Sub-molar uric acid non-enzymatic electrochemical sensor working electrode, preparation method and application

Abstract

The application belongs to the technical field of electrochemical sensors, and particularly relates to a working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor, a preparation method and application. The working electrode is a graphene / zinc sulfide heterojunction film, which comprises a substrate, the surface of the substrate is deposited with graphene, and a three-dimensional network ZnS skeleton is in-situ grown on the surface of the graphene, and the contact surface of the three-dimensional network ZnS skeleton and the graphene is a heterojunction interface. The application in-situ grows a unique three-dimensional network zinc sulfide skeleton on the surface of the graphene, the structure forms a continuous conductive network in a macroscopic view, and the ZnS network morphology provides abundant multistage pores and more active sites in a microscopic view, realizes rapid and high-sensitivity detection of a picomolar uric acid in a biological fluid, can meet the needs of sub-nanomolar uric acid monitoring, and has good stability.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, specifically relating to a working electrode, preparation method, and application of a sub-nanomolar uric acid enzyme-free electrochemical sensor. Background Technology

[0002] Uric acid (UA) is one of the end products of purine metabolism and an important small-molecule metabolic indicator in human body fluids (such as blood and urine). Numerous studies have shown that abnormal uric acid levels are closely related to the development of various diseases, including gout, cardiovascular disease, and metabolic syndrome. Particularly in neurodegenerative diseases or early metabolic abnormalities, fluctuations in uric acid concentration are often extremely small. Furthermore, due to the high cost of extracting biological body fluid samples, high-sensitivity sensors can effectively reduce the amount of biological samples consumed, significantly reducing sampling difficulty and patient discomfort. Therefore, developing ultra-high sensitivity sensors capable of detecting extremely low concentrations of uric acid is of great significance for disease evolution research and early diagnosis, real-time health monitoring, and dynamic physiological process analysis.

[0003] Traditional uric acid electrochemical sensors often rely on bioenzymes such as uricase to modify electrodes and improve selectivity. While enzyme-based electrochemical sensors possess good specificity, the high environmental sensitivity of bioactive substances limits their practical lifespan. Enzyme-free electrochemical sensors, by employing inorganic or carbon-based materials with high conductivity and electrochemical activity to replace enzymes, can achieve electrochemical recognition of uric acid without relying on biological macromolecules. However, current non-enzyme sensors mostly depend on noble metals or transition metal oxides, which suffer from bottlenecks such as high cost, poor conductivity, or susceptibility to interference at physiological potentials.

[0004] Graphene, as a sp² hybrid two-dimensional carbon material with a zero bandgap (Dirac cone) band structure, possesses excellent electrical properties and a high specific surface area, providing an ideal electron transport channel for electrochemical sensing and showing great application potential in the field of small molecule detection. However, the intrinsic chemical inertness of graphene's surface leads to a lack of sufficient electrochemical active sites, and the tendency for stacking between layers further reduces the effective reaction area, limiting its specific adsorption of uric acid molecules and resulting in low electrochemical sensing sensitivity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a working electrode, preparation method, and application for a sub-nanomolar uric acid enzyme-free electrochemical sensor. This invention grows a unique three-dimensional network of zinc sulfide framework in situ on the graphene surface. This structure forms a continuous conductive network macroscopically, and the ZnS network morphology microscopically provides abundant hierarchical channels and more active sites, enabling rapid and highly sensitive detection of picomolar uric acid in biological fluids, thus meeting the needs of sub-nanomolar uric acid monitoring.

[0006] This invention is specifically achieved through the following technical solutions: The first objective of this invention is to provide a working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor. The working electrode is a graphene / zinc sulfide heterojunction film, comprising a substrate on which graphene is deposited, and a three-dimensional network ZnS framework is grown in situ on the surface of the graphene. The contact surface between the three-dimensional network ZnS framework and the graphene is a heterojunction interface.

[0007] A second objective of this invention is to provide a method for preparing the working electrode of the above-mentioned sub-nanomolar uric acid enzyme-free electrochemical sensor, comprising the following steps: Graphene was deposited on a substrate using a chemical vapor deposition process. Sulfur powder is placed as a sulfur source in the upstream low-temperature zone of a tubular furnace, while zinc powder and a substrate sample with graphene deposited are placed in the downstream high-temperature zone. By controlling the furnace temperature and carrier gas flow rate, the sulfur powder and zinc powder sublimate into vapor, which is then transported to the graphene surface under the propulsion of the carrier gas. Under suitable temperature and time, the sulfur vapor and zinc vapor generate a zinc sulfide film in situ on the graphene surface, thereby forming a graphene / ZnS heterostructure, which serves as the working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor.

[0008] Preferably, the carrier gas is an inert gas with a pressure of 700 Torr and a flow rate of 100 sccm.

[0009] Preferably, the temperature of the downstream high-temperature zone is controlled at 550℃~600℃, and the temperature of the upstream low-temperature zone is controlled at 160℃~200℃, with the reaction time being 1 hour.

[0010] Preferably, the substrate sample with graphene deposited is placed at one end of an alumina crucible with the graphene side facing down, and then placed in a tube furnace.

[0011] The third objective of this invention is to provide a sub-nanomolar level enzyme-free electrochemical sensor for uric acid. In phosphate buffered saline (PBS), the aforementioned working electrode is used as a self-supporting working electrode, a platinum needle is used as a counter electrode, and Ag / AgCl is used as a reference electrode. The electrode working area is defined on a graphene / zinc sulfide film through micro-nano fabrication, and gold leads and a silicon dioxide passivation layer are prepared. Subsequently, a PDMS microfluidic cavity is prepared on the surface using a mold for injecting the solution to be tested.

[0012] The fourth objective of this invention is to provide the application of the aforementioned sub-nanomolar uric acid enzyme-free electrochemical sensor in uric acid detection. Differential pulse voltammetry is used as the detection method, with the scanning potential range set to -0.2V to 0.6V, pulse amplitude of 200mV, pulse width of 20ms, and pulse period of 100ms. The sample to be detected is added to the electrode working area, and the relative peak current change is calculated. Based on the relative peak current change and the uric acid concentration standard curve, the uric acid concentration of the sample to be detected is obtained.

[0013] The relative current response ΔI was calculated by measuring the peak current in uric acid solutions of different concentrations. pn =(I pn - I p0 ) / I p0 ΔI pn A calibration curve was plotted against uric acid concentration, and the linear detection range and detection limit of the sensor were determined by linear fitting. The lowest detection limit for uric acid was 100 pM.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a sub-nanomolar level enzyme-free electrochemical sensor for uric acid, comprising a graphene / zinc sulfide heterojunction film, including a substrate on which graphene is deposited, and a three-dimensional network ZnS framework grown in situ on the graphene surface. The contact surface between the three-dimensional network ZnS framework and the graphene is the heterojunction interface. This invention's graphene / zinc sulfide heterojunction sub-nanomolar level enzyme-free electrochemical sensor for uric acid has the following main performance characteristics: 1. Large specific surface area and excellent electrical conductivity This invention involves in-situ growth of zinc sulfide on graphene, constructing a three-dimensional network framework composed of interwoven nanodendritic structures, forming a multi-level porous structure that significantly increases the effective area for electrochemical reactions. The network ZnS / graphene heterostructure not only provides a continuous conductive network and a three-dimensional open reaction interface but also effectively reduces charge transport and diffusion resistance, enabling the sensor to exhibit rapid response, high sensitivity, wide linear range, and sub-nanomolar detection limit in uric acid detection, while maintaining good stability and inter-device repeatability.

[0015] 2. Wide linear detection range and low detection limit The peak current response of uric acid at different concentrations was detected using a differential pulse voltammetry (DPV) method with phosphate-buffered saline (PBS) as the electrolyte and a scanning potential of -0.2 to 0.6 V (relative to the Ag / AgCl reference electrode). The sensor exhibited a good logarithmic linear peak current response to uric acid in the concentration range of 100 pM to 1 mM, with high linear correlation and a detection limit as low as 100 pM, which can meet the needs of sub-nanomolar uric acid monitoring.

[0016] 3. Good selectivity and anti-interference ability A comparative test was conducted with 1 μM uric acid against common interfering substances such as 1 mM ascorbic acid, glucose, and hydrogen peroxide. The results showed that the relative current response of the sensor of the present invention to uric acid was significantly higher than that of the above interfering substances. It has good anti-interference ability against multiple coexisting components and can achieve selective identification of extremely low concentrations of uric acid in complex systems.

[0017] 4. Excellent repeatability and stability Multiple graphene / zinc sulfide heterojunction electrodes prepared using the same process were repeatedly measured with uric acid solutions of the same concentration. The deviations in their relative current responses were small, indicating good repeatability between the devices. Long-term testing showed that the electrodes maintained relatively stable responses after multiple uses and a certain storage time, making them suitable for practical detection.

[0018] As can be seen from the above performance, the sensor of the present invention has the combined advantages of wide linear range, low detection limit, high sensitivity and good selectivity in the detection of extremely low concentration uric acid. Moreover, the preparation method is simple and easy to implement, making it very suitable for large-scale production applications. Attached Figure Description

[0019] Figure 1 The graph shows the DPV response curves of the sensor based on the graphene / zinc sulfide heterojunction of Example 1 in a small molecule uric acid solution with a concentration of 100 pM to 1 mM.

[0020] Figure 2 The graph shows the relative current response of the sensor based on the graphene / zinc sulfide heterojunction of Example 1 in a uric acid small molecule solution with a concentration of 100 pM to 1 mM.

[0021] Figure 3 This is a comparison of the relative current response of the sensor sample based on the graphene / zinc sulfide heterostructure electrode of Example 1 in the presence of interfering substances such as 1 mM ascorbic acid, glucose, and hydrogen peroxide under the condition of 1 μM uric acid.

[0022] Figure 4 The image shows a scanning electron microscope (SEM) image of the sapphire-based graphene / zinc sulfide heterojunction prepared in Example 1.

[0023] Figure 5 This diagram illustrates the testing mechanism for detecting uric acid using an electrochemical uric acid sensor. Detailed Implementation

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention. Unless otherwise specified, the experimental methods and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials described are commercially available.

[0025] This invention proposes an enzyme-free electrochemical sensor for sub-nanomolar uric acid detection using a graphene / zinc sulfide heterostructure. Zinc sulfide (ZnS), a typical wide-bandgap II-VI semiconductor, possesses excellent chemical stability, and its unique band structure, when combined with graphene to form a heterostructure, effectively modulates the interfacial electron transport barrier. This invention utilizes a controlled process to grow a unique three-dimensional network of zinc sulfide framework in situ on the graphene surface. This structure macroscopically forms a continuous conductive network, while microscopically, the ZnS network morphology provides abundant hierarchical channels and more active sites, thereby achieving higher sensitivity and a lower detection limit in the electrochemical detection of uric acid.

[0026] The following description, in conjunction with the accompanying drawings, illustrates specific embodiments of the present invention. The experimental conditions and process parameters given in the examples are preferred solutions; unless otherwise specified, they can be performed according to conventional practices in the art or the recommended conditions of relevant reagent and equipment manufacturers.

[0027] Example 1 A working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor includes a substrate, on which graphene is deposited. A three-dimensional network ZnS framework is grown in situ on the graphene surface, and the interface between the three-dimensional network ZnS framework and the graphene is a heterojunction interface. The specific preparation method includes the following steps:

[0028] Step 1: High-quality sapphire-based graphene was selected as the growth platform, and graphene was deposited on the sapphire substrate using low-pressure chemical vapor deposition (CVD). Specific deposition conditions were as follows: After evacuating the reaction system, a 1:5 mixture of hydrogen and argon was introduced, and the control system maintained a constant pressure of 500 Torr; subsequently, the temperature was increased to 1400°C at a rate of 10°C / min, and methane precursor gas was introduced. After approximately 5 minutes of continuous growth, the gas supply was stopped, and the sample was allowed to cool naturally to room temperature under a protective atmosphere. The prepared sapphire / graphene sample was placed at one end of an alumina crucible, graphene side down, downstream of the high-temperature zone in the center of the tube furnace. Zinc powder was placed at the other end of the crucible as a zinc source; a crucible containing sulfur powder was placed upstream in the low-temperature zone as a sulfur source.

[0029] Step 2: After sealing the furnace tube, first evacuate to remove residual gas, then introduce argon gas to stabilize the system pressure at 700 Torr, and set the Ar flow rate to 100 sccm. Subsequently, increase the temperature at a rate of 15℃ / min to raise the downstream area to 550℃, while simultaneously adjusting the temperature at the upstream sulfur powder location to 160℃, and perform sulfurization for 1 hour.

[0030] Under the above conditions, sulfur powder and zinc powder sublimate to generate sulfur vapor and zinc vapor, respectively. These vapors, carried by a carrier gas, enter the downstream high-temperature zone and react on the graphene surface to form a zinc sulfide nanofilm, achieving in-situ growth of zinc sulfide on graphene. After the reaction is complete, heating is turned off and the mixture is allowed to cool naturally to room temperature under argon protection. The sample is then removed to obtain a sapphire-substrate graphene / zinc sulfide heterostructure film, which is used as the working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor.

[0031] Figure 4 The image shows a SEM image of the graphene / zinc sulfide heterostructure obtained in Example 1. It can be observed that a three-dimensional network of zinc sulfide layer is formed on the graphene surface, which is composed of a large number of slender nanocrystals that overlap and interweave with each other. The mesh distribution is relatively uniform, and the surface roughness is significantly increased compared with that of single-layer graphene.

[0032] Example 2 A working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor includes a substrate, on which graphene is deposited. A three-dimensional network ZnS framework is grown in situ on the graphene surface, and the interface between the three-dimensional network ZnS framework and the graphene is a heterojunction interface. The specific preparation method includes the following steps:

[0033] Step 1: Graphene was deposited on a sapphire substrate using low-pressure chemical vapor deposition (CVD). Specific deposition conditions were as follows: After evacuating the reaction system, a 1:5 mixture of hydrogen and argon was introduced, maintaining a constant pressure of 500 Torr. The temperature was then increased to 1400°C at a rate of 10°C / min, and methane precursor gas was introduced. Growth was continued for approximately 5 minutes, after which the gas supply was stopped, and the sample was allowed to cool naturally to room temperature under a protective atmosphere. The sapphire / graphene sample was placed at one end of an alumina crucible, graphene side down, downstream of the high-temperature zone in the center of the tube furnace. Zinc powder was placed at the other end of the crucible as a zinc source. A crucible containing sulfur powder was placed upstream in the low-temperature zone as a sulfur source.

[0034] Step 2: After sealing the furnace tube, first evacuate to remove residual gas, then introduce argon gas to stabilize the system pressure at around 700 Torr, and set the Ar flow rate to 100 sccm. Subsequently, increase the temperature at a rate of 20℃ / min to raise the downstream area to 600℃, while simultaneously adjusting the temperature at the upstream sulfur powder location to 200℃ to allow the sulfur powder to fully sublimate and sulfidate for 1 hour.

[0035] Under the above conditions, sulfur powder and zinc powder sublimate to generate sulfur vapor and zinc vapor, respectively. These vapors, carried by a carrier gas, enter the downstream high-temperature zone and react on the graphene surface to form a zinc sulfide nanofilm, achieving in-situ growth of zinc sulfide on graphene. After the reaction is complete, heating is turned off and the mixture is allowed to cool naturally to room temperature under argon protection. The sample is then removed to obtain a sapphire-substrate graphene / zinc sulfide heterostructure film, which is used as the working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor.

[0036] Example 3 A working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor includes a substrate, on which graphene is deposited. A three-dimensional network ZnS framework is grown in situ on the graphene surface, and the interface between the three-dimensional network ZnS framework and the graphene is a heterojunction interface. The specific preparation method includes the following steps:

[0037] Step 1: Graphene was deposited on a sapphire substrate using low-pressure chemical vapor deposition (CVD). Specific deposition conditions were as follows: After evacuating the reaction system, a 1:5 mixture of hydrogen and argon was introduced, maintaining a constant pressure of 500 Torr. The temperature was then increased to 1400°C at a rate of 10°C / min, and methane precursor gas was introduced. Growth was continued for approximately 5 minutes, after which the gas supply was stopped, and the sample was allowed to cool naturally to room temperature under a protective atmosphere. The sapphire / graphene sample was placed at one end of an alumina crucible, graphene side down, downstream of the high-temperature zone in the center of the tube furnace. Zinc powder was placed at the other end of the crucible as a zinc source. A crucible containing sulfur powder was placed upstream in the low-temperature zone as a sulfur source.

[0038] Step 2: After sealing the furnace tube, first evacuate to remove residual gas, then introduce argon gas to stabilize the system pressure at 700 Torr, and set the Ar flow rate to 100 sccm. Subsequently, increase the temperature at a rate of 15℃ / min to raise the downstream area to 570℃, while simultaneously adjusting the temperature at the upstream sulfur powder location to 180℃, and perform sulfurization for 1 hour.

[0039] Under the above conditions, sulfur powder and zinc powder sublimate to generate sulfur vapor and zinc vapor, respectively. These vapors, carried by a carrier gas, enter the downstream high-temperature zone and react on the graphene surface to form a zinc sulfide nanofilm, achieving in-situ growth of zinc sulfide on graphene. After the reaction is complete, heating is turned off and the mixture is allowed to cool naturally to room temperature under argon protection. The sample is then removed to obtain a sapphire-substrate graphene / zinc sulfide heterostructure film, which is used as the working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor.

[0040] The sapphire substrate graphene / zinc sulfide heterostructure thin films of the present invention can be prepared according to the methods described in Examples 1 to 3 above, and their properties are similar. Below, we will only use the material prepared in Example 1 as an example, and use it as the working electrode for a sub-nanomolar level enzyme-free uric acid electrochemical sensor to prepare an electrochemical sensor for detecting uric acid.

[0041] Application Example 1 The preparation method of a sub-nanomolar level enzyme-free electrochemical sensor for uric acid includes the following steps: A multi-step micro / nano fabrication process was used to construct a micro-electrochemical sensor from the prepared graphene / zinc sulfide heterojunction film. The specific steps are as follows: Photolithography and etching define the working area: Using a graphene / zinc sulfide heterojunction film as the working electrode, photoresist is spin-coated onto the surface of the graphene / zinc sulfide heterojunction film. After high-temperature soft baking and annealing, electron beam lithography is used to transfer the working area pattern to the film surface and then develop and cure it. Subsequently, plasma etching technology (100W power etching for 60s) is used to precisely etch the patterned graphene / zinc sulfide heterojunction as the electrode working area. Finally, residual photoresist is removed by standing in a solvent.

[0042] Gold lead electrode fabrication: The lead pattern is aligned and transferred to the sample surface by a secondary electron beam lithography process. After exposure and development, a gold (Au) thin film with a thickness of about 20 nm is deposited by electron beam evaporation. After removing the resist, the gold electrode lead is formed.

[0043] Passivation layer deposition: A distributed silicon dioxide (SiO2) layer with a thickness of about 10 nm is deposited as an insulating passivation layer in a specific non-working area by three electron beam lithography alignments and atomic layer deposition (ALD) process.

[0044] Microfluidic cavity integration: Polydimethylsiloxane (PDMS) liquid was deposited and solidified on a template using microjet technology, and the resulting PDMS structure with microcavities was obtained after peeling. This structure was precisely aligned and mounted on the surface of the sensor's working area to construct a complete microcavity structure that can be used to inject PBS buffer and uric acid test solution.

[0045] Using a platinum needle as the counter electrode and Ag / AgCl as the reference electrode, an electrochemical sensor for uric acid was obtained.

[0046] The following section describes the use of the electrochemical sensor prepared above to detect uric acid concentration. (1) Electrochemical detection method for sub-nanomolar uric acid To improve the detection sensitivity of trace uric acid, this invention employs differential pulse voltammetry (DPV) as the detection method. The prepared sensor is connected to an electrochemical workstation, with the operating mode set to DPV, the scan potential range set to -0.2 to 0.6 V, the pulse amplitude 200 mV, the pulse width 20 ms, and the pulse period 100 ms. The testing mechanism is as follows: Figure 5 As shown, 100 μL of PBS buffer was first added to the effective area of ​​the working electrode (approximately 5 mm × 5 mm) to obtain the baseline peak current I near the uric acid oxidation peak in the PBS-only system. p0 Subsequently, a series of uric acid standard solutions of different concentrations (100 pM to 1 mM) were prepared. These uric acid standard solutions were then added dropwise to the working electrode, and the corresponding peak current I was recorded in solutions of different uric acid concentrations. pn and press ΔI pn =(I pn -I p0 ) / I p0 Calculate the relative current response. Let ΔI pn A calibration curve is plotted against uric acid concentration. Linear fitting can then be used to determine the sensor's linear detection range and detection limit (e.g., ...). Figure 1 and Figure 2 (As shown). By comparing the relative peak current changes of unknown samples measured under the same DPV conditions, the concentration of the uric acid sample to be tested can be determined. The DPV testing method can effectively suppress the double-layer capacitance current and significantly improve the signal-to-noise ratio of low-concentration uric acid signals, which is particularly beneficial for the detection of trace uric acid at the nanomolar and even picomolar levels.

[0047] With the above test parameters remaining unchanged, selectivity can also be examined. Several common interfering substances (such as ascorbic acid, glucose, hydrogen peroxide, etc.) were selected, and solutions with a concentration of 1 mM were prepared respectively. Under the same test conditions, their respective relative current responses ΔI were measured. rn The response was compared with that of uric acid, and the results are illustrated in the figure below. Figure 3 As shown, this reflects the sensor's ability to distinguish uric acid in the presence of interfering substances.

[0048] (2) Performance analysis of electrochemical sensors Figure 1 The DPV response curves of the graphene / zinc sulfide (Gr / ZnS) heterojunction electrode for uric acid are shown in the concentration range of 100 pM to 1 mM. With the increase of uric acid concentration, the oxidation peak current shows a significant upward trend. Figure 2 The relative current response of the graphene / zinc sulfide (Gr / ZnS) heterojunction electrode for uric acid sensing was demonstrated. Within the range of 100 pM to 1 mM, the relative current response of the graphene / zinc sulfide electrode exhibited a good linear relationship with increasing uric acid concentration, demonstrating a wide linear detection range and a detection limit for uric acid as low as 100 pM.

[0049] Figure 3 The present invention provides a sensor for uric acid 1 The relative current response of the electrode based on the graphene / zinc sulfide heterostructure to uric acid, glucose, hydrogen peroxide, and other interfering substances at a concentration of M was compared. The results showed that the response signal of the electrode based on the graphene / zinc sulfide heterostructure to uric acid was significantly higher than that of other interfering substances, indicating that the sensor still has good selectivity and anti-interference ability in the presence of multiple coexisting substances.

[0050] Compared with the traditional method of modifying the supporting electrode by drop coating or impregnation, the sensor of this invention utilizes a graphene / zinc sulfide heterostructure with a mesh morphology as a self-supporting working electrode, which has high device consistency and yield, and stable structure, which is conducive to large-scale preparation. The mesh ZnS framework constructs a large-area heterojunction interface and multi-level channels on the graphene surface, which not only takes advantage of the high carrier mobility of graphene, but also significantly improves the electrochemical effective area and surface catalytic activity of zinc sulfide. As a result, the sensor exhibits faster response speed, higher signal output level and better overall performance in trace uric acid detection.

[0051] In the fabrication process of the working electrode of this invention, relevant parameters and conditions have a significant impact on the quality of the graphene / zinc sulfide heterostructure film, specifically: Synergistic regulation of temperature gradients in two zones: This invention controls the upstream sulfur source temperature at 160℃~200℃, and the downstream zinc source and substrate temperature at 550℃~600℃. If the temperature is too high, it can easily disrupt the lattice continuity of graphene or cause ZnS crystallization to occur too rapidly, resulting in large particle agglomeration; if the temperature is too low, it cannot provide the activation energy required for the sublimation of sulfur and zinc powder and for in-situ heterogeneous nucleation on the inert surface of graphene. This dual-temperature gradient is a crucial prerequisite for ensuring the formation of the three-dimensional network of hierarchical porous structures in ZnS.

[0052] Kinetic regulation of gas-phase transport: The reaction proceeds at an inert gas pressure of approximately 700 Torr and a carrier gas flow rate of 100 sccm. This pressure and flow rate balance the transport rates of zinc and sulfur vapors with the interfacial concentration distribution. Deviations from these parameters lead to vapor supersaturation or insufficient supply, thus preventing the formation of a macroscopically continuous conductive nanodendritic framework.

[0053] The substrate is placed face down in a tube furnace using an inverted spatial layout. This design effectively prevents the direct settling of larger impurities or unreacted clusters in the gas flow, allowing precursor gas molecules to undergo controlled in-situ reactions at the graphene interface through slow diffusion. This improves the compactness of the heterojunction interface and the reproducibility of the experiments, demonstrating significant process exploration and creative contributions.

[0054] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, it is intended to include any modifications and variations that fall within the scope of the claims and their equivalents.

Claims

1. A working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor, characterized in that, The graphene / zinc sulfide heterojunction film includes a substrate on which graphene is deposited, and a three-dimensional network ZnS framework is grown in situ on the surface of the graphene. The contact surface between the three-dimensional network ZnS framework and the graphene is a heterojunction interface.

2. A method for preparing a working electrode for a sub-nanomolar uric acid enzyme-free electrochemical sensor according to claim 1, characterized in that, Includes the following steps: Graphene was deposited on a substrate using a chemical vapor deposition process. Sulfur powder was placed as a sulfur source in the upstream low-temperature zone of the tubular furnace, and zinc powder and a substrate sample with graphene deposited were placed in the downstream high-temperature zone. By controlling the furnace temperature and carrier gas flow rate, sulfur powder and zinc powder are sublimated to form vapors, which are then transported to the graphene surface under the propulsion of the carrier gas. The sulfur vapor and zinc vapors generate a zinc sulfide film in situ on the graphene surface, thereby forming a graphene / ZnS heterostructure, which serves as the working electrode for a sub-nanomolar level enzyme-free electrochemical sensor for uric acid.

3. The preparation method according to claim 2, characterized in that, The temperature in the downstream high-temperature zone is controlled at 550℃~600℃, and the temperature in the upstream low-temperature zone is controlled at 160℃~200℃, and the reaction is carried out for 1 hour.

4. The preparation method according to claim 2, characterized in that, The carrier gas is an inert gas with a pressure of 700 Torr and a flow rate of 100 sccm.

5. The preparation method according to claim 2, characterized in that, The substrate sample with graphene deposited was placed at one end of an alumina crucible with the graphene side facing down and placed in a tube furnace.

6. A sub-nanomolar level enzyme-free electrochemical sensor for uric acid, characterized in that, Using the working electrode described in claim 1 as a self-supporting working electrode, a platinum needle as a counter electrode, and Ag / AgCl as a reference electrode, the electrode working area is defined on a graphene / zinc sulfide film by micro-nano fabrication, and gold leads and a silicon dioxide passivation layer are prepared. Subsequently, a PDMS microfluidic cavity is prepared on the surface using a mold for injecting the solution to be tested.

7. The application of a sub-nanomolar uric acid enzyme-free electrochemical sensor according to claim 6 in uric acid detection.

8. The application according to claim 7, characterized in that, Differential pulse voltammetry was used as the detection method. The scanning potential range was set to -0.2V to 0.6V, the pulse amplitude was 200mV, the pulse width was 20ms, and the pulse period was 100ms. The sample to be tested was dropped into the electrode working area, and the relative peak current change was calculated. Based on the relative peak current change and the uric acid concentration standard curve, the uric acid concentration of the sample to be tested was obtained.

9. The application according to claim 7, characterized in that, The minimum detection limit for uric acid is 100 pM.

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