to reach optimal catalytic activity. Therefore, combining nanomaterials with strong photothermal conversion capabilities with enzymes as working electrodes for electrochemical sensors, using light-assisted methods, can significantly improve the sensitivity of electrochemical sensors, enabling precise clinical testing with high specificity, high sensitivity, and rapid response.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity, comprising a reference electrode, a counter electrode, and a working electrode, which is connected to an electrochemical workstation when in use. The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity also comprises an insulating substrate, a graphene nanowall, an enzyme probe, a polymer film, and a sample loading tank.

[0008] A working electrode and a counter electrode are provided on the upper surface of the insulating substrate. The graphene nanowall is provided on the upper surface of the working electrode for receiving light energy and converting the light energy into heat energy. The enzyme probe is connected to the graphene nanowall via a connecting molecule and serves as an identification probe. The polymer film is provided on the outer surface of the graphene nanowall and the enzyme probe and serves as a protective layer. The sample loading groove is provided on the upper surface of the insulating substrate and surrounds the working electrode and the counter electrode for loading the sample of the object to be tested.

[0009] In one embodiment of the present invention, the material of the insulating substrate is selected from one or more of polyimide, polyethylene terephthalate, polydimethylsiloxane or silicon / silicon dioxide;

[0010] The reference electrode is a silver / silver chloride electrode;

[0011] The counter electrode and the working electrode are both patterned electrodes with a thickness of 5 to 1000 nm;

[0012] The material of the counter electrode is selected from one or more of conductive metals or conductive metal polymers;

[0013] The material of the working electrode is selected from one or more of conductive metals or conductive metal polymers.

[0014] In one embodiment of the present invention, the conductive metal is selected from one of gold, silver, copper, nickel, titanium, chromium or aluminum.

[0015] In one embodiment of the present invention, the graphene nanowall is a graphene nanowall treated with oxygen plasma (to increase the surface active sites of the graphene nanowall), and has a thickness of 0.1 to 50 μm.

[0016] In one embodiment of the present invention, the graphene nanowall material treated with oxygen plasma will increase in temperature and stabilize in the range of 30 to 40°C after absorbing light. This temperature is exactly the condition for the highest enzyme catalytic activity, so that the rate of electrochemical reaction of the analyte under enzyme catalysis reaches the fastest, thereby obtaining the maximum electrochemical reaction current signal.

[0017] In one embodiment of the present invention, the linker molecule is selected from one of an organic small molecule or a gold nanoparticle;

[0018] The enzyme probe is an oxidase, hydrolase or transferase for detecting a target;

[0019] The enzyme probe is selected from one or more of glucose oxidase, catalase, urate oxidase, lactate oxidase, acetylcholinesterase, choline oxidase, sarcosine oxidase, alkaline phosphatase or ethanolamine transferase;

[0020] The material of the polymer film is selected from one of chitosan, perfluorosulfonic acid or polydopamine;

[0021] The material of the sample loading groove is selected from one of polydimethylsiloxane, epoxy resin or polyurethane.

[0022] In one embodiment of the present invention, the organic molecule is selected from one of 1-pyrenebutyric acid succinimidyl ester, biotin or polyethylene glycol.

[0023] A second object of the present invention is to provide a method for preparing the above-mentioned graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity, comprising the following steps:

[0024] (A1) preparing a working electrode and a counter electrode on an insulating substrate, and preparing a reference electrode;

[0025] (A2) placing a graphene nanowall on the upper surface of the working electrode prepared in step (A1), modifying the linker molecule after treating the graphene nanowall with oxygen plasma, fixing the enzyme probe to the linker molecule, and then coating the enzyme probe and the graphene nanowall surface with a polymer. Finally, placing a sample loading groove on the upper surface of the insulating substrate and on the outside of the working electrode and the counter electrode to obtain a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity.

[0026] In one embodiment of the present invention, the linker molecule modification method involves immersing the electrochemical sensor device with the graphene nanowall in an organic small molecule solution or spraying it with gold nanoparticles, thereby modifying the linker molecule on the graphene nanowall surface. The enzyme probe is then reacted with the linker molecule through a chemical reaction, thereby ensuring that the enzyme probe is modified on the graphene nanowall surface. Alternatively, the enzyme probe can be loaded onto the graphene nanowall surface by physical adsorption (dropping an enzyme solution onto the graphene nanowall surface, where it automatically adsorbs due to intermolecular van der Waals forces) or encapsulation (dropping an enzyme onto the graphene wall surface and then encapsulating it with a film such as chitosan).

[0027] In one embodiment of the present invention, the enzyme probe fixation method is specifically as follows: direct modification, the enzyme probe is prepared into a solution with a concentration of 1 to 100 U per milliliter, the electrochemical sensor device with graphene nanowalls is immersed in the enzyme probe solution with a concentration of 1 to 100 U per milliliter at room temperature for 2 to 12 hours, and then rinsed with buffer.

[0028] In the present invention, the graphene nano-wall electrochemical sensor with photothermal enhanced enzyme activity is stored at a low temperature with a humidity of 50 to 90% when not in use.

[0029] The third object of the present invention is to provide a method for detecting a target in an analyte using the above-mentioned graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity, the method being a cyclic voltammetry test method, comprising the following steps:

[0030] (B1) Connecting the counter electrode, reference electrode, and working electrode to an electrochemical workstation and irradiating the graphene nanowall with light;

[0031] (B2) adding a buffer solution dropwise onto the surface of the graphene nanowall, scanning a given working electrode voltage range, and starting the test solution test when no redox peak appears in the cyclic voltammetry curve;

[0032] (B3) adding a sample solution onto the surface of the graphene nanowall, causing the target substance in the sample to undergo an oxidation-reduction reaction under the catalysis of the enzyme probe, resulting in a corresponding electrochemical current signal being generated on the surface of the graphene nanowall;

[0033] 10 to 30 seconds after adding the test solution, perform a cyclic voltammetry scan and compare it with the cyclic voltammetry curve of the solution without adding the test solution, and use the change of the redox current peak as the signal;

[0034] (B4) When the redox peak appears, it indicates that the target is detected; when the redox peak does not appear, it indicates that the target is not detected.

[0035] In one embodiment of the present invention, in step (B1), during the light irradiation process, the wavelength of light is 360 to 1500 nm;

[0036] In step (B3), during the redox reaction, the wavelength of light is 360 to 1500 nm.

[0037] A fourth object of the present invention is to provide a method for detecting a target in an analyte using the above-mentioned graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity, the method being a current-time test method comprising the following steps:

[0038] (C1) Connecting the counter electrode, reference electrode, and working electrode to an electrochemical workstation and irradiating the graphene nanowall with light;

[0039] (C2) Buffer solution is added to the surface of the graphene nanowall, and a fixed working electrode voltage is given to keep the current constant. The current change caused by the current noise is recorded as ΔI 非待测 , and use this as the signal response value of the non-DUT;

[0040] (C3) The target solution is added to the surface of the graphene nanowall. The target undergoes an oxidation-reduction reaction under the catalysis of the enzyme probe, causing the corresponding electrochemical current signal to be generated on the surface of the graphene nanowall. When the current-time test mode is used, after the target solution is added, the current response reaches equilibrium and stability over time, and the current change value is read: ΔI 待测 , and use this as the signal response value of the object to be measured;

[0041] (C4) When ΔI 待测 Greater than 3ΔI 非待测 When , it indicates that the target is detected; when ΔI 待测 Less than or equal to 3ΔI 非待测 , indicating that the target was not detected.

[0042] In one embodiment of the present invention, in step (C1), during the light irradiation process, the wavelength of light is 360 to 1500 nm;

[0043] In step (C3), during the redox reaction, the wavelength of light is 360 to 1500 nm.

[0044] In the present invention, the graphene nanowall in the graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity is subjected to oxygen plasma treatment in order to increase the active sites of the graphene nanowall, thereby connecting with more linker molecules and further connecting with more enzyme probes; when in use, the graphene nanowall is irradiated with light, and the graphene nanowall absorbs light energy and converts it into heat energy, thereby raising the ambient temperature to 30-40°C, so that the enzyme modified on the surface of the graphene nanowall is in a state of highest catalytic activity, thereby improving the efficiency of the electrochemical reaction of the analyte and achieving the purpose of improving sensitivity.

[0045] Graphene nanowall electrochemical sensors with photothermal enhanced enzyme activity offer the advantages of high sensitivity, high specificity, and portability. During detection, the sample is added to the working electrode surface of the electrochemical sensor, causing the enzyme modified on the sensor to catalyze the analyte, resulting in an electrochemical reaction.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] In the graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity of the present invention, the graphene nanowall absorbs light energy to increase the temperature of the material itself and the surrounding area to a range of 30-40°C, thereby placing the enzyme modified on the surface of the graphene nanowall in a state of highest catalytic activity, improving the efficiency of the electrochemical reaction of the analyte and achieving the purpose of improving sensitivity.

[0048] The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity of the present invention has the advantages of ultrahigh sensitivity (detection limit of attomole level), high specificity, portability and low cost, and broadens the detection and application scope of traditional electrochemical sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a schematic diagram of the partial structure of the graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity described in Example 1; the numbers in the figure are: 1, insulating substrate; 2, working electrode; 3, counter electrode; 4, sample loading tank; 5, graphene nanowall; 6, enzyme probe.

[0050] Figure 2 This is a schematic diagram of the structure of the graphene nanowall and oxidase in the graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity described in Example 1; the numbers in the figure are: 5, graphene nanowall; 6, enzyme probe.

[0051] Figure 3 This is a temperature comparison diagram of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity described in Example 1 before and after illumination;

[0052] Figure 4 This is the redox peak current-concentration response curve of the electrochemical sensor detecting glucose in Example 3;

[0053] Figure 5 This is the cyclic voltammetry curve of the electrochemical sensor detecting glucose in Example 3;

[0054] Figure 6 Schematic diagram of specific test of glucose detection by electrochemical sensor in Example 3;

[0055] Figure 7 Schematic diagram of the stability test of the electrochemical sensor for detecting glucose in Example 3;

[0056] Figure 8 This is the cyclic voltammetry curve of the electrochemical sensor detecting lactic acid in Example 5;

[0057] Figure 9 Schematic diagram of the specificity test of the electrochemical sensor for detecting lactic acid in Example 5;

[0058] Figure 10 This is the cyclic voltammetry curve of the electrochemical sensor for detecting uric acid in Example 7;

[0059] Figure 11 Schematic diagram of the specific test of uric acid detection by the electrochemical sensor in Example 7;

[0060] Figure 12 This is the cyclic voltammetry curve of the electrochemical sensor detecting acetylcholine in Example 11;

[0061] Figure 13 Schematic diagram of the specificity test of the electrochemical sensor for detecting acetylcholine in Example 11. DETAILED DESCRIPTION

[0062] The present invention provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity, comprising a reference electrode, a counter electrode and a working electrode, which is connected to an electrochemical workstation when in use. The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity also comprises an insulating substrate, a graphene nanowall, an enzyme probe, a polymer film and a sample loading tank.

[0063] A working electrode and a counter electrode are provided on the upper surface of the insulating substrate. The graphene nanowall is provided on the upper surface of the working electrode for receiving light energy and converting the light energy into heat energy. The enzyme probe is connected to the graphene nanowall via a connecting molecule and serves as an identification probe. The polymer film is provided on the outer surface of the graphene nanowall and the enzyme probe and serves as a protective layer. The sample loading groove is provided on the upper surface of the insulating substrate and surrounds the working electrode and the counter electrode for loading the sample of the object to be tested.

[0064] In one embodiment of the present invention, the material of the insulating substrate is selected from one or more of polyimide, polyethylene terephthalate, polydimethylsiloxane or silicon / silicon dioxide;

[0065] The reference electrode is a silver / silver chloride electrode;

[0066] The counter electrode and the working electrode are both patterned electrodes with a thickness of 5 to 1000 nm;

[0067] The material of the counter electrode is selected from one or more of conductive metals or conductive metal polymers;

[0068] The material of the working electrode is selected from one or more of conductive metals or conductive metal polymers.

[0069] In one embodiment of the present invention, the conductive metal is selected from one of gold, silver, copper, nickel, titanium, chromium or aluminum.

[0070] In one embodiment of the present invention, the graphene nanowall is a graphene nanowall treated with oxygen plasma (to increase the surface active sites of the graphene nanowall), and has a thickness of 0.1 to 50 μm.

[0071] In one embodiment of the present invention, the graphene nanowall material treated with oxygen plasma will increase in temperature and stabilize in the range of 30 to 40°C after absorbing light. This temperature is exactly the condition for the highest enzyme catalytic activity, so that the rate of electrochemical reaction of the analyte under enzyme catalysis reaches the fastest, thereby obtaining the maximum electrochemical reaction current signal.

[0072] In one embodiment of the present invention, the linker molecule is selected from one of an organic small molecule or a gold nanoparticle;

[0073] The enzyme probe is an oxidase, hydrolase or transferase for detecting a target;

[0074] The enzyme probe is selected from one or more of glucose oxidase, catalase, urate oxidase, lactate oxidase, acetylcholinesterase, choline oxidase, sarcosine oxidase, alkaline phosphatase or ethanolamine transferase;

[0075] The material of the polymer film is selected from one of chitosan, perfluorosulfonic acid or polydopamine;

[0076] The material of the sample loading groove is selected from one of polydimethylsiloxane, epoxy resin or polyurethane.

[0077] In one embodiment of the present invention, the organic molecule is selected from one of 1-pyrenebutyric acid succinimidyl ester, biotin or polyethylene glycol.

[0078] The present invention provides a method for preparing the above-mentioned graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity, comprising the following steps:

[0079] (A1) preparing a working electrode and a counter electrode on an insulating substrate, and preparing a reference electrode;

[0080] (A2) placing a graphene nanowall on the upper surface of the working electrode prepared in step (A1), modifying the linker molecule after treating the graphene nanowall with oxygen plasma, fixing the enzyme probe to the linker molecule, and then coating the enzyme probe and the graphene nanowall surface with a polymer. Finally, placing a sample loading groove on the upper surface of the insulating substrate and on the outside of the working electrode and the counter electrode to obtain a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity.

[0081] In one embodiment of the present invention, the linker molecule modification method involves immersing the electrochemical sensor device with the graphene nanowall in an organic small molecule solution or spraying it with gold nanoparticles, thereby modifying the linker molecule on the graphene nanowall surface. The enzyme probe is then reacted with the linker molecule through a chemical reaction, thereby ensuring that the enzyme probe is modified on the graphene nanowall surface. Alternatively, the enzyme probe can be loaded onto the graphene nanowall surface by physical adsorption (dropping an enzyme solution onto the graphene nanowall surface, where it automatically adsorbs due to intermolecular van der Waals forces) or encapsulation (dropping an enzyme onto the graphene wall surface and then encapsulating it with a film such as chitosan).

[0082] In one embodiment of the present invention, the enzyme probe fixation method is specifically as follows: direct modification, the enzyme probe is prepared into a solution with a concentration of 1 to 100 U per milliliter, the electrochemical sensor device with graphene nanowalls is immersed in the enzyme probe solution with a concentration of 1 to 100 U per milliliter at room temperature for 2 to 12 hours, and then rinsed with buffer.

[0083] In the present invention, the graphene nano-wall electrochemical sensor with photothermal enhanced enzyme activity is stored at a low temperature with a humidity of 50 to 90% when not in use.

[0084] The present invention provides a method for detecting a target in an analyte using the above-mentioned graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity. The method is a cyclic voltammetry test method, comprising the following steps:

[0085] (B1) Connecting the counter electrode, reference electrode, and working electrode to an electrochemical workstation and irradiating the graphene nanowall with light;

[0086] (B2) adding a buffer solution dropwise onto the surface of the graphene nanowall, scanning a given working electrode voltage range, and starting the test solution test when no redox peak appears in the cyclic voltammetry curve;

[0087] (B3) adding a sample solution onto the surface of the graphene nanowall, causing the target substance in the sample to undergo an oxidation-reduction reaction under the catalysis of the enzyme probe, resulting in a corresponding electrochemical current signal being generated on the surface of the graphene nanowall;

[0088] 10 to 30 seconds after adding the test solution, perform a cyclic voltammetry scan and compare it with the cyclic voltammetry curve of the solution without adding the test solution, and use the change of the redox current peak as the signal;

[0089] (B4) When the redox peak appears, it indicates that the target is detected; when the redox peak does not appear, it indicates that the target is not detected.

[0090] In one embodiment of the present invention, in step (B1), during the light irradiation process, the wavelength of light is 360 to 1500 nm;

[0091] In step (B3), during the redox reaction, the wavelength of light is 360 to 1500 nm.

[0092] The present invention provides a method for detecting a target in an analyte using the above-mentioned graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity. The method is a current-time test method, comprising the following steps:

[0093] (C1) Connecting the counter electrode, reference electrode, and working electrode to an electrochemical workstation and irradiating the graphene nanowall with light;

[0094] (C2) Buffer solution is added to the surface of the graphene nanowall, and a fixed working electrode voltage is given to keep the current constant. The current change caused by the current noise is recorded as ΔI 非待测 , and use this as the signal response value of the non-DUT;

[0095] (C3) The target solution is added to the surface of the graphene nanowall. The target undergoes an oxidation-reduction reaction under the catalysis of the enzyme probe, causing the corresponding electrochemical current signal to be generated on the surface of the graphene nanowall. When the current-time test mode is used, after the target solution is added, the current response reaches equilibrium and stability over time, and the current change value is read: ΔI 待测 , and use this as the signal response value of the object to be measured;

[0096] (C4) When ΔI 待测 Greater than 3ΔI 非待测 When , it indicates that the target is detected; when ΔI 待测 Less than or equal to 3ΔI 非待测 , indicating that the target was not detected.

[0097] In one embodiment of the present invention, in step (C1), during the light irradiation process, the wavelength of light is 360 to 1500 nm;

[0098] In step (C3), during the redox reaction, the wavelength of light is 360 to 1500 nm.

[0099] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0100] In the description of the present invention, unless otherwise expressly specified or limited, the terms "connected," "connected," and "fixed" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integration; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention in specific circumstances.

[0101] In the present invention, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Furthermore, a first feature being "above," "above," and "above" a second feature may include the first feature being directly above or obliquely above the second feature, or may simply mean that the first feature is higher in level than the second feature. A first feature being "below," "below," and "below" a second feature may include the first feature being directly below or obliquely below the second feature, or may simply mean that the first feature is lower in level than the second feature.

[0102] In the description of this embodiment, terms such as "upper," "lower," "left," and "right" are used to refer to positions or locations based on those shown in the accompanying drawings. These terms are intended solely to facilitate description and simplify operation, and are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used solely for descriptive purposes and have no special meaning.

[0103] In the following examples, unless otherwise specified, the reagents are all commercially available reagents, and the detection, operation means and methods used are all conventional detection, operation means and methods in the art.

[0104] Example 1

[0105] This embodiment provides a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity, such as Figure 1-2 As shown, it includes a reference electrode, a counter electrode 3 and a working electrode 2. When in use, the reference electrode, the counter electrode 3 and the working electrode 2 are connected to the electrochemical workstation; the graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity also includes an insulating substrate 1, a graphene nanowall 5, an enzyme probe 6, a polymer film and a sample loading groove 4. The working electrode 2 and the counter electrode 3 are provided on the upper surface of the insulating substrate 1. The graphene nanowall 5 is provided on the upper surface of the working electrode 2 for receiving light energy and converting the light energy into heat energy; the enzyme probe 6 is connected to the graphene nanowall 5 through a connecting molecule and serves as an identification probe; the polymer film is provided on the outer surface of the graphene nanowall 5 and the enzyme probe 6 as a protective layer; the sample loading groove 4 is provided on the upper surface of the insulating substrate 1 and surrounds the working electrode 2 and the counter electrode 3 for loading the sample of the object to be tested.

[0106] Furthermore, the material of the insulating substrate 1 is selected from one or more of polyimide, polyethylene terephthalate, polydimethylsiloxane or silicon / silicon dioxide; the reference electrode is a silver / silver chloride electrode; the counter electrode 3 and the working electrode 2 are both patterned electrodes with a thickness of 5 to 1000 nm; the material of the counter electrode 3 is selected from one or more of conductive metals or conductive metal polymers; the material of the working electrode 2 is selected from one or more of conductive metals or conductive metal polymers; preferably, the conductive metal is selected from one of gold, silver, copper, nickel, titanium, chromium or aluminum.

[0107] Furthermore, the graphene nanowall 5 is a graphene nanowall treated with oxygen plasma (increasing the surface active sites of the graphene nanowall), and its thickness is 0.1 to 50 μm; after absorbing light, the temperature of the graphene nanowall material treated with oxygen plasma will rise and stabilize in the range of 30 to 40°C ( Figure 3), this temperature is the condition for the highest enzyme catalytic activity, which makes the rate of electrochemical reaction of the analyte under enzyme catalysis reach the fastest, thereby obtaining the maximum electrochemical reaction current signal.

[0108] Furthermore, the connecting molecule is selected from one of organic small molecules or gold nanoparticles; the organic molecule is selected from one of 1-pyrenebutyric acid succinimidyl ester, biotin or polyethylene glycol; the enzyme probe 6 is a target oxidase for detecting the target; preferably, the enzyme probe 6 is selected from one of glucose oxidase, catalase, urate oxidase, lactate oxidase, acetylcholine oxidase or sarcosine oxidase; the material of the polymer film is selected from one of chitosan, perfluorosulfonic acid or polydopamine; the material of the sample loading groove 4 is selected from one of polydimethylsiloxane, epoxy resin or polyurethane.

[0109] Example 2

[0110] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0111] (S1) Patterned working electrodes (gold: thickness 25 nm) and counter electrodes (gold: thickness 25 nm) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0112] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0113] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0114] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank was placed above the working electrode, and then 100 μL of glucose oxidase solution (concentration of 50 U) was added. After incubation for 6 hours, the solution was washed with phosphate buffer solution, and finally 10 μL of perfluorosulfonic acid solution was drop-coated on the surface of the graphene nanowall and glucose oxidase. The solution was allowed to dry to form a film, thereby obtaining a graphene nanowall electrochemical sensor for detecting photothermal enhanced enzyme activity of glucose.

[0115] Example 3

[0116] This example provides an application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 2 in detecting glucose using a cyclic voltammetry test method.

[0117] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity prepared in Example 1 to an electrochemical workstation, and irradiating the graphene nanowall with 980 nm light (50 mW light power) for one minute;

[0118] (R2) 40 μL of PBS solution was added to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.4 V. When no redox peak appeared in the cyclic voltammetry test, glucose detection was started;

[0119] (R3) During the test, first use it, then add 40 μL of glucose sample on the surface of the graphene nanowall, and then irradiate the graphene nanowall with white light for 1 minute. The test concentration is 3×10 -18 mol / L to 3×10 -3 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0120] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0121] Figure 4 The redox peak current-concentration response curve of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity in Example 3 for detecting glucose is shown in FIG. Figure 4 It can be found that the electrochemical sensor constructed in Example 2 has a very wide response range to glucose.

[0122] Figure 5 The cyclic voltammetry curve of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity in Example 3 is shown in FIG. Figure 5 It can be found that with the assistance of light, the sensitivity of the electrochemical sensor is greatly improved.

[0123] Figure 6 The specific detection of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity of Example 3 is achieved by Figure 6 It can be found that the electrochemistry has significant specificity in glucose detection (the control group is PBS, KCl, ascorbic acid, uric acid, and acetylcholine; the experimental group is glucose).

[0124] Figure 7 The stability test of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity of Example 3 was conducted. Figure 7It can be found that the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity can still respond significantly to glucose (concentration of 30 μM) after being stored for 29 days and 75 days.

[0125] Example 4

[0126] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0127] (S1) Patterned working electrodes (silver: 25 nm thickness) and counter electrodes (gold: 25 nm thickness) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0128] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0129] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0130] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank was placed above the working electrode, and then 100 μL of lactate oxidase solution (concentration of 50 U) was added. After incubation for 6 hours, the solution was washed with phosphate buffer solution, and finally 10 μL of perfluorosulfonic acid solution was drop-coated on the graphene nanowall and lactate oxidase surface. After drying to form a film, a graphene nanowall electrochemical sensor for detecting photothermal enhanced enzyme activity of lactic acid was obtained.

[0131] Example 5

[0132] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 4 in the detection of lactose, using a cyclic voltammetry test method.

[0133] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 4 to an electrochemical workstation, and irradiating the graphene nanowall with a 1550 nm near-infrared laser (with an optical power of 50 mW) for one minute;

[0134] (R2) 40 μL of PBS solution was added to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.4 V. When no redox peak appeared during the cyclic voltammetry test, lactic acid detection was started;

[0135] (R3) During the test, the graphene nanowall was first used, and then 40 μL of a sample containing lactic acid was added to the surface of the graphene nanowall. The graphene nanowall was then irradiated with white light for 1 minute. The test concentration ranged from 1×10 -15 mol / L to 1×10 -4 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0136] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0137] Figure 8 This is the cyclic voltammetry curve of the electrochemical sensor. With the assistance of light, the detection limit of the electrochemical sensor has been greatly improved, and 1 femtomolar lactic acid solution can be detected.

[0138] Figure 9 This is the specific test data of the electrochemical sensor. Its response current to lactic acid is much larger than that of the control group (the control groups are NaCl, KCl, serotonin, dopamine, creatine, glucose, and uric acid).

[0139] Example 6

[0140] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0141] (S1) Patterned working electrodes (copper: 25 nm thickness) and counter electrodes (gold: 25 nm thickness) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0142] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0143] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0144] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank was placed above the working electrode, and then 100 μL of urate oxidase solution (concentration of 50 U) was added. After incubation for 6 hours, the solution was washed with phosphate buffer solution. Finally, 10 μL of perfluorosulfonic acid solution was drop-coated on the surface of the graphene nanowall and urate oxidase, and the solution was allowed to dry to form a film, thereby obtaining a graphene nanowall electrochemical sensor for detecting uric acid by photothermal enhancement of enzyme activity.

[0145] Example 7

[0146] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 6 in the detection of uric acid using a cyclic voltammetry test method.

[0147] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 6 to an electrochemical workstation, and irradiating the graphene nanowall with a 650 nm laser (with an optical power of 50 mW) for one minute;

[0148] (R2) 40 μL of PBS solution was added to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.4 V. When no redox peak appeared during the cyclic voltammetry test, uric acid detection was started;

[0149] (R3) During the test, 40 μL of uric acid sample was added to the surface of the graphene nanowall, and then the graphene nanowall was irradiated with white light for 1 minute. The test concentration ranged from 5×10 -15 mol / L to 5×10 -4 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0150] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0151] Figure 10 This is the cyclic voltammetry curve of the electrochemical sensor. With the assistance of light, the detection limit of the electrochemical sensor has been greatly improved, and 5 femtomolar uric acid solution can be detected.

[0152] Figure 11 This is the specific test data of the electrochemical sensor. Its response current to uric acid is much larger than that of the control group (the control groups are NaCl, KCl, serotonin, dopamine, creatine, glucose, and lactic acid).

[0153] Example 8

[0154] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0155] (S1) Patterned working electrodes (nickel: 25 nm thick) and counter electrodes (gold: 25 nm thick) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0156] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0157] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0158] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank is placed above the working electrode, and then 100 μL of sarcosine oxidase solution (concentration of 50 U) is added. After incubation for 6 hours, it is washed with phosphate buffer solution, and finally 10 μL of perfluorosulfonic acid solution is drop-coated on the surface of the graphene nanowall and sarcosine oxidase. After it is dried to form a film, a graphene nanowall electrochemical sensor for detecting photothermal enhanced enzyme activity of sarcosine is obtained.

[0159] Example 9

[0160] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 8 in detecting sarcosine using a current-time test method.

[0161] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 8 to an electrochemical workstation, and irradiating the graphene nanowall with a 1200 nm laser (with an optical power of 50 mW) for one minute;

[0162] (R2) 40 μL of PBS solution was added to the surface of the graphene nanowall. A fixed working electrode voltage was given to keep the current constant. The current change caused by the current noise was recorded as ΔI 非待测 , and use this as the signal response value of the non-DUT;

[0163] (R3) Add a sample containing sarcosine (test concentration ranges from 1×10 -15 moles / liter (fM) to 1×10 -4mol / L (0.1mM) (final concentration), the target (sarcosine) undergoes a redox reaction under the catalysis of sarcosine oxidase, causing a corresponding electrochemical current signal to be generated on the surface of the graphene nanowall. When using the current-time test mode, after adding a blood sample containing sarcosine, the current response reaches equilibrium and stability over time, and the current change value is read: ΔI 待测 , and use this as the signal response value of creatine;

[0164] (R4) When ΔI 待测 Greater than 3ΔI 非待测 When , it indicates that the target: sarcosine is detected; when ΔI 待测 Less than or equal to 3ΔI 非待测 , indicating that the target was not detected.

[0165] In this example, the test concentration of sarcosine was 1×10 -15 mol / L to 1×10 -4 mol / L (final concentration) (wherein, the concentration was tested from low to high, and the adjacent test concentration multiple was 10 times), the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity could detect sarcosine.

[0166] Example 10

[0167] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0168] (S1) Patterned working electrodes (titanium: 25 nm thick) and counter electrodes (gold: 25 nm thick) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0169] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0170] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0171] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank is placed above the working electrode, and then 100 μL of a mixed solution of acetylcholinesterase and choline oxidase is added (wherein the volume ratio of acetylcholinesterase solution to choline oxidase solution is 1:1, and the concentrations of both are 50 U). After incubation for 6 hours, the mixture is washed with phosphate buffer solution, and finally 10 μL of perfluorosulfonic acid solution is drop-coated on the surface of the graphene nanowall and the acetylcholinesterase and choline oxidase, and the mixture is allowed to dry to form a film, thereby obtaining a graphene nanowall electrochemical sensor for detecting photothermal enhanced enzyme activity of acetylcholine.

[0172] Example 11

[0173] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 10 in detecting acetylcholine using a cyclic voltammetry test method.

[0174] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 10 to an electrochemical workstation, and irradiating the graphene nanowall with a 600 nm laser (with an optical power of 50 mW) for one minute;

[0175] (R2) 40 μL of PBS solution was added to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.7 V. When no redox peak appeared during the cyclic voltammetry test, acetylcholine detection was started;

[0176] (R3) During the test, first use it, then add 40 μL of sample containing acetylcholine on the surface of the graphene nanowall, and then irradiate the graphene nanowall with white light for 1 minute. The test concentration ranges from 2×10 -10 mol / L to 2×10 -4 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0177] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0178] Figure 12 This is the cyclic voltammetry curve of the electrochemical sensor. With the assistance of light, the detection limit of the electrochemical sensor has been greatly improved, and 200 pmoles of acetylcholine solution can be detected.

[0179] Figure 13 This is the specific test data of the electrochemical sensor. Its response current to acetylcholine is much larger than that of the control group (the control groups are KCl, ascorbic acid, glucose, dopamine, and uric acid).

[0180] Example 12

[0181] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0182] (S1) Patterned working electrodes (aluminum: thickness 25 nm) and counter electrodes (gold: thickness 25 nm) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0183] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0184] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0185] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank is placed above the working electrode, and then 100 μL of catalase solution (concentration of 50 U) is added. After incubation for 6 hours, it is washed with phosphate buffer solution, and finally 10 μL of perfluorosulfonic acid solution is drop-coated on the graphene nanowall and catalase surface. After it is dried to form a film, a graphene nanowall electrochemical sensor for detecting photothermal enhanced enzyme activity of hydrogen peroxide is obtained.

[0186] Example 13

[0187] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 12 in detecting hydrogen peroxide using a cyclic voltammetry test method.

[0188] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 12 to an electrochemical workstation, and irradiating the graphene nanowall with an 800 nm laser (50 mW optical power) for 45 seconds;

[0189] (R2) 40 μL of PBS solution was added dropwise to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.7 V. When no redox peak appeared during the cyclic voltammetry test, hydrogen peroxide detection was started;

[0190] (R3) During the test, first use, then add 40 μL of a sample containing hydrogen peroxide on the surface of the graphene nanowall, and then irradiate the graphene nanowall with white light for 1 minute. The test concentration ranges from 3×10 -15 mol / L to 3×10 -4 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0191] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0192] Example 14

[0193] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0194] (S1) Patterned working electrodes (chromium: thickness 25 nm) and counter electrodes (gold: thickness 25 nm) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0195] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0196] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0197] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank is placed above the working electrode, and then 100 μL of alkaline phosphatase solution (concentration of 50 U) is added. After incubation for 6 hours, it is washed with phosphate buffer solution, and finally 10 μL of perfluorosulfonic acid solution is drop-coated on the graphene nanowall and alkaline phosphatase surface. After it is dried to form a film, a graphene nanowall electrochemical sensor for detecting the photothermal enhanced enzyme activity of lauryl alcohol monophosphate is obtained.

[0198] Example 15

[0199] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 14 in the detection of lauryl alcohol phosphate using a cyclic voltammetry test method.

[0200] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 14 to an electrochemical workstation, and irradiating the graphene nanowall with an 850 nm laser (50 mW optical power) for 45 seconds;

[0201] (R2) 40 μL of PBS solution was added dropwise to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.7 V. When no redox peak was observed during the cyclic voltammetry test, the lauryl alcohol phosphate monoester detection was started.

[0202] (R3) During the test, the sample containing phosphate monoester was first added to the surface of the graphene nanowall, and then the graphene nanowall was irradiated with white light for 1 minute. The test concentration ranged from 5×10 -15 mol / L to 5×10 -4 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0203] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0204] Example 16

[0205] This embodiment provides a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity and a preparation method thereof.

[0206] (S1) Patterned working electrodes (chromium: 25 nm thickness) and counter electrodes (gold: 50 nm thickness) were prepared on polyimide substrates using laser direct write photolithography, and a reference electrode was also prepared.

[0207] (S2) After step (S1), a 10-μm-thick graphene nanowall is prepared on a 25-μm-thick copper foil using chemical vapor deposition. The copper foil is dissolved by wet etching, and the prepared graphene nanowall is transferred onto the working electrode. The graphene nanowall is then patterned using micro-nanofabrication technology.

[0208] (S3) After step (S2), the product of step (S2) was treated in an oxygen plasma cleaner for 1 minute; then, it was immersed in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 6 hours, and then rinsed with ethanol and ultrapure water, respectively, and finally dried with nitrogen;

[0209] (S4) After step (S3), a polydimethylsiloxane (PDMS) tank was placed above the working electrode, and then 100 μL of ethanolamine transferase solution (concentration of 50 U) was added. After incubation for 6 hours, the solution was washed with phosphate buffer solution. Finally, 10 μL of perfluorosulfonic acid solution was drop-coated on the surface of the graphene nanowall and the ethanolamine transferase, and the solution was allowed to dry to form a film, thereby obtaining a graphene nanowall electrochemical sensor for detecting the photothermal enhanced enzyme activity of alanine.

[0210] Example 17

[0211] This example provides the application of the graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity prepared in Example 16 in the detection of alanine using a cyclic voltammetry test method.

[0212] (R1) Connecting the three electrodes of the graphene nanowall electrochemical sensor for photothermal enhanced enzyme activity prepared in Example 16 to an electrochemical workstation, and irradiating the graphene nanowall with a 900 nm laser (50 mW optical power) for 45 seconds;

[0213] (R2) 40 μL of PBS solution was added to the surface of the graphene nanowall, and the scanning voltage range was adjusted to -0.1 to 0.7 V. When no redox peak appeared during the cyclic voltammetry test, alanine detection was started;

[0214] (R3) During the test, the sample containing alanine was first added to the surface of the graphene nanowall, and then the graphene nanowall was irradiated with white light for 1 minute. The test concentration ranged from 5×10 -13 mol / L to 5×10 -3 mol / L (final concentration); the concentrations were tested from low to high, and the multiples of adjacent test concentrations were 10 times.

[0215] Each concentration of the test sample is added to the surface of the graphene nanowall, and a cyclic voltammetry test is performed directly to read the current signal of the redox peak. The signal is read as the change in the redox peak current.

[0216] Table 1 Types of enzyme probes and test target markers used to prepare devices in the above examples

[0217]

[0218]

[0219] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity, comprising a reference electrode, a counter electrode, and a working electrode, which is connected to an electrochemical workstation when in use, characterized in that: The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity also includes an insulating substrate, a graphene nanowall, an enzyme probe, a polymer film and a sample loading slot. A working electrode and a counter electrode are provided on the upper surface of the insulating substrate. The graphene nanowall is provided on the upper surface of the working electrode for receiving light energy and converting the light energy into heat energy. The enzyme probe is connected to the graphene nanowall via a connecting molecule and serves as an identification probe. The polymer film is provided on the outer surface of the graphene nanowall and the enzyme probe and serves as a protective layer. The sample loading groove is provided on the upper surface of the insulating substrate and surrounds the working electrode and the counter electrode for loading the sample of the object to be tested.

2. The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity according to claim 1, characterized in that: The counter electrode and the working electrode are both patterned electrodes with a thickness of 5 to 1000 nm.

3. The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity according to claim 1, characterized in that: The graphene nano-wall is a graphene nano-wall treated by oxygen plasma, and has a thickness of 0.1 to 50 μm.

4. The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity according to claim 1, characterized in that: The linking molecule is selected from one of organic small molecules or gold nanoparticles.

5. The graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity according to claim 1, characterized in that: The enzyme probe is an oxidase, a hydrolase or a transferase for detecting a target.

6. A method for preparing a graphene nanowall electrochemical sensor for photothermal enhancement of enzyme activity according to any one of claims 1 to 5, characterized in that: The following steps are involved: (A1) preparing a working electrode and a counter electrode on an insulating substrate; (A2) placing a graphene nanowall on the upper surface of the working electrode prepared in step (A1), modifying the linker molecule after treating the graphene nanowall with oxygen plasma, fixing the enzyme probe to the linker molecule, and then coating the enzyme probe and the graphene nanowall surface with a polymer. Finally, placing a sample loading groove on the upper surface of the insulating substrate and on the outside of the working electrode and the counter electrode to obtain a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity.

7. A method for detecting a target in an analyte using a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity as claimed in any one of claims 1 to 5, characterized in that: The method is a cyclic voltammetry test method, comprising the following steps: (B1) Connecting the counter electrode, reference electrode, and working electrode to an electrochemical workstation and irradiating the graphene nanowall with light; (B2) adding a buffer solution dropwise onto the surface of the graphene nanowall, scanning a given working electrode voltage range, and starting the test solution test when no redox peak appears in the cyclic voltammetry curve; (B3) adding a sample solution onto the surface of the graphene nanowall, causing the target substance in the sample to undergo an oxidation-reduction reaction under the catalysis of the enzyme probe, resulting in a corresponding electrochemical current signal being generated on the surface of the graphene nanowall; 10 to 30 seconds after adding the test solution, perform a cyclic voltammetry scan and compare it with the cyclic voltammetry curve of the solution without adding the test solution, and use the change of the redox current peak as the signal; (B4) When the redox peak in step (B3) appears, it indicates that the target is detected; when the redox peak in step (B3) does not appear, it indicates that the target is not detected.

8. The method for detecting a target in an analyte using a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity according to claim 7, wherein in step (B1), during the light irradiation process, the wavelength of the light is 360 to 1500 nm; In step (B3), during the redox reaction, the wavelength of light is 360 to 1500 nm.

9. A method for detecting a target substance in an analyte using a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity as claimed in any one of claims 1 to 5, characterized in that: The method is a current-time test method, comprising the following steps: (C1) Connecting the counter electrode, reference electrode, and working electrode to an electrochemical workstation and irradiating the graphene nanowall with light; (C2) Buffer solution is added to the surface of the graphene nanowall, and a fixed working electrode voltage is given to keep the current constant. The current change caused by the current noise is recorded as ΔI 非待测 , and use this as the signal response value of the non-DUT; (C3) The target solution is added to the surface of the graphene nanowall. The target undergoes an oxidation-reduction reaction under the catalysis of the enzyme probe, causing the corresponding electrochemical current signal to be generated on the surface of the graphene nanowall. When the current-time test mode is used, after the target solution is added, the current response reaches equilibrium and stability over time, and the current change value is read: ΔI 待测 , and use this as the signal response value of the object to be measured; (C4) When ΔI 待测 Greater than 3ΔI 非待测 When ΔI 待测 Less than or equal to 3ΔI 非待测 , indicating that the target was not detected.

10. The method for detecting a target in an analyte using a graphene nanowall electrochemical sensor with photothermal enhanced enzyme activity according to claim 9, wherein in step (C1), during the light irradiation process, the wavelength of the light is 360 to 1500 nm; In step (C3), during the redox reaction, the wavelength of light is 360 to 1500 nm.

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