GO / MXene composite material based on ultrathin two-dimensional heterostructure, electrochemical sensor and application of GO / MXene composite material

Abstract

The invention belongs to the technical field of medical instruments, and particularly discloses a graphene oxide (GO) / MXene composite material based on an ultrathin two-dimensional heterostructure, an electrochemical sensor and application thereof. On the basis of specific enzyme reaction of tartrate-resistant acid phosphatase (TRACP-5b), an electrochemical detection method based on an ultrathin two-dimensional heterostructure material system is designed and is used for sensitively detecting the content of TRACP-5b in human gingival crevicular fluid (GCF), so that real-time monitoring of human oral periodontitis is realized. Therefore, the electrochemical biosensor based on the GO / MXene material of the ultrathin two-dimensional heterostructure is constructed, the constructed electrochemical sensing system can realize effective detection of TRACP-5b based on the high electron transmission capability and catalytic activity of the material, the lowest limit of detection (LOD) is 18.6 ng / mL, and a new thought is provided for clinical diagnosis and real-time monitoring of periodontitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical devices, and particularly relates to a GO / MXene composite material based on an ultrathin two-dimensional heterostructure, an electrochemical sensor and applications thereof. Background Art

[0002] The recent Global Burden of Disease study showed that severe periodontitis is the sixth most prevalent disease globally, with a total prevalence of 11.2% and approximately 743 million people affected. As an inflammatory lesion, the pathological features of periodontitis are the interweaving of inflammation and repair, osteogenesis and osteoclastogenesis processes, and the progression is accompanied by the formation and deepening of periodontal pockets and the resorption of alveolar bone, and the resorption of alveolar bone is the main risk factor for tooth loosening and exfoliation. Currently, the clinical diagnostic techniques for periodontitis include clinical periodontal pocket probing and traditional medical imaging methods (such as X-ray films, CT, etc.). However, the former is time-consuming in operation and the results are affected by the examiner's experience and skills, and the latter is costly and has a radiation risk to patients, and neither can frequently perform real-time monitoring and early risk assessment of the alveolar bone resorption of treated patients. Therefore, a method that can be used for rapid clinical detection or accurately predict the severity of severe periodontitis and the status of alveolar bone resorption is urgently needed.

[0003] Gingival crevicular fluid (GCF) is the only fluid that directly exudes from body fluids, and the changes in its content and composition are closely related to periodontal tissues, which can provide important quantitative indicators for the evaluation of periodontal health status. Since GCF can be sampled repeatedly and the sampling is non-invasive, which can minimize discomfort to the greatest extent, GCF is a very promising source of diagnostic information at present, which helps to improve the efficiency and accuracy of clinical diagnosis and improve the irreversible periodontal damage caused by the untimely clinical diagnosis and treatment. In addition, 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 have shown that biomarkers related to root resorption in human GCF are considered to be possible periodontitis monitoring indicators, specifically including TRACP-5b, dentin phosphoprotein, cementum protein-1, etc.

[0004] TRACP-5b is an active enzyme secreted by osteoclasts, mainly derived from osteoclasts with bone resorption function. Its expression level and activity are positively correlated with bone resorption in the body. Therefore, the content of TRACP-5b in serum has been used internationally as a marker to measure bone resorption and osteoclast activity. The activity level of this enzyme does not change significantly during the day and night and is not affected by eating, so samples can be collected for detection at any time of the day. Periodontitis is closely related to bone resorption and the number of osteoclasts. Relevant studies have shown that during the progression of periodontitis, the expression level of TRACP-5b will increase significantly. Therefore, TRACP-5b is expected to be a potential biomarker for periodontitis and can be used to diagnose the severity of periodontitis.

[0005] Traditional detection methods of TRACP-5b usually perform quantitative or semi-quantitative detection and analysis through enzyme-linked immunosorbent assay (ELISA), Western-blot, and proteomics methods. These methods have high detection time and economic costs and low yields, and they also cannot achieve real-time monitoring of periodontal diseases, so they still have great limitations in clinical applications.

[0006] Currently, there are few studies on TRACP-5b in GCF based on electrochemical technology. Electrochemical sensors are widely concerned in the sensor field because of their advantages such as simple operation, low cost, fast analysis speed, and small size, and are suitable for on-site rapid detection under various requirements. Now there have been relevant reports showing that the method based on electrochemical sensors can achieve highly sensitive real-time monitoring of the human oral microenvironment in a low-cost and non-invasive way. However, due to the low expression level of TRACP-5b in GCF and the complex components in GCF, this brings challenges to the detection of TRACP-5b. Therefore, it is necessary to construct a high-performance electrochemical sensing system with high sensitivity and strong specificity for the detection of TRACP-5b. Summary of the Invention

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

[0008] As a single-layer material exfoliated from graphite oxide, graphene oxide (GO) has characteristics such as a large specific surface area, abundant functional groups, and a special delocalized electron arrangement, endowing it with a strong affinity for common carbon-based ring materials in biomolecules and drugs, and making it commonly used in fields such as medicine, biology, and electrochemical sensing. However, GO lacks certain electron transport capabilities. Research shows that the degree of oxidation of GO has a significant impact on its conductivity. When the degree of oxidation exceeds 20%, the conductivity will drop sharply; when used as a sensitive material, GO will also cause a decrease in electron transport capabilities and a reduction in the number of surface active groups due to severe aggregation between flakes, resulting in its actual electrochemical performance often being lower than expected. Therefore, the electrochemical application of GO has certain limitations. MXene materials are two-dimensional materials with high conductivity, a large specific surface area, and abundant surface groups. Relevant reports have confirmed that they exhibit efficient, stable, and tunable catalytic performance in the electrocatalytic reaction of phenolic-related markers, so they have good application prospects in electrochemical detection. However, MXene materials will also have problems such as relatively low capacitance, blocked electron transport, and low stability of electrode materials due to stacking in practical applications. The composite material with an ultrathin two-dimensional heterostructure constructed by GO and MXene in the present invention can effectively improve the material stacking problem compared with a single sensitive material, and the heterostructure material has a higher electron transport efficiency, can effectively improve the catalytic activity, effectively overcome the application defects of GO and MXene, and lay a key foundation for the highly sensitive electrochemical detection of TRACP-5b enzyme.

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

[0010] In the first aspect, the present invention provides a GO / MXene composite material based on an ultrathin two-dimensional heterostructure, which is a composite material with the characteristics of an ultrathin two-dimensional heterostructure formed by an ultrathin single-layer graphene material and an ultrathin single-layer MXene material through vertical stacking or in-plane growth; the graphene material is graphene oxide or reduced graphene oxide; the MXene material is Ti 3 C 2 、Nb 2 C, Ti 2 C or V 2 C.

[0011] Further, the mass ratio range of the graphene to the MXene is 1:(1 - 5). When the mass ratio of the graphene to the MXene exceeds the defined range, the excess graphene or MXene nanosheets may affect the formation of the two-dimensional heterostructure, which is not conducive to the application of electrochemical sensing. In addition, as the thickness of the two-dimensional material increases, the 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 the MXene is 2 nm to 6 nm.

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

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

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

[0015] S13. Charge-modify the two-dimensional ultrathin single-layer MXene material with polyethyleneimine (PEI);

[0016] S14. Add the two-dimensional ultrathin single-layer graphene dispersion liquid to the charge-modified two-dimensional ultrathin single-layer MXene dispersion liquid, and react to obtain a graphene / MXene composite material with the characteristics of an ultrathin two-dimensional heterostructure.

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

[0018] S11. Under the condition of an ice-water bath, slowly immerse 1 g of MAX phase material powder into 10 - 20 mL of HF solution, and stir for 5 - 10 min; then raise the temperature to 40 - 60 °C and continue to stir for 12 - 24 h; after the reaction is completed, centrifuge at a speed of 3000 - 5500 rpm for 5 - 10 min, wash the obtained precipitate with deionized water until neutral, and then vacuum-dry the precipitate at 40 - 60 °C for 12 - 24 h to obtain a multi-layer MXene material;

[0019] S12. Immerse 5 - 10 mg of the above-mentioned MXene material powder into 25 - 50 mL of DMSO solution, and continuously stir at room temperature for 18 - 24 h; after the reaction is completed, wash with deionized water to remove the excess DMSO; place the obtained dispersion in an ice-water bath and ultrasonicate for 1 - 2 h, then centrifuge at a speed of 3000 - 5500 rpm for 1 - 2 h; after centrifugation, pour out the upper-layer dispersion, and the obtained dispersion is the two-dimensional ultrathin single-layer MXene material at this time;

[0020] S13. Uniformly disperse the above-mentioned two-dimensional ultrathin single-layer MXene material powder in an appropriate amount of deionized water, and add 5 - 10 mL of PEI to modify the charge of the MXene material; then ultrasonicate the dispersion for 5 - 10 min, wash with deionized water to remove the excess PEI and redisperse it in deionized water to form a stable hydrogel;

[0021] S14. Add the two-dimensional ultrathin single-layer graphene dispersion to the charge-modified MXene dispersion under vigorous stirring, and continuously stir at room temperature for 6 - 24 h; the obtained material will be vacuum-dried at 40 - 60 °C for 12 - 24 h to obtain a graphene / MXene composite material with the characteristics of an ultrathin two-dimensional heterostructure.

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

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

[0024] A counter electrode (CE), which is a carbon electrode provided on the other side of the working electrode and is used to form a current loop with the WE during electrochemical measurement to ensure the continuous progress of the electrochemical reaction on the working electrode;

[0025] A reference electrode (RE), which is an Ag / AgCl electrode provided on one side of the working electrode and is used to provide a known and stable reference potential for electrochemical measurement;

[0026] Wires (conductive), which are respectively connected to the bottoms of the RE, WE, and CE and are used to form a complete current loop for each electrode of the sensor during electrochemical measurement.

[0027] Further, the preparation method of the disposable electrochemical sensor includes the following steps:

[0028] S21. Uniformly print Ag / AgCl ink on PET and dry it to obtain the printed RE.

[0029] S22. Uniformly print conductive carbon paste on the PET printed with RE and dry it to obtain the printed WE and CE.

[0030] S23. Uniformly print conductive silver paste on the PET printed with RE, WE and CE and dry it to obtain the printed wire.

[0031] S24. Prepare an ink by mixing GO / MXene composite material with absolute ethanol and Nafion solution, and uniformly drop-coat the ink on the surface of WE to complete the preparation of the disposable electrochemical sensor.

[0032] More specifically, the preparation method of the disposable electrochemical sensor includes the following steps (where the diluents used in steps S21, S22, and S23 can use conventional commercially available ink diluents, conductive carbon paste diluents, and conductive silver paste diluents):

[0033] S21. Weigh 5 - 10 g of Ag / AgCl ink and dilute it with 5 - 10 mL of diluent. Then use a screen printing machine to uniformly print the diluted Ag / AgCl ink on PET and dry it at room temperature to obtain the printed RE.

[0034] S22. Weigh 4 - 8 g of conductive carbon paste and dilute it with 5 - 10 mL of diluent. Use a screen printing machine to uniformly print the diluted conductive carbon paste on the above-mentioned PET printed with RE and dry it at room temperature to obtain the printed WE and CE.

[0035] S23. Weigh 5 - 15 g of conductive silver paste and dilute it with 5 - 20 mL of diluent. Use a screen printing machine to uniformly print the diluted conductive silver paste on the above-mentioned PET printed with RE, WE and CE and dry it at room temperature to obtain the printed wire.

[0036] S24. Take 5 - 10 mg of GO / MXene composite material and mix it with 100 - 500 μL of absolute ethanol and 20 - 100 mL of Nafion solution to prepare a uniform ink; take 2 - 10 μL of the above ink and uniformly drop-coat it on the surface of WE of the electrochemical sensor to complete the preparation of the disposable electrochemical sensor.

[0037] In a third aspect, the present invention provides the application of the above disposable electrochemical sensor in detecting the content of TRACP-5b in human GCF. Based on the principle that TRACP-5b enzyme catalyzes the generation of the enzymatic reaction product 1-naphthol from inert sodium 1-naphthyl phosphate monosodium salt under specific conditions, the electrochemical sensor of the present invention can highly sensitively detect 1-naphthol, thereby realizing the accurate quantification of TRACP-5b and achieving the purpose of diagnosing and real-time monitoring of periodontitis.

[0038] Further, the application method is to insert the disposable electrochemical sensor into a handheld electrochemical sensor device for use. The handheld electrochemical sensor device includes a disposable electrochemical sensor, a control circuit board, and a display. The wire end of the disposable electrochemical sensor is connected to the control circuit board, and the display is electrically connected to the control circuit board.

[0039] During detection, first prepare a TRACP-5b gradient solution, react it with sodium 1-naphthyl phosphate monosodium salt, and then drop-coat it onto the electrode surface. The corresponding current value is measured through the control circuit board, and a standard curve is established between the TRACP-5b concentration and the current value. Then, the sample to be tested is reacted with sodium 1-naphthyl phosphate monosodium salt and drop-coated onto the electrode surface, and the measured current value is brought into the standard curve to obtain the content of TRACP-5b in the sample to be tested.

[0040] The present invention has the following beneficial effects:

[0041] In order to enable the designed electrochemical sensitive material with ultra-thin two-dimensional heterostructure characteristics to have excellent electrochemical response ability to TRACP-5b, the present invention uses a single-layer ultra-thin MXene material and a single-layer ultra-thin GO as two parts for constructing the heterostructure. The single-layer MXene nanosheets are charge-modified by PEI, and a GO / MXene material with an ultra-thin two-dimensional heterostructure is constructed by electrostatic adsorption. In this way, not only can the disadvantages of insufficient conductivity of GO and easy stacking of MXene be compensated, but also the heterostructure can provide a faster electron transport channel in the electrochemical reaction, improving the reaction efficiency.

[0042] Through relevant characterization techniques and electrochemical testing methods, the electron transport ability of the obtained material and its highly sensitive electrochemical response to 1-naphthol and TRACP-5b were verified. According to the test results, a highly sensitive response of TRACP-5b can be achieved in a wide concentration range of 40 ng / mL to 1000 ng / mL under the electrochemical sensing method, and the lowest detection limit (LOD) for TRACP-5b is 18.6 ng / mL. At the same time, the reproducibility and stability of the electrochemical sensing system were also tested to verify its practical application ability.

[0043] The present invention also develops a handheld electrochemical sensor device 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.

[0044] In summary, the electrochemical sensor device based on the GO / MXene composite material of the present invention can effectively and real-time monitor periodontitis by sensitively and accurately detecting TRACP-5b. Brief Description of the Drawings

[0045] Figure 1 : Schematic diagram for detecting TRACP-5b in the present invention.

[0046] Figure 2 : Ti in Example 1 3 AlC 2 Morphology diagram. In the figure, a is the scanning electron microscope (SEM) image of the MAX phase material Ti 3 AlC 2 , b is the SEM image of the multi-layer MXene phase material Ti 3 C 2 , c is the SEM image of the single-layer MXene phase material Ti 3 C 2 .

[0047] Figure 3 : Characterization test results of the GO / MXene composite material in Example 1. In the figure, a is the transmission electron microscope (TEM) image of the single-layer MXene material Ti 3 C 2 , b is the TEM image of the single-layer GO, c is the TEM image after the combination of Ti 3 C 2 and GO, d is the morphology imaging and corresponding lattice fringes of the single-layer MXene material Ti 3 C 2 under the high-resolution transmission electron microscope (HR-TEM), e is the morphology of the single-layer GO under the HR-TEM imaging, f is the heterogeneous structure morphology of GO / MXene under the HR-TEM imaging, g is the TEM morphology of GO / MXene, h is the high-angle annular dark-field transmission electron microscope (HAADF-TEM) image of the TEM morphology of GO / MXene shown in g, and i is the elemental analysis result of the morphology shown in h.

[0048] Figure 4 : Schematic structural 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.

[0049] Figure 5: Schematic diagram of circuit connection detection of the handheld electrochemical sensor device in Example 2 and the corresponding mobile phone detection interface.

[0050] Figure 6 : Electrochemical impedance (EIS) test result graph of the disposable electrochemical sensor in Example 3. The inset is the equivalent circuit model fitted according to the EIS results.

[0051] Figure 7 : Cyclic voltammetry (CV) test result graph of the disposable electrochemical sensor in Example 3.

[0052] Figure 8 : Differential pulse voltammetry (DPV) test result graph of the disposable electrochemical sensor in Example 3.

[0053] Figure 9 : a is 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. b is the linear equation constructed for 1-naphthol in the concentration range of 10 to 100 nM and its corresponding current values by the disposable electrochemical sensor.

[0054] Figure 10 : a is 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 TRACP-5b at each concentration and its corresponding current response value. b is the linear equation constructed for TRACP-5b in the concentration range of 500 to 1000 ng / mL and its corresponding current values by the disposable electrochemical sensor in Example 4.

[0055] Figure 11 : a is the sensitivity test of the disposable electrochemical sensor in Example 4 for TRACP-5b in the concentration range of 40 to 400 ng / mL, where the inset is an enlarged view of TRACP-5b at each concentration and its corresponding current response value. b is the linear equation constructed for TRACP-5b in the concentration range of 40 to 400 ng / mL and its corresponding current values by the disposable electrochemical sensor in Example 4.

[0056] Figure 12 : Result graph of the reproducibility test of the disposable electrochemical sensor in Example 4.

[0057] Figure 13 : Result graph of the stability test of the disposable electrochemical sensor in Example 4.

[0058] Figure 14:EIS test result graph of the disposable electrochemical sensor modified with monolayer MXene material in Comparative Example 1. The inset is the equivalent circuit model fitted according to the EIS results.

[0059] Figure 15 :CV test result graph of the disposable electrochemical sensor modified with monolayer MXene material in Comparative Example 1.

[0060] Figure 16 :EIS test result graph of the disposable electrochemical sensor modified with monolayer GO in Comparative Example 2. The inset is the equivalent circuit model fitted according to the EIS results.

[0061] Figure 17 :CV test result graph of the disposable electrochemical sensor modified with monolayer GO in Comparative Example 2. Detailed implementation manners

[0062] Based on the principle that TRACP-5b enzyme catalyzes the generation of the enzymatic reaction product 1-naphthol from inert sodium 1-naphthyl phosphate under specific conditions (as shown in Figure 1 ), an electrochemical method for highly sensitive detection of 1-naphthol was constructed to accurately quantify TRACP-5b. In terms of the preparation of the electrode material, through the electrostatic adsorption of PEI-modified MXene material and GO, a GO / MXene composite material with the characteristics of an ultrathin two-dimensional heterostructure was constructed. Based on this composite material, an electrochemical sensor device was constructed to achieve rapid and sensitive detection of TRACP-5b, aiming at the real-time monitoring of periodontitis.

[0063] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0064] Example 1: Preparation of GO / MXene composite material

[0065] This example provides a GO / MXene composite material based on a heterostructure. The GO / MXene composite material is a composite material with the characteristics of an ultrathin two-dimensional heterostructure formed by the vertical stacking or in-plane growth of an ultrathin single-layer graphene material and an ultrathin single-layer MXene material. The graphene material is GO, and the MXene material is Ti 3 C 2 . The preparation method of this composite material is as follows:

[0066] S11. Under the condition of an ice-water bath, slowly immerse 1 g of MAX phase material powder into 15 mL of HF solution and stir for 10 min; then raise the temperature to 40 °C and continue stirring for 24 h; after the reaction is completed, centrifuge at a speed of 3500 rpm for 10 min. Wash the obtained precipitate with deionized water until neutral, and then vacuum-dry the precipitate at 60 °C for 24 h to obtain a multi-layer MXene material;

[0067] S12. Immerse 5 mg of the above MXene material powder into 50 mL of DMSO solution and continuously stir at room temperature for 24 h; after the reaction is completed, wash with deionized water to remove the excess DMSO; place the obtained dispersion under the condition of an ice-water bath and ultrasonicate for 1 h, and then centrifuge at a speed of 3500 rpm for 1 h; after centrifugation, pour out the upper-layer dispersion. At this time, the obtained dispersion is a two-dimensional ultrathin single-layer MXene material;

[0068] S13. Uniformly disperse the above two-dimensional ultrathin single-layer MXene material powder in an appropriate amount of deionized water, and add 5 mL of PEI to modify the charge of the MXene material; then ultrasonicate the dispersion for 5 min, wash the excess PEI with deionized water and redisperse it in deionized water to form a stable hydrogel;

[0069] S14. Add the two-dimensional ultrathin single-layer GO dispersion to the modified MXene dispersion under vigorous stirring and continuously stir at room temperature for 6 h; the obtained material will be vacuum-dried at 60 °C for 12 h to obtain a GO / MXene composite material with the characteristics of an ultrathin two-dimensional heterostructure. The mass ratio range of GO to MXene in this composite material is 1:1; the thickness range of the described GO and MXene is about 2 nm.

[0070] Characterization and testing:

[0071] The synthesis method of the multi-layer MXene material Ti 3 C 2 in this example is based on the selective etching of the Al layer of its precursor MAX phase Ti 3 AlC 2 to obtain. The SEM characterization images of MAX phase Ti 3 AlC 2 and the etched Ti 3 C 2 are shown in Figure 2 a and Figure 2 b respectively. Ti 3 AlC 2 shows a characteristic morphology of close packing and the iconic accordion morphology of multi-layer Ti 3 C 2 after the Al layer is etched, indicating that HF has an effect on Ti3 AlC 2 Effective etching of. Subsequently, multi-layered Ti was intercalated by the organic solvent dimethyl sulfoxide (DMSO). 3 C 2 was exfoliated to obtain monolayer Ti 3 C 2 nanosheets ( Figure 2 c).

[0072] Figure 3 Figure a shows the TEM image of the obtained monolayer MXene material Ti 3 C 2 . It clearly shows that the monolayer Ti 3 C 2 is in an isolated flake structure with clear edges. Figure 3 The TEM image of b shows the characteristic wrinkled morphology of GO. Figure 3 Figure c shows the morphological image after the combination of the two materials. It can be clearly seen that there is a wrinkled structure similar to GO at the bottom layer and a semi-transparent material morphology similar to Ti 3 C 2 at the upper layer. Further, the morphologies of GO, MXene, and GO / MXene were characterized by HR-TEM, HAADF-TEM, and elemental analysis. Figure 3 Figures d and Figure 3 e clearly show the lattice fringes of the monolayer Ti 3 C 2 nanosheets and the disordered amorphous structure of monolayer GO. The lattice fringe gap of Ti 3 C 2 is about 0.4 nm. Figure 3 Figure f shows the heterostructure morphology of GO / MXene under HR-TEM imaging. It can be seen from the figure that in this morphology, the Ti 3 C 2 nanosheets with clear lattice fringes overlap with GO with amorphous structure characteristics, forming a well-connected two-dimensional layered heterostructure. Figure 3 Figures g and 3h are the TEM morphology and the corresponding HAADF-TEM image of GO / MXene, respectively, clearly showing the heterostructure morphology formed by the stacking of two materials with different morphologies. Figure 3 The elemental analysis results shown in Figure i indicate that the Ti element is only uniformly distributed in the upper layer of the shown heterostructure, indicating that the upper layer is the monolayer MXene material Ti 3 C 2 , and the lower layer is monolayer GO, further demonstrating the successful construction of the GO / MXene two-dimensional heterostructure morphology.

[0073] Example 2: Preparation of disposable electrochemical sensors

[0074] Based on the GO / MXene composite material prepared in Example 1, this embodiment further prepares a disposable electrochemical sensor, the structure of which is as Figure 4 shown, including a PET substrate and a plurality of detection units arranged 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 coated with a GO / MXene composite material on the surface; the CE is a carbon electrode arranged on the other side of the WE; the RE is an Ag / AgCl electrode arranged on one side of the WE; the wires are respectively connected to the bottoms of the RE, WE, and CE. The preparation method of this electrochemical sensor in this embodiment is as follows:

[0075] S21. Weigh 5 g of Ag / AgCl ink and use a screen printing machine to evenly print the diluted Ag / AgCl ink on PET, and dry it at room temperature to obtain the printed RE;

[0076] S22. Weigh 5 g of conductive carbon paste and use a screen printing machine to evenly print the diluted conductive carbon paste on the PET printed with RE above, and dry it at room temperature to obtain the printed WE and CE;

[0077] S23. Weigh 5 g of conductive silver paste and use a screen printing machine to evenly print the diluted conductive silver paste on the PET printed with RE, WE, and CE above, and dry it at room temperature to obtain the printed wire;

[0078] S24. Take 5 mg of GO / MXene composite material and configure it into a uniform ink with 100 μL of absolute ethanol and 20 μL of Nafion solution; take 2 μL of the above ink and evenly drop it on the surface of the WE of the electrochemical sensor to complete the preparation of the disposable electrochemical sensor.

[0079] When the disposable electrochemical sensor prepared in this embodiment is used, it is inserted into a handheld electrochemical sensor device for detection, as Figure 5 shown. The handheld electrochemical sensor device includes a disposable electrochemical sensor, a control circuit board, and a display. The wire end of the disposable electrochemical sensor is connected to the control circuit board, and the display is electrically connected to the control circuit board and is used to display the detection result of the TRACP-5b content, which can be a mobile phone. The method for detecting an actual sample with this sensor device is as follows:

[0080] First, prepare a TRACP-5b gradient solution, react it with sodium 1-naphthyl phosphate monosodium salt, and then drop-coat it onto the electrode surface. A series of current values are measured through the control circuit board, and a standard curve is established between the TRACP-5b concentration and the current value. Then, add the substrate sodium 1-naphthyl phosphate monosodium salt to the gingival crevicular fluid sample to be tested for reaction. After the reaction is completed, drop the reaction solution onto the electrode surface, measure the current value through the control circuit board, and substitute this current value into the standard curve to obtain the content of TRACP-5b in the sample to be tested.

[0081] Example 3: Electrochemical Performance Test of Disposable Electrochemical Sensor

[0082] In this example, the disposable electrochemical sensor prepared in Example 2 was used as the test object to verify the electron transfer ability of the GO / MXene composite material and its electrochemical response ability to 1-naphthol. The specific electrochemical performance test methods and results are as follows:

[0083] (1) Take 50 μL of 0.1 M [Fe(CN) 6 3- / 4- solution and uniformly drop-coat it on the disposable electrochemical sensor. Ensure that the solution covers the WE, RE, and CE during drop-coating, and then perform EIS testing. The EIS testing conditions are a frequency of 10 5 to 0.1 Hz and an amplitude of 5 mV.

[0084] The EIS test results are as Figure 6 shown, where SPE is a disposable electrochemical sensor without any modified material. The inset shows the equivalent circuit model fitted according to the EIS results, which consists of a double-layer capacitor (C dl ), a solution resistor (R S ), a charge transfer resistor (R ct ), and a Warburg element (Z W ). The semicircle in the high-frequency region and the inclined straight line feature in the low-frequency region of the two electrochemical sensors are clearly shown in the EIS results, indicating that the above electrode reactions include both charge transfer and diffusion mechanisms. Compared with the blank SPE, the SPE modified with the GO / MXene composite material has a smaller semicircle region radius in the high-frequency region, and at the same time, 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.

[0085] ​(2) 50 μL of a KOH blank solution with a pH of 10 and the same KOH solution containing 1-naphthol sample were evenly drop-coated on a disposable electrochemical sensor. Ensure that the solution covers the WE, RE, and CE during drop-coating, and then perform CV testing. The CV testing conditions were carried out at a scanning rate of 60 mV / s within a potential range of -0.6 to 0.6 V vs. Ag / AgCl.

[0086] The CV test results are as Figure 7 shown, which clearly indicate that GO / MXene has an obvious response to 1-naphthol, and its electrochemical oxidation potential is approximately around 0.26 V vs. Ag / AgCl.

[0087] (3) 50 μL of a KOH blank solution with a pH of 10 and the same KOH solution containing 1-naphthol sample were evenly drop-coated on a disposable electrochemical sensor. Ensure that the solution covers the WE, RE, and CE during drop-coating, and then perform DPV testing. The DPV testing conditions were carried out with an amplitude of 50 mV, a pulse width of 200 ms, a step height of 5 mV, a step time of 500 ms, and a scanning rate of 10 mV / s.

[0088] The DPV test results are as Figure 8 shown, and the results verify the response ability of GO / MXene to 1 μM 1-naphthol at 0.25 V vs. Ag / AgCl. Compared with the blank solution (KOH solution with pH = 10 without 1-naphthol), the electrochemical response signal of the GO / MXene material to 1-naphthol is 6.59 μA.

[0089] (4) The sensitivity test results of the electrochemical sensor based on the GO / MXene composite material to 1-naphthol are as Figure 9 shown. The electrochemical test method is chronoamperometry, which demonstrates the response ability of the GO / MXene electrochemical sensor to 1-naphthol at concentrations from 10 to 100 nM. The results show that at a potential of 0.25 V vs. Ag / AgCl, the current value increases with the increase in the concentration of 1-naphthol ( Figure 9 a). Figure 9 b shows the linear relationship between the 1-naphthol concentration and the current value. This correlation can be expressed by the linear equation I = 0.019x + 1.249, where the determination coefficient is R 2 = 0.950, indicating a good linear fitting degree. At the same time, the LOD was calculated based on three times the signal-to-noise ratio and is 9.162 nM, indicating that the constructed GO / MXene electrochemical sensing system has an excellent response ability to 1-naphthol.

[0090] Example 4: Effect test of disposable electrochemical sensor for detecting TRACP-5b

[0091] In this embodiment, the method for the sensor to perform TRACP-5b determination is as follows:

[0092] (1) Take Tris-HCl buffer with pH = 5 and a concentration of 0.1 M as the reaction background, dilute TRACP-5b to the required concentration, and prepare the specific enzyme reaction substrate of TRACP-5b, sodium 1-naphthyl phosphate, under the same conditions.

[0093] (2) Take 5 μL of the TRACP-5b solution and 45 μL of the substrate into a centrifuge tube. After thoroughly pipetting and mixing in an ice-water bath, immediately place it in a 37 °C water bath and incubate for 30 min.

[0094] (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 the substrate solution to obtain the test blank solution.

[0095] (4) Take 50 μL of the above test solution and the blank solution and evenly drop-coat them on the disposable electrochemical sensor. Ensure that the test solution covers the WE, RE, and CE during drop-coating. Then use Figure 5 a circuit for testing.

[0096] Based on the above method, sensitivity, reproducibility, and stability tests were conducted on the sensor detection. The test results are as follows:

[0097] (1) Sensitivity test of the electrochemical sensor based on the GO / MXene material for TRACP-5b:

[0098] In this embodiment, the electrochemical sensor detection system prepared based on the GO / MXene composite material and the disposable electrochemical sensor can be used to sensitively detect the concentration of TRACP-5b in human GCF. Figure 10 a shows the current response of the electrochemical sensor based on GO / MXene to TRACP-5b in the concentration range of 500 ng / mL to 1000 ng / mL. The electrochemical test current value increases with the increase in the concentration of TRACP-5b, indicating that within this concentration range, the electrochemical response signal is positively correlated with the concentration of TRACP-5b. At the same time, the current value has a good linear relationship with the concentration of TRACP-5b ( Figure 10 b), and its linear standard curve is represented by the equation I = 0.007x - 1.685, where the determination coefficient R 2 = 0.916. Figure 11 a shows the electrochemical test of the electrochemical sensor based on GO / MXene to TRACP-5b in the lower concentration range of 40 ng / mL to 400 ng / mL. Its good sensing linear relationship is as Figure 11As shown in b, the linear equation is expressed as I = 0.005x + 0.119, and the determination coefficient R 2 = 0.994, and its LOD is 18.6 ng / mL, indicating that the constructed GO / MXene electrochemical detection system has the ability to highly sensitively detect TRACP-5b.

[0099] (2) Reproducibility test of the electrochemical sensor based on GO / MXene material:

[0100] In this embodiment, for the electrochemical sensing and detection system prepared based on GO / MXene composite material and disposable electrochemical sensor, to evaluate the reproducibility of this sensing system, 10 prepared disposable GO / MXene electrodes were randomly selected, and TRACP-5b with a concentration of 500 ng / mL was used as the detection object for electrochemical testing. Among Figure 12 it was observed that all the selected electrodes showed stable current signals, indicating that the constructed electrochemical sensing system has good reproducibility.

[0101] (3) Stability test of the electrochemical sensor based on GO / MXene material:

[0102] In this embodiment, for the electrochemical sensor detection system prepared based on GO / MXene composite material and disposable electrochemical sensor, to evaluate the stability of this sensing system, the constructed GO / MXene electrode was directly exposed to the daily environment for a one-month monitoring to observe the current signal fluctuation of the electrochemical detection system for TRACP-5b with a concentration of 500 ng / mL. Figure 13 It shows that within 10 days, the current response of the sensing electrode is almost the same as that of the original electrode; after 10 days, the current value gradually decreases, which may be due to the partial oxidation of MXene in GO / MXene, thus destroying the heterostructure and resulting in the reduction of the electrode performance. Generally speaking, the constructed GO / MXene sensor has good reproducibility and stability.

[0103] Comparative Example 1

[0104] It is basically the same as Example 1, except that the sensitive material prepared for the disposable electrochemical sensor is different. Specifically, only MXene is used as the sensitive material for electrochemical detection. The morphological characteristics of single-layer MXene are basically the same as Figure 3 a. In addition, the response of MXene to 1-naphthol was tested by EIS and CV techniques, and the results are respectively as Figure 14 and Figure 15As shown. Specifically, the EIS results analysis is basically the same as that of Example 3, but the radius of the semicircle 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 the conductivity and electron transfer efficiency of MXene are poorer than those of GO / MXene. The CV results show that compared with the ultrathin two-dimensional heterostructure GO / MXene ( Figure 7 ), the electrochemical response of MXene to 1-naphthol at the same concentration is weaker, indicating that pure MXene nanosheets are not suitable for this application.

[0105] Comparative Example 2

[0106] Basically the same as Example 1, the only difference is that the sensitive material prepared for the disposable electrochemical sensor is different. Specifically, only GO is used as the sensitive material for electrochemical detection. The morphological characteristics of monolayer GO are basically the same as Figure 3 b. The response of GO to 1-naphthol was also tested by EIS and CV techniques, and the results are shown in Figure 16 and Figure 17 respectively. Specifically, the EIS results are similar to those of Comparative Example 1. Compared with the GO / MXene composite material, the conductivity and electron transfer efficiency of GO are poorer. The CV results show that compared with the ultrathin two-dimensional heterostructure GO / MXene, the electrochemical response of GO to 1-naphthol at the same concentration is weaker, and at the same time, the oxidation potential increases to 0.29 V vs. AgCl, reflecting the disadvantage of low electron transfer rate of GO, indicating that pure GO nanosheets are not suitable for this application.

[0107] This specific implementation manner is only an interpretation of the present invention and not a limitation thereof. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A GO / MXene composite material based on an ultra-thin two-dimensional heterogeneous structure, characterized in that: The GO / MXene composite material is a composite material with ultra-thin two-dimensional heterogeneous structure characteristics formed by vertical stacking or in-plane growth of an ultra-thin single-layer graphene material and an ultra-thin single-layer MXene material; the graphene material is graphene oxide or reduced graphene oxide; the MXene material is Ti3C2, Nb2C, Ti2C or V2C.

2. The ultra-thin two-dimensional heterostructured GO / MXene composite material according to claim 1, characterized in that: The mass ratio of the graphene material to MXene is in the range of 1:(1-5); the thickness of the graphene material to MXene is in the range of 2nm-6nm.

3. The ultra-thin two-dimensional heterostructured GO / MXene composite material according to claim 2, characterized in that: The preparation method comprises the following steps: S11. reacting the MAX phase material with HF to generate a multilayer MXene material; S12. reacting the multilayer MXene material with DMSO to generate a two-dimensional ultra-thin single-layer MXene material; S13. Charge modification of two-dimensional ultra-thin monolayer MXene materials using PEI; S14. Add the two-dimensional ultra-thin single-layer graphene material dispersion into the charge-modified MXene dispersion to react and obtain a GO / MXene composite material having ultra-thin two-dimensional heterogeneous structure characteristics.

4. The GO / MXene composite material having ultra-thin two-dimensional heterogeneous structure according to claim 3, characterized in that: The preparation method comprises the following steps: S11. In an ice-water bath, slowly immerse 1 g of MAX phase material powder in 10-20 mL of HF solution and stir for 5-10 min; then heat to 40-60 ° C and continue stirring for 12-24 h; after the reaction is completed, centrifuge at 3000-5500 rpm for 5-10 min, wash the resulting precipitate with deionized water until neutral, and then vacuum dry the precipitate at 40-60 ° C for 12-24 h to obtain a multilayer MXene material; S12. 5-10 mg of the above MXene material powder is immersed in 25-50 mL of DMSO solution and stirred continuously at room temperature for 18-24 h; after the reaction is completed, the excess DMSO is removed by washing with deionized water; the obtained dispersion is placed in an ice water bath and ultrasonicated for 1-2 h, and then centrifuged at a speed of 3000-5500 rpm for 1-2 h; after the centrifugation is completed, the upper layer of the dispersion is poured out, and the obtained dispersion is a two-dimensional ultra-thin single-layer MXene material; S13. The two-dimensional ultra-thin single-layer MXene material powder is uniformly dispersed in an appropriate amount of deionized water, and 5 to 10 mL of PEI is added to charge-modify the MXene material; the dispersion is then ultrasonically treated for 5 to 10 minutes, excess PEI is washed with deionized water and redispersed in deionized water to form a stable hydrosol; S14. The two-dimensional ultra-thin single-layer graphene dispersion is added to the charge-modified MXene dispersion under vigorous stirring, and the stirring is continued at room temperature for 6 to 24 hours; the obtained material is vacuum dried at 40 to 60°C for 12 to 24 hours to obtain a composite material with ultra-thin two-dimensional heterogeneous structure characteristics.

5. A disposable electrochemical sensor, characterized in that: It includes a PET substrate and at least one detection unit arranged on the surface of the PET substrate, wherein the detection unit includes the following components: The working electrode WE is a carbon electrode coated with the GO / MXene composite material based on the ultra-thin two-dimensional heterostructure according to any one of claims 1 to 4. During electrochemical measurement, current will pass through the electrode and drive the specified chemical process, and 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 and is used to form a current loop with the WE in electrochemical measurement, thereby ensuring that the electrochemical reaction on the WE can continue; The reference electrode RE is an Ag / AgCl electrode disposed on one side of the WE and is used to provide a known and stable reference potential for electrochemical measurements; The wires are connected to the bottom ends of the RE, WE and CE respectively, and are used to enable the electrodes of the sensor to form a complete current loop in electrochemical measurement.

6. The disposable electrochemical sensor according to claim 5, characterized in that: The preparation method comprises the following steps: S21. The Ag / AgCl ink is evenly printed on the PET and dried to obtain a printed RE; S22. The conductive carbon paste is evenly printed on the PET printed with the reference electrode, and dried to obtain the printed WE and CE; S23. The conductive silver paste is evenly printed on the PET printed with RE, WE and CE, and the printed conductor is obtained by drying; S24. Prepare ink by mixing GO / MXene composite material with anhydrous ethanol and Nafion solution, and evenly drop the ink on the WE surface to complete the preparation of disposable electrochemical sensor.

7. The disposable electrochemical sensor according to claim 6, characterized in that: The preparation method comprises the following steps: S21. Weigh 5 to 10 g of Ag / AgCl ink and dilute it with 5 to 10 mL of diluent, then use a screen printer to evenly print the diluted Ag / AgCl ink on PET, and dry it at room temperature to obtain a 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 printer to evenly print the diluted conductive carbon paste on the PET printed with the reference electrode, and dry it at room temperature to obtain the printed WE and CE; S23. Weigh 5 to 15 g of conductive silver paste and dilute it with 5 to 20 mL of diluent, use a screen printer to evenly print the diluted conductive silver paste on the PET printed with RE, WE and CE, and dry it at room temperature to obtain the printed conductor; S24. Take 5-10 mg of GO / MXene composite material, mix it with 100-500 μL of anhydrous ethanol and 20-100 mL of Nafion solution to form a uniform ink; take 2-10 μL of the above ink and evenly drop it on the WE surface of the electrochemical sensor to complete the preparation of the disposable electrochemical sensor.

8. Use of the disposable electrochemical sensor according to any one of claims 5 to 7 in detecting the content of TRACP-5b in human gingival crevicular fluid.

9. The use according to claim 8, characterized in that: The application method is to insert the disposable electrochemical sensor into a handheld electrochemical sensor device for use, wherein the handheld electrochemical sensor device comprises a disposable electrochemical sensor, a control circuit board and a display, wherein the lead end 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 detection, a TRACP-5b gradient solution is first prepared, which is reacted with 1-naphthyl phosphate monosodium salt and then dripped onto the electrode surface. The corresponding current value is measured by a control circuit board, and a standard curve is established between the TRACP-5b concentration and the current value. Then, the sample to be tested is reacted with 1-naphthyl phosphate monosodium salt and then dripped onto the electrode surface. The measured current value is brought into the standard curve to obtain the TRACP-5b content in the sample to be tested.

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