An electrochemical sensor based on an ultrathin two-dimensional heterostructure GO / MXene composite material and its application

Abstract

This invention belongs to the field of medical device technology, specifically disclosing an ultrathin two-dimensional heterostructure graphene oxide (GO) / MXene composite material, an electrochemical sensor, and their applications. Based on the specific enzymatic reaction of tartrate-resistant acid phosphatase (TRACP-5b), this invention designs an electrochemical detection method based on an ultrathin two-dimensional heterostructure material system for sensitive detection of TRACP-5b content in human gingival crevicular fluid (GCF), thereby achieving real-time monitoring of periodontitis in the human oral cavity. To this end, this invention constructs an electrochemical biosensor based on an ultrathin two-dimensional heterostructure GO / MXene material. Based on the high electron transport capacity and catalytic activity of this material, the constructed electrochemical sensing system can effectively detect TRACP-5b, with a limit of detection (LOD) of 18.6 ng / mL, providing a new approach for the clinical diagnosis and real-time monitoring of periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of medical device technology, specifically relating to a GO / MXene composite material based on an ultrathin two-dimensional heterostructure, an electrochemical sensor, and its applications. Background Technology

[0002] Recent global burden of disease studies indicate that severe periodontitis is the sixth most prevalent disease worldwide, with an overall prevalence of 11.2%, affecting approximately 743 million people. As an inflammatory condition, periodontitis is characterized by an interplay of inflammation and repair, bone formation and resorption. Its progression is accompanied by the deepening of periodontal pockets and alveolar bone resorption, which is a major risk factor for tooth loosening and loss. Current clinical diagnostic techniques for periodontitis include clinical periodontal pocket probing and traditional medical imaging methods (such as X-rays and CT scans). However, the former is time-consuming and its results are influenced by the examiner's experience and skill, while the latter is costly and poses radiation risks to patients. Neither method allows for frequent real-time monitoring and early risk assessment of alveolar bone resorption in treated patients. Therefore, a method for rapid clinical detection or accurate prediction of the severity of severe periodontitis and alveolar bone resorption is urgently needed.

[0003] Gingival crevicular fluid (GCF) is the only fluid that directly exudes from body fluids. Its content and composition are closely related to periodontal tissues, providing important quantitative indicators for evaluating periodontal health. Because GCF sampling is repeatable and non-invasive, minimizing discomfort, it is a promising source of diagnostic information, helping to improve the efficiency and accuracy of clinical diagnosis and mitigate irreversible periodontal damage caused by delayed clinical diagnosis and treatment. Furthermore, in in vitro osteoclast experiments, the activity level of tartrate-resistant acid phosphatase (TRACP-5b) can sensitively reflect the number and activity of osteoclasts in GCF. Related reports indicate that biomarkers related to root resorption in human GCF are considered potential indicators for periodontitis monitoring, specifically including TRACP-5b, dentin phosphoprotein, and cementumin-1.

[0004] TRACP-5b is an active enzyme secreted by osteoclasts, primarily derived from osteoclasts with bone resorption function. Its expression level and activity are positively correlated with bone resorption, therefore, serum TRACP-5b levels are currently used internationally as a biomarker for measuring bone resorption and osteoclast activity. The enzyme's activity level does not significantly change between day and night and is unaffected by food intake, allowing for sample collection and testing at any time of day. Periodontitis is closely related to bone resorption and osteoclast count. Studies have shown that TRACP-5b expression levels significantly increase during the progression of periodontitis; therefore, TRACP-5b holds promise as a potential biomarker for diagnosing the severity of periodontitis.

[0005] Traditional methods for detecting TRACP-5b typically involve quantitative or semi-quantitative analysis using enzyme-linked immunosorbent assay (ELISA), Western blotting, and proteomics. These methods are time-consuming and costly, with low returns, and they also cannot achieve real-time monitoring of periodontal disease, thus having significant limitations in clinical applications.

[0006] Currently, there is limited research on TRACP-5b in GCF based on electrochemical technology. Electrochemical sensors, due to their advantages of ease of operation, low cost, fast analysis speed, and small size, are suitable for rapid on-site detection in various applications and have attracted widespread attention in the sensor field. Recent reports indicate that electrochemical sensor-based methods can achieve highly sensitive real-time monitoring of the human oral microenvironment in a low-cost, non-invasive manner. However, the low expression level of TRACP-5b in GCF and the complex composition of GCF pose challenges to its detection. Therefore, there is a need to construct a high-sensitivity and highly specific high-performance electrochemical sensing system for the detection of TRACP-5b. Summary of the Invention

[0007] The purpose of this invention is to provide a GO / MXene composite material based on an ultrathin two-dimensional heterostructure, an electrochemical sensor and its application, which can perform highly sensitive and specific detection of TRACP-5b in human GCF to achieve real-time monitoring of patients with periodontitis.

[0008] Graphene oxide (GO), a monolayer material exfoliated from graphite oxide, possesses a large specific surface area, abundant functional groups, and a unique delocalized electron arrangement, endowing it with a strong affinity for carbon-based ring materials commonly found in biomolecules and drugs. This makes it frequently used in medicine, biology, and electrochemical sensing. However, GO lacks sufficient electron transport capacity. Studies have shown that the degree of oxidation of GO significantly affects its conductivity; when the oxidation degree exceeds 20%, the conductivity drops sharply. When used as a sensing material, severe aggregation between GO flakes further reduces electron transport capacity and the number of surface active groups, often resulting in lower-than-expected electrochemical performance. Therefore, the electrochemical applications of GO are somewhat limited. MXene materials, on the other hand, are two-dimensional materials with high conductivity, large specific surface area, and abundant surface groups. Reports have confirmed their efficient, stable, and tunable catalytic performance in the electrocatalytic reactions of phenol-related markers, thus showing promising application prospects in electrochemical detection. However, in practical applications, MXene materials can suffer from problems such as relatively low capacitance, impaired electron transport, and low electrode material stability due to stacking. This invention presents a composite material with an ultrathin two-dimensional heterostructure constructed from GO and MXene. Compared to single sensitive materials, this effectively improves the material stacking problem, and the heterostructure material exhibits higher electron transport efficiency, effectively enhancing catalytic activity. This overcomes the application shortcomings of GO and MXene, laying a crucial foundation for achieving highly sensitive electrochemical detection of the TRACP-5b enzyme.

[0009] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a GO / MXene composite material based on an ultrathin two-dimensional heterostructure, which is a composite material with ultrathin two-dimensional heterostructure characteristics formed by vertical stacking or in-plane growth of ultrathin monolayer graphene material and ultrathin monolayer MXene material; wherein the graphene material is graphene oxide or reduced graphene oxide; and the MXene material is Ti3C2, Nb2C, Ti2C or V2C.

[0011] Furthermore, the mass ratio of graphene to MXene ranges from 1:(1~5). When the mass ratio of graphene to MXene exceeds the specified range, excess graphene or MXene nanosheets may affect the formation of the two-dimensional heterostructure, which is detrimental to the application of electrochemical sensing. In addition, as the thickness of the two-dimensional material increases, the charge carriers in the formed heterostructure will be more affected by interface scattering and lattice defects, thereby affecting the electrochemical performance of the composite material. Therefore, the thickness range of the graphene and MXene is 2 nm to 6 nm.

[0012] Furthermore, the preparation method of GO / MXene composite material based on ultrathin two-dimensional heterostructure includes the following steps:

[0013] S11. React the MAX phase material (the precursor material of MXene material) with hydrofluoric acid (HF), and selectively etch the precursor MAX phase based on HF to generate multilayer MXene material;

[0014] S12. Reaction of multilayer MXene material with dimethyl sulfoxide (DMSO) is performed to exfoliate the multilayer MXene material with DMSO to obtain a two-dimensional ultrathin monolayer MXene material. DMSO can be inserted between MXene layers, and the sulfur atoms in its molecular structure can form hydrogen bonds with functional groups (such as hydroxyl and carboxyl groups) on the surface of MXene, thereby breaking the van der Waals forces between the layers and separating the MXene layers into monolayer or a few layers of nanosheets.

[0015] S13. Charge modification of two-dimensional ultrathin monolayer MXene material using polyethyleneimine (PEI);

[0016] S14. A two-dimensional ultrathin monolayer graphene dispersion is added to a charge-modified two-dimensional ultrathin monolayer MXene dispersion to obtain a graphene / MXene composite material with ultrathin two-dimensional heterostructure characteristics.

[0017] More specifically, the preparation method of GO / MXene composite materials based on ultrathin two-dimensional heterostructure features includes the following steps:

[0018] S11. Under ice-water bath conditions, 1 g of MAX phase material powder was slowly immersed into 10-20 mL of HF solution and stirred for 5-10 min; then the temperature was raised to 40-60 ℃ and stirring was continued for 12-24 h; after the reaction was completed, the mixture was centrifuged at 3000-5500 rpm for 5-10 min, and the resulting precipitate was washed with deionized water until neutral, and then vacuum dried at 40-60 ℃ for 12-24 h to obtain multilayer MXene material;

[0019] S12. Immerse 5-10 mg of the above MXene material powder in 25-50 mL of DMSO solution and stir continuously at room temperature for 18-24 h. After the reaction is complete, wash with deionized water to remove excess DMSO. Place the resulting dispersion in an ice-water bath and sonicate for 1-2 h, then centrifuge at 3000-5500 rpm for 1-2 h. After centrifugation, pour off the upper dispersion. The resulting dispersion is the two-dimensional ultrathin monolayer MXene material.

[0020] S13. The above two-dimensional ultrathin monolayer MXene material powder is uniformly dispersed in an appropriate amount of deionized water, and 5-10 mL of PEI is added to modify the charge of the MXene material; then the dispersion is ultrasonically treated for 5-10 min, and the excess PEI is washed with deionized water and redispersed in deionized water to form a stable hydrosol.

[0021] S14. The two-dimensional ultrathin monolayer graphene dispersion is added to the charge-modified MXene dispersion under vigorous stirring, and the mixture is stirred continuously at room temperature for 6 to 24 h. The resulting material is then vacuum dried at 40 to 60 °C for 12 to 24 h to obtain a graphene / MXene composite material with ultrathin two-dimensional heterostructure characteristics.

[0022] In a second aspect, the present invention provides a disposable electrochemical sensor comprising a polyethylene terephthalate (PET) substrate and at least one detection unit disposed on the surface of the PET substrate, the detection unit comprising the following components:

[0023] The working electrode (WE) is a carbon electrode with the surface coated with the above-mentioned GO / MXene composite material based on an ultrathin two-dimensional heterostructure. During electrochemical measurement, current will pass through the electrode and drive the specified chemical process. Its potential change directly reflects the characteristics and rate of the electrochemical reaction.

[0024] The counter electrode (CE) is a carbon electrode located on the other side of the working electrode. In electrochemical measurements, it is used to form a current loop with the WE, thereby ensuring that the electrochemical reaction on the working electrode continues.

[0025] The reference electrode (RE) is an Ag / AgCl electrode disposed on one side of the working electrode to provide a known and stable reference potential for electrochemical measurements.

[0026] Conductors are connected to the bottom of RE, WE, and CE respectively, to form a complete current loop for each electrode of the sensor in electrochemical measurements.

[0027] Furthermore, the method for preparing the disposable electrochemical sensor includes the following steps: S21. Uniformly printing Ag / AgCl ink onto PET and drying to obtain the printed RE;

[0028] S22. The conductive carbon paste is uniformly printed onto the PET printed with RE, and dried to obtain the printed WE and CE;

[0029] S23. The conductive silver paste is uniformly printed onto the PET printed with RE, WE and CE, and dried to obtain the printed wire;

[0030] S24. Prepare an ink by mixing GO / MXene composite material with anhydrous ethanol and Nafion solution. Apply the ink evenly to the WE surface to complete the preparation of a disposable electrochemical sensor.

[0031] More specifically, the method for preparing the disposable electrochemical sensor includes the following steps (wherein the diluents used in steps S21, S22, and S23 can be conventional commercially available ink diluents, conductive carbon paste diluents, and conductive silver paste diluents):

[0032] S21. Weigh 5-10 g of Ag / AgCl ink and dilute it with 5-10 mL of thinner. Then, use a screen printing machine to evenly print the diluted Ag / AgCl ink onto PET and dry it at room temperature to obtain the printed RE.

[0033] S22. Weigh 4 to 8 g of conductive carbon paste and dilute it with 5 to 10 mL of diluent. Use a screen printing machine to evenly print the diluted conductive carbon paste onto the PET printed with RE. Dry at room temperature to obtain the printed WE and CE.

[0034] S23. Weigh 5 ~ 15 g of conductive silver paste and dilute it with 5 ~ 20 mL of diluent. Use a screen printing machine to evenly print the diluted conductive silver paste onto the PET printed with RE, WE and CE. Dry at room temperature to obtain the printed wire.

[0035] S24. Take 5 ~ 10 mg of GO / MXene composite material and mix it with 100 ~ 500 μL of anhydrous ethanol and 20 ~ 100 μL of Nafion solution to prepare a uniform ink; take 2 ~ 10 μL of the above ink and uniformly drop it onto the WE surface of the electrochemical sensor to complete the preparation of the disposable electrochemical sensor.

[0036] Thirdly, this invention provides the application of the aforementioned disposable electrochemical sensor in detecting the TRACP-5b content in human GCF. Based on the principle that the TRACP-5b enzyme catalyzes the formation of the enzyme product 1-naphthol from inert 1-naphthyl phosphate monosodium salt under specific conditions, the electrochemical sensor of this invention can detect 1-naphthol with high sensitivity, thereby achieving accurate quantification of TRACP-5b and realizing the purpose of diagnosis and real-time monitoring of periodontitis.

[0037] Furthermore, the application method involves inserting the disposable electrochemical sensor into a handheld electrochemical sensor device, which includes a disposable electrochemical sensor, a control circuit board, and a display. The lead wire of the disposable electrochemical sensor is connected to the control circuit board, and the display is electrically connected to the control circuit board.

[0038] During the test, a TRACP-5b gradient solution was first prepared, reacted with 1-naphthyl phosphate monosodium salt, and then dropped onto the electrode surface. The corresponding current value was measured by the control circuit board, and a standard curve was established between the TRACP-5b concentration and the current value. Then, the sample to be tested was reacted with 1-naphthyl phosphate monosodium salt and dropped onto the electrode surface. The measured current value was then substituted into the standard curve to obtain the TRACP-5b content in the sample to be tested.

[0039] The present invention has the following beneficial effects:

[0040] To enable the designed electrochemically sensitive material with an ultrathin two-dimensional heterostructure to exhibit excellent electrochemical response to TRACP-5b, this invention uses a monolayer of ultrathin MXene material and a monolayer of ultrathin GO as the two components to construct the heterostructure. The monolayer MXene nanosheets were charged and modified by PEI, and a GO / MXene material with an ultrathin two-dimensional heterostructure was constructed via electrostatic adsorption. This approach not only overcomes the shortcomings of insufficient conductivity in GO and the tendency of MXene to stack, but also provides a faster electron transport channel in the electrochemical reaction, thereby improving reaction efficiency.

[0041] The electron transport capability and highly sensitive electrochemical response to 1-naphthol and TRACP-5b of the obtained material were verified using relevant characterization techniques and electrochemical testing methods. According to the test results, the electrochemical sensing method can achieve a highly sensitive response to TRACP-5b over a wide concentration range from 40 ng / mL to 1000 ng / mL, with a limit of detection (LOD) of 18.6 ng / mL for TRACP-5b. Furthermore, the reproducibility and stability of the electrochemical sensing system were tested to verify its practical application capability.

[0042] This invention also developed a handheld electrochemical sensor based on GO / MXene materials and a disposable electrochemical sensor for highly sensitive and multi-channel real-time detection of TRACP-5b in human GCF.

[0043] In summary, the electrochemical sensor based on GO / MXene composite material of this invention can achieve effective real-time monitoring of periodontitis through the sensitive and accurate detection of TRACP-5b. Attached Figure Description

[0044] Figure 1 : Schematic diagram of the principle of TRACP-5b detection in this invention.

[0045] Figure 2 The image shows the morphology of Ti3AlC2 in Example 1. In the image, a is a scanning electron microscope (SEM) image of Ti3AlC2, a is a SEM image of Ti3C2, a multilayer MXene phase material, and a single-layer MXene phase material, Ti3C2.

[0046] Figure 3 Characterization results of the GO / MXene composite material in Example 1. In the figure, a is a transmission electron microscope (TEM) image of monolayer MXene material Ti3C2, b is a TEM image of monolayer GO, c is a TEM image after Ti3C2 and GO are combined, d is the morphology and corresponding lattice fringes of monolayer MXene material Ti3C2 under high resolution transmission electron microscopy (HR-TEM), e is the morphology of monolayer GO under HR-TEM imaging, f is the heterostructure morphology of GO / MXene under HR-TEM imaging, g is the TEM morphology of GO / MXene, h is the high-angle annular dark-field transmission electron microscopy (HAADF-TEM) image of the TEM morphology of GO / MXene shown in g, and i is the elemental analysis results of the morphology shown in h.

[0047] Figure 4 : Schematic diagram of the disposable electrochemical sensor in Example 2. In the figure, A is the reference electrode (RE), B is the working electrode (WE), C is the counter electrode (CE), and D is the wire.

[0048] Figure 5 Example 2: Circuit connection test diagram of the handheld electrochemical sensor and corresponding mobile phone test interface.

[0049] Figure 6 : Electrochemical impedance spectroscopy (EIS) test results of the disposable electrochemical sensor in Example 3. The inset shows the equivalent circuit model fitted based on the EIS results.

[0050] Figure 7 Figure 3: Cyclic voltammetry (CV) test results of the disposable electrochemical sensor in Example 3.

[0051] Figure 8 Figure 3: Differential pulse voltammetry (DPV) test results of the disposable electrochemical sensor in Example 3.

[0052] Figure 9Image a shows the sensitivity test of the disposable electrochemical sensor in Example 3 for 1-naphthol in the concentration range of 10 to 100 nM. The inset is an enlarged view of 1-naphthol at each concentration and its corresponding current response value. Image b shows the linear equation constructed by the disposable electrochemical sensor for 1-naphthol in the concentration range of 10 to 100 nM and its corresponding current value.

[0053] Figure 10 Image a shows the sensitivity test of the disposable electrochemical sensor in Example 4 for TRACP-5b in the concentration range of 500 to 1000 ng / mL. The inset is an enlarged view of each concentration of TRACP-5b and its corresponding current response value. Image b shows the linear equation constructed by the disposable electrochemical sensor in Example 4 for TRACP-5b in the concentration range of 500 to 1000 ng / mL and its corresponding current value.

[0054] Figure 11 Image a shows the sensitivity test of the disposable electrochemical sensor in Example 4 for TRACP-5b in the concentration range of 40 to 400 ng / mL, with the inset showing a magnified view of each concentration of TRACP-5b and its corresponding current response value. Image b shows the linear equation constructed by the disposable electrochemical sensor in Example 4 for TRACP-5b and its corresponding current value in the concentration range of 40 to 400 ng / mL.

[0055] Figure 12 Figure 4 shows the results of the reproducibility test of the disposable electrochemical sensor in Example 4.

[0056] Figure 13 Figure 4: Results of stability test of disposable electrochemical sensor in Example 4.

[0057] Figure 14 EIS test results of the disposable electrochemical sensor modified with monolayer MXene material in Comparative Example 1. The inset shows the equivalent circuit model fitted based on the EIS results.

[0058] Figure 15 : CV test results of the disposable electrochemical sensor modified with monolayer MXene material in Comparative Example 1.

[0059] Figure 16 EIS test results of the disposable electrochemical sensor modified with monolayer GO in Comparative Example 2. The inset shows the equivalent circuit model fitted based on the EIS results.

[0060] Figure 17 Figure 2: CV test results of the disposable electrochemical sensor modified with monolayer GO in Comparative Example 2. Detailed Implementation

[0061] This invention is based on the principle that TRACP-5b enzyme catalyzes the formation of the enzyme product 1-naphthol from inert 1-naphthyl phosphate monosodium salt under specific conditions (e.g., Figure 1 As shown in the figure, a highly sensitive electrochemical method for the detection of 1-naphthol was constructed to accurately quantify TRACP-5b. Regarding electrode material preparation, a GO / MXene composite material with an ultrathin two-dimensional heterostructure was constructed through the electrostatic adsorption of PEI-modified MXene with GO. An electrochemical sensor device was built based on this composite material to achieve rapid and sensitive detection of TRACP-5b, thus enabling real-time monitoring of periodontitis.

[0062] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0063] Example 1: Preparation of GO / MXene composite material

[0064] This embodiment provides a GO / MXene composite material based on a heterostructure. The GO / MXene composite material is a composite material with ultrathin two-dimensional heterostructure characteristics formed by vertical stacking or in-plane growth of ultrathin monolayer graphene and ultrathin monolayer MXene materials; the graphene material is GO; and the MXene material is Ti3C2. The preparation method of this composite material is as follows:

[0065] S11. Under ice-water bath conditions, 1 g of MAX phase material powder was slowly immersed into 15 mL of HF solution and stirred for 10 min; then the temperature was raised to 40 ℃ and stirring was continued for 24 h; after the reaction was completed, the mixture was centrifuged at 3500 rpm for 10 min, the resulting precipitate was washed with deionized water until neutral, and then vacuum dried at 60 ℃ for 24 h to obtain multilayer MXene material;

[0066] S12. Immerse 5 mg of the above MXene material powder in 50 mL of DMSO solution and stir continuously at room temperature for 24 h. After the reaction is complete, wash with deionized water to remove excess DMSO. Place the resulting dispersion in an ice-water bath and sonicate for 1 h, then centrifuge at 3500 rpm for 1 h. After centrifugation, pour off the upper dispersion. The resulting dispersion is the two-dimensional ultrathin monolayer MXene material.

[0067] S13. The above two-dimensional ultrathin monolayer MXene material powder is uniformly dispersed in an appropriate amount of deionized water, and 5 mL of PEI is added to modify the charge of the MXene material. After that, the dispersion is ultrasonically treated for 5 min, and the excess PEI is washed with deionized water and redispersed in deionized water to form a stable hydrosol.

[0068] S14. A two-dimensional ultrathin monolayer GO dispersion was added to a modified MXene dispersion under vigorous stirring, and the mixture was stirred continuously at room temperature for 6 h. The resulting material was then vacuum dried at 60 °C for 12 h to obtain a GO / MXene composite material with an ultrathin two-dimensional heterostructure. The mass ratio of GO to MXene in this composite material was 1:1; the thickness of the GO and MXene was approximately 2 nm.

[0069] Characterization tests:

[0070] The synthesis method of the multilayer MXene material Ti3C2 in this embodiment is based on the selective etching of the Al layer of its precursor MAX phase Ti3AlC2 by HF. The SEM characterization images of the MAX phase Ti3AlC2 and the etched Ti3C2 are shown below. Figure 2 a and Figure 2 As shown in b, Ti3AlC2 exhibits a closely packed morphology and the characteristic accordion-like morphology of multilayer Ti3C2 after the Al layer is etched, indicating the effective etching of Ti3AlC2 by HF. Subsequently, the multilayer Ti3C2 was exfoliated using the organic solvent dimethyl sulfoxide (DMSO) intercalation method to obtain monolayer Ti3C2 nanosheets. Figure 2 c).

[0071] Figure 3 Image a shows a TEM image of the obtained monolayer MXene material Ti3C2, which clearly shows that the monolayer Ti3C2 is an isolated sheet-like structure with sharp edges. Figure 3 The TEM image of b shows the unique folded morphology of GO. Figure 3 Image c shows the morphology of the composite material, clearly revealing a GO-like folded structure in the bottom layer and a Ti3C2-like translucent material morphology in the upper layer. Furthermore, the morphologies of GO, MXene, and GO / MXene were characterized using HR-TEM, HAADF-TEM, and elemental analysis. Figure 3 d and Figure 3 The lattice fringes of the monolayer Ti3C2 nanosheets and the disordered amorphous structure of the monolayer GO can be clearly seen. The lattice fringes of Ti3C2 have an interstic gap of about 0.4 nm. Figure 3 f represents the heterostructure morphology of GO / MXene under HR-TEM imaging. As can be seen from the figure, in this morphology, Ti3C2 nanosheets with clear lattice fringes overlap with GO with amorphous structural features, forming a well-connected two-dimensional layered heterostructure. Figure 3g and 3h are the TEM morphology of GO / MXene and the corresponding HAADF-TEM images, respectively, clearly showing the heterostructure morphology formed by the stacking of two materials with different morphologies. Figure 3 The elemental analysis results shown in i indicate that Ti is only uniformly distributed in the upper layer of the heterostructure, indicating that the upper layer is a single-layer MXene material Ti3C2 and the lower layer is a single-layer GO, further demonstrating the successful construction of the GO / MXene two-dimensional heterostructure morphology.

[0072] Example 2: Fabrication of a disposable electrochemical sensor

[0073] This embodiment further prepares a disposable electrochemical sensor based on the GO / MXene composite material prepared in Example 1, the structure of which is as follows. Figure 4 As shown, the device includes a PET substrate and multiple detection units disposed on the surface of the PET substrate. Each detection unit includes A. a reference electrode RE, B. a working electrode WE, C. a counter electrode CE, and D. a wire. The WE is a carbon electrode with a surface coated with a GO / MXene composite material; the CE is a carbon electrode disposed on the other side of the WE; the RE is an Ag / AgCl electrode disposed on one side of the WE; and the wires are respectively connected to the bottom ends of the RE, WE, and CE. The fabrication method of this electrochemical sensor in this embodiment is as follows:

[0074] S21. Weigh 5 g of Ag / AgCl ink and use a screen printing machine to evenly print the diluted Ag / AgCl ink onto PET. Dry at room temperature to obtain the printed RE.

[0075] S22. Weigh 5 g of conductive carbon paste and use a screen printing machine to evenly print the diluted conductive carbon paste onto the PET printed with RE. Dry at room temperature to obtain the printed WE and CE.

[0076] S23. Weigh 5 g of conductive silver paste and use a screen printing machine to evenly print the diluted conductive silver paste onto the PET printed with RE, WE and CE. Dry at room temperature to obtain the printed wire.

[0077] S24. Take 5 mg of GO / MXene composite material and mix it with 100 μL of anhydrous ethanol and 20 μL of Nafion solution to prepare a uniform ink; take 2 μL of the above ink and uniformly drop it onto the WE surface of the electrochemical sensor to complete the preparation of the disposable electrochemical sensor.

[0078] The disposable electrochemical sensor prepared in this embodiment is inserted into a handheld electrochemical sensor device for detection during use, such as... Figure 5As shown, the handheld electrochemical sensor includes a disposable electrochemical sensor, a control circuit board, and a display. The lead wire of the disposable electrochemical sensor is connected to the control circuit board, and the display is electrically connected to the control circuit board to display the TRACP-5b content detection results. It can be a mobile phone. The method for actual sample detection using this sensor is as follows:

[0079] First, a gradient solution of TRACP-5b was prepared, reacted with sodium 1-naphthyl phosphate, and then drop-coated onto the electrode surface. A series of current values ​​were measured using a control circuit board, and a standard curve was established between the TRACP-5b concentration and the current value. Next, sodium 1-naphthyl phosphate was added to the gingival crevicular fluid sample to be tested for reaction. After the reaction was complete, the reaction solution was drop-coated onto the electrode surface, and the current value was measured using the control circuit board. This current value was then used to calculate the TRACP-5b content in the sample.

[0080] Example 3: Electrochemical performance testing of a disposable electrochemical sensor

[0081] This embodiment uses the disposable electrochemical sensor prepared in Example 2 as the test object to verify the electron transport capability of the GO / MXene composite material and its electrochemical response to 1-naphthol. The specific electrochemical performance testing methods and results are as follows:

[0082] (1) Take 50 μL 0.1 M [Fe(CN)6] 3- / 4- The solution was uniformly drop-coated onto the disposable electrochemical sensor, ensuring coverage of WE, RE, and CE during drop-coating, before EIS testing. The EIS testing conditions were a frequency of 10⁻⁶. 5 The frequency is 0.1 Hz, and the amplitude is 5 mV.

[0083] EIS test results are as follows Figure 6 As shown, SPE is a disposable electrochemical sensor without any modification to the material. The inset shows the equivalent circuit model fitted based on EIS results, which consists of a double-layer capacitor (C0). dl ), a solution resistor (R) S ), a charge transfer resistor (R) ct ) and a Warburg element (Z) W The EIS results clearly show the semi-circular characteristics of the two electrochemical sensors in the high-frequency region and the inclined linear characteristics in the low-frequency region, indicating that the above electrode reactions all involve charge transfer and diffusion mechanisms. Compared with the blank SPE, the SPE modified with GO / MXene composite material has a smaller semi-circular radius in the high-frequency region, while GO / MXene has a larger slope in the low-frequency region, which indicates that the GO / MXene material has good conductivity and electron transfer efficiency.

[0084] (2) Take 50 μL of KOH blank solution with pH 10 and the same KOH solution containing 1-naphthol sample and uniformly drop them onto the disposable electrochemical sensor. When dropping, ensure that the solution covers WE, RE and CE, and then perform CV test. The CV test conditions are performed at a scan rate of 60 mV / s in the potential range of -0.6 to 0.6 V vs. Ag / AgCl.

[0085] CV test results as follows Figure 7 As shown, it can be clearly demonstrated that GO / MXene has a significant response to 1-naphthol, with an electrochemical oxidation potential of approximately 0.26 V vs. Ag / AgCl.

[0086] (3) Take 50 μL of KOH blank solution with pH 10 and the same KOH solution containing 1-naphthol sample and uniformly drop them onto the disposable electrochemical sensor. When dropping, ensure that the solution covers WE, RE and CE, and then perform DPV test. The DPV test conditions are 50 mV amplitude, 200 ms pulse width, 5 mV step height, 500 ms step time and 10 mV / s scan rate.

[0087] DPV test results are as follows Figure 8 As shown, the results validate the responsiveness of GO / MXene to 1 μM 1-naphthol at 0.25 V vs. Ag / AgCl. Compared to the blank solution (KOH solution with pH = 10 without 1-naphthol), the electrochemical response signal of GO / MXene to 1-naphthol is 6.59 μA.

[0088] (4) The sensitivity test results of the electrochemical sensor based on GO / MXene composite material to 1-naphthol are as follows: Figure 9 As shown, the electrochemical testing method was chronoamperometry, demonstrating the response capability of the GO / MXene electrochemical sensor to 1-naphthol concentrations ranging from 10 to 100 nM. The results indicate that at a potential of 0.25 V vs. Ag / AgCl, the current value increases with increasing 1-naphthol concentration. Figure 9 a). Figure 9 b shows the linear relationship between 1-naphthol concentration and current value. This correlation can be represented by the linear equation I = 0.019 x + 1.249, with a coefficient of determination of R² = 0.950, indicating a good linear fit. Meanwhile, the LOD, calculated based on a three-fold signal-to-noise ratio, is 9.162 nM, demonstrating that the constructed GO / MXene electrochemical sensing system exhibits excellent response to 1-naphthol.

[0089] Example 4: Performance test of disposable electrochemical sensor for detecting TRACP-5b

[0090] The specific method for TRACP-5b measurement using the sensor in this embodiment is as follows:

[0091] (1) Using a Tris-HCl buffer solution with pH = 5 and a concentration of 0.1 M as the reaction background, TRACP-5b was diluted to the required concentration, and the specific enzyme reaction substrate 1-naphthalene phosphate monosodium salt of TRACP-5b was prepared under the same conditions.

[0092] (2) Take 5 μL of TRACP-5b solution and 45 μL of substrate into a centrifuge tube, mix thoroughly by blowing under an ice-water bath, and then immediately place it in a 37 ℃ water bath for 30 min.

[0093] (3) Add 10 μL of 20% KOH to the above reaction solution to terminate the enzyme reaction. The resulting solution is the test solution. Repeat the above steps with 50 μL of substrate solution to obtain the test blank solution.

[0094] (4) Take 50 μL of the above test solution and blank solution and uniformly drop-coat them onto the disposable electrochemical sensor, ensuring that the test solution covers WE, RE, and CE during drop-coating. Then use... Figure 5 The circuit was tested.

[0095] Based on the above method, the sensor detection sensitivity, repeatability, and stability were tested, and the test results are as follows:

[0096] (1) Sensitivity test of TRACP-5b by an electrochemical sensor based on GO / MXene material:

[0097] The electrochemical sensor detection system prepared in this embodiment based on GO / MXene composite material and disposable electrochemical sensor can be used to sensitively detect the concentration of TRACP-5b in human GCF. Figure 10 This paper demonstrates the current response of an electrochemical sensor based on GO / MXene to TRACP-5b in the concentration range of 500 ng / mL to 1000 ng / mL. The electrochemical current value increases with increasing TRACP-5b concentration, indicating a positive correlation between the electrochemical response signal and TRACP-5b concentration within this concentration range. Furthermore, the current value exhibits a good linear relationship with TRACP-5b concentration. Figure 10 (b) Its linear standard curve is represented by the equation I = 0.007 x - 1.685, where the coefficient of determination R² = 0.916. Figure 11This demonstrates the electrochemical testing performance of a GO / MXene-based electrochemical sensor on TRACP-5b at low concentrations ranging from 40 ng / mL to 400 ng / mL. Its good sensing linearity is shown in Figure 1. Figure 11 As shown in b, the linear equation is expressed as I = 0.005 x + 0.119, the coefficient of determination R² = 0.994, and its LOD is 18.6 ng / mL, indicating that the constructed GO / MXene electrochemical detection system has the ability to detect TRACP-5b with high sensitivity.

[0098] (2) Reproducibility test of the electrochemical sensor based on GO / MXene material:

[0099] In this embodiment, an electrochemical sensing detection system based on GO / MXene composite material and a disposable electrochemical sensor was prepared. To evaluate the reproducibility of this sensing system, 10 prepared disposable GO / MXene electrodes were randomly selected, and electrochemical tests were performed using TRACP-5b at a concentration of 500 ng / mL as the detection target. Figure 12 The study observed that all selected electrodes exhibited stable current signals, indicating that the constructed electrochemical sensing system has good reproducibility.

[0100] (3) Stability testing of the electrochemical sensor based on GO / MXene material:

[0101] In this embodiment, an electrochemical sensor detection system based on GO / MXene composite material and disposable electrochemical sensor was prepared. To evaluate the stability of the sensing system, the constructed GO / MXene electrode was directly exposed to the daily environment for a month for monitoring to observe the current signal fluctuation of the electrochemical detection system in response to a concentration of 500 ng / mL TRACP-5b. Figure 13 The results show that the current response of the sensing electrode is almost identical to that of the original electrode within 10 days; after 10 days, the current value gradually decreases, which may be due to the partial oxidation of MXene in GO / MXene, which destroys the heterostructure and leads to a decrease in electrode performance. Overall, the constructed GO / MXene sensor exhibits good reproducibility and stability.

[0102] Comparative Example 1

[0103] The process is basically the same as in Example 1, except that the sensitive material used in the disposable electrochemical sensor is different; specifically, only MXene is used as the sensitive material for electrochemical detection. The morphological characteristics of the monolayer MXene are basically the same. Figure 3 a. In addition, the response of MXene to 1-naphthol was tested using EIS and CV techniques, and the results are as follows: Figure 14 and Figure 15 As shown. Specifically, the EIS results analysis is basically the same as in Example 3, but the radius of the semicircular region of MXene in the high-frequency region is larger than that of the GO / MXene composite material in the high-frequency region. At the same time, the slope of MXene in the low-frequency region is smaller, indicating that MXene has poorer conductivity and electron transfer efficiency compared to GO / MXene. CV results show that compared with the ultrathin two-dimensional heterostructure GO / MXene ( Figure 7 The weak electrochemical response of MXene to 1-naphthol at the same concentration indicates that simple MXene nanosheets are not suitable for this application.

[0104] Comparative Example 2

[0105] The process is basically the same as in Example 1, except that the sensitive material used in the disposable electrochemical sensor is different; specifically, only GO is used as the sensitive material for electrochemical detection. The morphological characteristics of the monolayer GO are basically the same. Figure 3 b. The response of GO to 1-naphthol was also tested using EIS and CV techniques, and the results are as follows: Figure 16 and Figure 17 As shown in the figure. Specifically, the EIS results are similar to those of Comparative Example 1, indicating that GO exhibits poorer conductivity and electron transfer efficiency compared to the GO / MXene composite. CV results show that, compared to the ultrathin two-dimensional heterostructure GO / MXene, GO has a weaker electrochemical response to the same concentration of 1-naphthol, while its oxidation potential increases to 0.29 V vs. AgCl, reflecting the low electron transfer rate of GO and suggesting that pure GO nanosheets are not suitable for this application.

[0106] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A GO / MXene composite material based on an ultrathin two-dimensional heterostructure, characterized in that, The GO / MXene composite material is a composite material with ultrathin two-dimensional heterostructure characteristics formed by vertically stacking ultrathin monolayer graphene material and ultrathin monolayer MXene material; the graphene material is graphene oxide or reduced graphene oxide; the MXene material is Ti3C2, Nb2C, Ti2C or V2C. The preparation method of the GO / MXene composite material includes the following steps: S11. React the MAX phase material with HF to generate a multilayer MXene material; S12. React multilayer MXene material with DMSO to generate two-dimensional ultrathin monolayer MXene material; S13. Charge modification of two-dimensional ultrathin monolayer MXene material using PEI; S14. A two-dimensional ultrathin monolayer graphene material dispersion was added to a charge-modified MXene dispersion, and the reaction was carried out to obtain a GO / MXene composite material with ultrathin two-dimensional heterostructure characteristics.

2. The ultrathin two-dimensional heterostructured GO / MXene composite material according to claim 1, characterized in that, The mass ratio of the graphene material to MXene ranges from 1:(1 ~ 5); the thickness of the graphene material and MXene ranges from 2 nm to 6 nm.

3. The GO / MXene composite material with ultrathin two-dimensional heterogeneous structure according to claim 1, characterized in that, Its preparation method includes the following steps: S11. Under ice-water bath conditions, 1 g of MAX phase material powder was slowly immersed into 10-20 mL of HF solution and stirred for 5-10 min; then the temperature was raised to 40-60 ℃ and stirring was continued for 12-24 h; after the reaction was completed, the mixture was centrifuged at 3000-5500 rpm for 5-10 min, and the resulting precipitate was washed with deionized water until neutral. The precipitate was then vacuum dried at 40-60 ℃ for 12-24 h to obtain multilayer MXene material. S12. Immerse 5-10 mg of the above MXene material powder in 25-50 mL of DMSO solution and stir continuously at room temperature for 18-24 h. After the reaction is complete, wash with deionized water to remove excess DMSO. Place the resulting dispersion in an ice-water bath and sonicate for 1-2 h, then centrifuge at 3000-5500 rpm for 1-2 h. After centrifugation, pour off the upper dispersion. The resulting dispersion is the two-dimensional ultrathin monolayer MXene material. S13. The above two-dimensional ultrathin monolayer MXene material powder is uniformly dispersed in an appropriate amount of deionized water, and 5-10 mL of PEI is added to modify the charge of the MXene material; then the dispersion is ultrasonically treated for 5-10 min, and the excess PEI is washed with deionized water and redispersed in deionized water to form a stable hydrosol. S14. The two-dimensional ultrathin monolayer graphene dispersion is added to the charge-modified MXene dispersion under vigorous stirring, and the mixture is stirred continuously at room temperature for 6 to 24 hours. The resulting material is then vacuum dried at 40 to 60 °C for 12 to 24 hours to obtain a composite material with ultrathin two-dimensional heterostructure characteristics.

4. A disposable electrochemical sensor, characterized in that, The device includes a PET substrate and at least one detection unit disposed on the surface of the PET substrate, the detection unit comprising the following components: The working electrode WE is a carbon electrode with a surface coated with the GO / MXene composite material based on an ultrathin two-dimensional heterostructure as described in any one of claims 1-3. During electrochemical measurement, current will pass through the electrode and drive the specified chemical process. Its potential change directly reflects the characteristics and rate of the electrochemical reaction. The counter electrode CE is a carbon electrode disposed on the other side of the WE, which is used to form a current loop with the WE in electrochemical measurements, thereby ensuring that the electrochemical reaction on the WE can continue. The reference electrode RE is an Ag / AgCl electrode disposed on the WE side, used to provide a known and stable reference potential for electrochemical measurements; The wires are connected to the bottom of RE, WE and CE respectively, to form a complete current loop for each electrode of the sensor in electrochemical measurements.

5. The disposable electrochemical sensor according to claim 4, characterized in that, Its preparation method includes the following steps: S21. Print Ag / AgCl ink evenly onto PET and dry to obtain the printed RE; S22. The conductive carbon paste is uniformly printed onto the PET plate with the reference electrode, and dried to obtain the printed WE and CE. S23. The conductive silver paste is uniformly printed onto the PET printed with RE, WE and CE, and dried to obtain the printed wire; S24. Prepare an ink by mixing GO / MXene composite material with anhydrous ethanol and Nafion solution. Apply the ink evenly to the WE surface to complete the preparation of a disposable electrochemical sensor.

6. The disposable electrochemical sensor according to claim 5, characterized in that, Its preparation method includes the following steps: S21. Weigh 5-10 g of Ag / AgCl ink and dilute it with 5-10 mL of thinner. Then, use a screen printing machine to evenly print the diluted Ag / AgCl ink onto PET and dry it at room temperature to obtain the printed RE. S22. Weigh 4 to 8 g of conductive carbon paste and dilute it with 5 to 10 mL of diluent. Use a screen printing machine to evenly print the diluted conductive carbon paste onto the PET plate with the reference electrode printed on it. Dry it at room temperature to obtain the printed WE and CE. S23. Weigh 5 ~ 15 g of conductive silver paste and dilute it with 5 ~ 20 mL of diluent. Use a screen printing machine to evenly print the diluted conductive silver paste onto the PET printed with RE, WE and CE. Dry at room temperature to obtain the printed wire. S24. Take 5 ~ 10 mg of GO / MXene composite material and mix it with 100 ~ 500 μL of anhydrous ethanol and 20 ~ 100 μL of Nafion solution to prepare a uniform ink; take 2 ~ 10 μL of the above ink and uniformly drop it onto the WE surface of the electrochemical sensor to complete the preparation of the disposable electrochemical sensor.

7. The application of the disposable electrochemical sensor according to any one of claims 4-6 in detecting the TRACP-5b content in human gingival crevicular fluid.

8. The application according to claim 7, characterized in that, The application method involves inserting the disposable electrochemical sensor into a handheld electrochemical sensor device, which includes a disposable electrochemical sensor, a control circuit board, and a display. The lead wire of the disposable electrochemical sensor is connected to the control circuit board, and the display is electrically connected to the control circuit board. During the test, a TRACP-5b gradient solution was first prepared, reacted with 1-naphthyl phosphate monosodium salt, and then dropped onto the electrode surface. The corresponding current value was measured by the control circuit board, and a standard curve was established between the TRACP-5b concentration and the current value. Then, the sample to be tested was reacted with 1-naphthyl phosphate monosodium salt and dropped onto the electrode surface. The measured current value was then substituted into the standard curve to obtain the TRACP-5b content in the sample to be tested.

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