Ethyl glucuronide electrochemical biosensor based on MXene nanomaterials and preparation method and detection method thereof

Abstract

The present invention discloses an ethyl glucuronide electrochemical biosensor based on MXene nanomaterials and a preparation method thereof, wherein the AuNPs-Nb4C3Tx complex is fixed on the electrode surface through a bridging agent chitosan, and then a histidine-tagged recombinant protein G is bound to the surface of gold nanoparticles, and an EtG antibody is fixed on the electrode surface through a histidine-tagged recombinant protein G. Also disclosed is a method for detecting the sensor based on the competition between EtG and EtG-BSA for binding to EtG antibodies. The immunoelectrochemical biosensor of the present invention can achieve rapid, sensitive and specific detection of samples, with a low detection limit (0.11 ng / mL) and a wide detection range (1 ng / mL-100 μg / mL). The simple, label-free quantitative detection method of ethyl glucuronide based on electrochemical impedance spectroscopy of the present invention is simple to operate and the results are accurate and reliable.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoelectrochemical biosensors, and in particular to an ethyl glucuronide electrochemical biosensor based on MXene nanomaterials, and a preparation method and a detection method thereof. Background Art

[0002] Non-oxidative metabolites of ethanol (NMEs) are metabolites formed by nonoxidative metabolic pathways after ethanol enters the human body. Because they have a longer half-life and greater stability than ethanol and its oxidative metabolites, they hold great promise for detecting alcohol consumption, estimating the time of drinking, estimating the time of death after drinking, and distinguishing antemortem alcohol consumption from ethanol production during decomposition. Current strategies for detecting ethyl glucuronides include solid-phase extraction (SPE)-gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), and immunoassay kits. However, some techniques require D5 labeling of ethyl glucuronide (EtG), which poses health risks to operators and the environment. Other techniques require expensive or bulky instrumentation, complex procedures, lengthy detection times, or the need for skilled personnel, significantly limiting their application in the rapid detection of ethyl glucuronides. Electrochemical biosensors have attracted widespread attention and seen rapid development due to their advantages, including ease of operation, time-saving, low cost, miniaturization, high sensitivity, and high selectivity. Electrochemical technology has been applied in many fields. Therefore, the development of simple, portable, low-cost methods for the quantitative detection of ethyl glucuronide is of great significance.

[0003] The advent of nanotechnology has opened up new avenues for detecting biomolecules through signal amplification strategies. In 2011, researchers at Drexel University in the United States first reported the discovery of MXenes—two-dimensional transition metal carbides, nitrides, or carbonitrides. These materials represent a new class of two-dimensional nanomaterials derived from the MAX phase of a layered ceramic material by etching away the A element. The name reflects both their MAX phase origin and their graphene-like two-dimensional sheet structure. The chemical formula of MXene is Mn+1XnTx, where M represents an early transition metal element (Sc, Ti, V, Cr, Zr, Nb, Mo, etc.), X represents carbon and / or nitrogen, and Tx represents a surface end group (—O, —OH, —F, etc.). Due to the diverse composition and structure of the MAX phase, MXene materials derived from it have become one of the largest families of two-dimensional materials, with over 100 theoretically predicted and over 40 synthesized to date. Due to their large specific surface area and excellent physical and chemical properties, MXene nanomaterials have been widely studied and applied in various fields such as electrocatalysis, optics, and electrochemistry. Studies have shown that MXene has strong reducing ability and can reduce metal nanoparticles in the absence of reducing agents and stabilizers.

[0004] Gold nanoparticles are stable nanomaterials with excellent biocompatibility. They can conjugate with many biomolecules without altering their properties or activity. AuNPs-MXene nanocomposites and MXene / Ag composites exhibit excellent conductivity, electrocatalytic activity, and biocompatibility. Electrochemical biosensors are one of the most commonly used detection methods in scientific research, offering advantages such as excellent selectivity, high sensitivity, and simple operation. Currently, electrochemical-based biosensors have achieved the detection of a variety of biomarkers, and are becoming an increasingly reliable platform for bioassays. Summary of the Invention

[0005] The present invention aims to address the above-mentioned technical problems in EtG detection in the prior art and to provide an electrochemical biosensor for ethyl glucuronide based on MXene nanomaterials. This sensor utilizes competitive binding between EtG and EtG-BSA to quantitatively detect ethyl glucuronide. Experimental verification demonstrates that the detection results are accurate and reliable. Based on this, the present invention protects the following technical solutions:

[0006] A method for preparing an ethyl glucuronide electrochemical biosensor based on MXene nanomaterials comprises immobilizing an AuNPs-Nb4C3Tx complex on an electrode surface via a bridging agent, chitosan, then binding a histidine-tagged recombinant protein G to the surface of the gold nanoparticles, and immobilizing an EtG antibody on the electrode surface via the histidine-tagged recombinant protein G. The method comprises the following steps:

[0007] S1. Preparation of AuNPs-Nb4C3Tx / chitosan solution: Disperse the AuNPs-Nb4C3Tx composite material in chitosan solution and store for later use;

[0008] S2. Fixation of AuNPs-Nb4C3Tx: coat the electrode with the AuNPs-Nb4C3Tx / chitosan solution prepared in step S1 and let it dry;

[0009] S3, Combination of his-PG and AuNPs-Nb4C3Tx: The his-PG solution was coated on the electrode modified with AuNPs-Nb4C3Tx;

[0010] S4. Continue coating the electrode with EtG antibody, and then use blocking solution to block the non-specific binding sites on the electrode surface.

[0011] The preparation method of the AuNPs-Nb4C3Tx composite material is as follows: taking MXene nanomaterial Nb4C3Tx, adding it to deionized water, dispersing it evenly, then adding HAuCl4 solution, after sufficient reaction, centrifuging and washing, separating the supernatant and the precipitate, and the precipitate is the AuNPs-Nb4C3Tx composite material.

[0012] In the preparation method of the AuNPs-Nb4C3Tx composite material, the concentration of Nb4C3Tx is 0.5-2 mg / mL, preferably 0.5-1.5 mg / mL or 0.8-1.5 mg / mL or 1 mg / mL.

[0013] In the above technical solution, the EtG antibody is a monoclonal antibody, the blocking solution is bovine serum albumin, and the electrode is a gold electrode or a glassy carbon electrode.

[0014] In the above-mentioned technical solution for preparing the sensor, the concentration of the his-PG solution in step S3 is 15-25 μg / mL, preferably 17-23 μg / mL or 19-21 μg / mL.

[0015] In the above-mentioned technical solution for the preparation method of the sensor,

[0016] The antibody incubation time when coating the EtG antibody in step S4 is 2-4 hours, preferably 2.5-3.5 hours or 3 hours;

[0017] The concentration of the EtG antibody is 15-25 μg / mL, preferably 17-23 μg / mL or 19-21 μg / mL.

[0018] The ethyl glucuronide electrochemical biosensor prepared by any of the preparation methods described above.

[0019] A method for detecting ethyl glucuronide is to use the aforementioned ethyl glucuronide electrochemical biosensor for detection.

[0020] The detection method is based on the competition between EtG and EtG-BSA for binding to EtG antibodies. Each sample containing EtG is mixed with an EtG-BSA solution of known concentration and then detected using the above-mentioned ethyl glucuronide electrochemical biosensor. The method comprises the following steps:

[0021] 1) Establishing a standard curve equation: Prepare EtG standard solutions with different concentration gradients, mix the EtG standard solutions with EtG-BSA solution, and then add them dropwise to the electrode surface. Allow to react fully at room temperature, and measure the corresponding impedance signal using electrochemical impedance spectroscopy. Establish a standard curve equation based on the relationship between EtG concentration and impedance signal.

[0022] 2) The sample solution to be tested was mixed with the EtG-BSA solution and then added dropwise to the electrode surface. The mixture was allowed to react fully at room temperature, and the impedance signal was detected by electrochemical impedance spectroscopy.

[0023] 3) Substituting the impedance signal obtained in step 2) into the standard curve equation in step 1) to calculate the concentration of EtG in the sample solution to be tested.

[0024] Preferably,

[0025] In the step 1), the concentration of the EtG standard solution is 0.005 ng / mL to 520 μg / mL, preferably 0.01 ng / mL to 500 μg / mL, and preferably the concentration gradient of the EtG standard solution is 0.01 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, and 500 μg / mL;

[0026] In steps 1) and 2), the concentration of EtG-BSA is 3-7 μg / mL, preferably 4-6 μg / mL; the EtG standard solution or the test sample solution is mixed with the EtG-BSA solution in equal volumes;

[0027] In steps 1) and 2), the incubation time of the EtG / EtG-BSA mixed solution and the antibody is 40 to 60 minutes, preferably 40 to 50 minutes or 45 minutes;

[0028] The regression equation of the standard curve was Y = 256.7152-47.20321logC (ng / mL), the correlation coefficient was 0.96234, the detection limit was 0.11 ng / mL, the detection range was 1 ng / mL-100 μg / mL, Y represented the impedance value, and logC represented the logarithm of the EtG concentration.

[0029] The detection principle of the sensor of the present invention is as follows:

[0030] The AuNPs-Nb4C3Tx complex was immobilized on the electrode surface via the bridging agent chitosan. Histidine-tagged recombinant protein G (his-PG) ​​was firmly bound to the gold nanoparticle surface, forming an oriented layer that prepared the way for the antibody to be fixed to the gold electrode surface. EtG antibody was immobilized on the electrode surface via histidine-tagged recombinant protein G. The EtG antibody specifically binds to his-PG via its fragment crystalline region (Fc), forcing the EtG antibody to expose its antigen-binding site to the environment, thereby enhancing the recognition ability between the antigen Ag and the antibody Ab.

[0031] EtG is a small molecule with only one antigenic epitope, making it difficult to electrochemically measure it using a double-antibody sandwich method loaded with a signal molecule. Furthermore, due to its small molecular weight, the specific binding of the antibody to the antigen has little effect on electron transport capacity, resulting in minimal changes in the current signal, making it impossible to construct a biosensor for electrochemical detection of EtG. EtG-BSA effectively compensates for this smaller molecular weight drawback. Antibodies against ethyl glucuronide can simultaneously recognize EtG and EtG-BSA. When EtG and EtG-BSA compete for binding on the electrode surface, the change in current intensity during detection of different EtG concentrations is significantly increased. This detection method helps expand the detection range. Finally, the electrochemical impedance spectroscopy (EIS) signal of the electrode was tested and the quantitative results analyzed, thus establishing a label-free electrochemical quantitative detection method for ethyl glucuronide.

[0032] The beneficial effects of the present invention are:

[0033] The immunoelectrochemical biosensor of the present invention can achieve rapid, sensitive and specific detection of samples, has a low detection limit (0.11 ng / mL) and a wide detection range (1 ng / mL-100 μg / mL), and has a simple preparation method, low preparation cost, and portability. It has excellent stability and repeatability and has good market application prospects.

[0034] The simple, label-free, electrochemical impedance spectroscopy-based quantitative detection method for ethyl glucuronide of the present invention has simple operation, accurate and reliable results, and improves the detection sensitivity by using a method in which EtG and EtG-BSA compete for binding to antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope image of the nanocomposite material AuNPs-Nb4C3Tx.

[0036] Figure 2 Electrochemical impedance spectroscopy of the layer-by-layer assembled EtG biosensor.

[0037] Figure 3 This is the cyclic voltammogram of the layer-by-layer assembled EtG biosensor.

[0038] Figure 4 This is the ESD diagram of MXENE-Nb4C3Tx composited with gold nanoparticles.

[0039] Figure 5 is the Nyquist plot of different EtG concentrations.

[0040] Figure 6 This is the effect of different concentrations of MXENE-Nb4C3Tx on the detection signal.

[0041] Figure 7 It is the effect of antibody binding time on the detection signal.

[0042] Figure 8 Figure 3 is the effect of different concentrations of EtG-BSA on the detection signal.

[0043] Figure 9 is the effect of EtG-BSA binding time on the detection signal.

[0044] Figure 10 This is a specificity test of the sensor of the present invention.

[0045] Figure 11 This is a reproducible test of the sensing strategy at different concentrations of the EtG level to be tested.

[0046] Figure 12 It is a stability test of the sensing strategy.

[0047] Figure 13 is the response signal of different concentrations of EtG. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0049] The experimental methods in the following examples are conventional methods unless otherwise specified; the biological and chemical reagents used are conventional reagents in the art and are commercially available unless otherwise specified.

[0050] Main reagent sources:

[0051] Niobium carbide (Nb4C3Tx) MXene multilayer nanosheets (CAS No. 12069-94-2): brand XFNANO;

[0052] EtG-BSA (BSA-modified EtG antigen): Etbylgucuronide (EtG)-BSA Antigen, East Coast Bio (USA), specification - 1 mg, product number LAOO7;

[0053] EtG antibody: EthyLglucuronide (EtG) Antibod, EastCoast Bio (USA), specification 1 mg, product number HM128;

[0054] Histidine-tagged recombinant protein G (his-PG): Recombinant Protein A / GHis, Brand: Sangon (China), Product No. C610042-0001.

[0055] Example 1 Preparation of the EtG Immunoelectrochemical Biosensor of the Present Invention

[0056] Follow these steps:

[0057] (1) Preparation of AuNPs-Nb4C3Tx

[0058] First, 1 mg of MXene-Nb4C3Tx was added to 5 mL of deionized water and sonicated for 20 minutes to uniformly disperse it in the water. Then, 1 mL of freshly prepared 20 mmol / L aqueous HAuCl4 solution was added dropwise under stirring. After a full reaction of 5 minutes, the mixture was centrifuged and washed three times to separate the supernatant and precipitate. Finally, the precipitate was dispersed in 1 mL of 0.5% chitosan solution (0.05 g chitosan dissolved in 10 mL of 1% glacial acetic acid) and stored in a brown bottle at 4°C.

[0059] The successful synthesis of the nanocomposite AuNPs-Nb4C3Tx plays a vital role in the preparation of the sensor of the present invention. The morphology of the prepared AuNPs-Nb4C3Tx was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and EDS spectroscopy. Figure 1 、 Figure 4 As shown, the scanning electron microscopy results of the nanocomposite AuNPs-Nb4C3Tx showed that gold nanoparticles have been successfully composited onto the MXENE-Nb4C3Tx nanomaterial.

[0060] (2) Preparation of immunoelectrochemical biosensors

[0061] ① Fixation of AuNPs-Nb4C3Tx: 10 μL of the solution of AuNPs-Nb4C3Tx (1 ​​mg / mL) containing chitosan prepared in step (1) was dropped onto the surface of the gold disk electrode and allowed to dry naturally at room temperature.

[0062] The synthesized nanocomposite AuNPs-Nb4C3Tx was characterized by electrochemical impedance spectroscopy and electrochemical cyclic voltammetry data: electrochemical experiments were carried out in a standard three-electrode system. 3- / 4- Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed in a working solution of 10 mM PBS, pH 7.4 and 0.1 M potassium chloride. CV scans were performed in the potential range of -0.2 V to 0.6 V at a scan rate of 0.1 V / s. EIS was performed at 1 × 10 -1 to 1×10 5 Hz frequency range and 50mV amplitude.

[0063] The EIS graph reflects the resistance of the material, and the CV graph reflects the conductivity of the reaction. By comparing and analyzing the CV and EIS graphs of different materials, we can understand the conductivity of the material. The better the conductivity, the higher the sensitivity of the electrochemical sensor.

[0064] During the research, the conductivity of the electrode modified with nanomaterial MXENE-Nb4C3Tx alone and the electrode modified with gold nanocomposite MXENE-Nb4C3Tx were compared: the results showed that the charge transfer value of the bare gold electrode was relatively large ( Figure 2 and Figure 3 Curve a); When the bare gold electrode is modified with the nanomaterial MXENE-Nb4C3Tx, the semicircle diameter of the impedance curve decreases and the impedance decreases, and the redox peak of the CV curve increases significantly, indicating that the charge transfer ability increases ( Figure 2 and Figure 3 When the nano-gold is composited with MXENE-Nb4C3Tx and fixed on the bare gold electrode, the semicircle diameter of the curve is further reduced, the redox peak of the CV curve is further increased, and the charge transfer value is further increased ( Figure 2 and Figure 3 The results show that the conductivity of the electrode modified with gold nanocomposite MXENE-Nb4C3Tx is further increased compared with that of the electrode modified with MXENE-Nb4C3Tx alone. Figure 4 The green fluorescent spots represent the distribution of gold nanoparticles. These results indicate that the MXENE-Au nanocomposite has been successfully synthesized.

[0065] ② Binding of his-PG to AuNPs-Nb4C3Tx: 10 μL of a 20 μg / mL his-PG (histidine-tagged recombinant protein G) solution was added to the Au-Nb4C3T-modified gold electrode surface. The mixture was incubated overnight at 4°C and then rinsed with 10 mM PBS buffer. This step allows his-PG to firmly bind to the gold nanoparticle surface, forming an oriented layer that prepares the surface for the antibody to be fixed to the gold electrode.

[0066] ③ Binding of antibody to his-PG / AuNPs-Nb4C3Tx: The electrode obtained in step ② was incubated with 10 μL of 20 μg / mL ethyl glucuronide antibody (EtG antibody) at 4°C for 3 hours. After rinsing with 10 mM PBS buffer, the electrode was incubated with 10 μL of 0.25 wt% bovine serum albumin (BSA) at 4°C for 1 hour. Store at 4°C for later use. The EtG antibody specifically binds to his-PG through the fragment crystallization region (Fc), forcing the antibody (Ab) to expose its binding site to the environment, thereby improving the recognition and identification ability between the antigen (Ag) and Ab. That is, the immunoelectrochemical biosensor of the present invention is prepared.

[0067] To demonstrate the successful attachment of his-PG and the antibody to the gold electrode surface, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) data were used to characterize the AuNPs-Nb4C3Tx-modified gold electrode. First, 10 μL of 20 μg / mL his-PG was added to the AuNPs-Nb4C3Tx-modified gold electrode, incubated overnight at 4°C, and then rinsed with 10 mM PBS. EIS (electrochemical impedance spectroscopy) and CV (cyclic voltammetry, commonly used to calculate diffusion coefficients) data were measured. A new AuNPs-Nb4C3Tx-modified gold electrode was then added with 10 μL of 20 μg / mL his-PG and incubated overnight at 4°C. The resulting electrode was then incubated with 10 μL of 20 μg / mL ethyl glucuronide antibody at 4°C for 3 h, and EIS and CV data were measured. When his-PG is bound to the gold electrode, the semicircle diameter of the impedance curve increases, the impedance increases, and the redox peak of the CV curve decreases significantly, indicating that the charge transfer ability is reduced ( Figure 2 and Figure 3 When the antibody is connected to the electrode, the semicircle diameter of the curve increases further, the redox peak of the CV curve decreases further, and the charge transfer value decreases further ( Figure 2 and Figure 3 These results indicate that his-PG and antibodies were successfully attached to the gold electrode surface.

[0068] Example 2 Detection of ethyl glucuronide (EtG) using the sensor of the present invention

[0069] 1. Detection method

[0070] The sensor prepared in Example 1 was used for detection. The detection method was as follows: the gold disk electrode was rinsed with 10 mM PBS buffer, 5 μg / mL EtG-BSA was mixed with equal volumes of the sample to be tested, and 10 μL of the mixture was dropwise added to the electrode surface. The mixture was reacted at room temperature (RT, 25±2°C) for 45 minutes, and the corresponding electrochemical signal was measured. In this way, a portable ethyl glucuronide immunoelectrochemical biosensor system based on the competitive binding of EtG and EtG-BSA of the nanolayered material Nb4C3Tx was established.

[0071] The purchased ethyl glucuronide was diluted into different concentration gradients (0.01ng / mL, 1ng / mL, 10ng / mL, 100ng / mL, 1μg / mL, 10μg / mL, 100μg / mL, 500μg / mL) to simulate the test solution, and the sensor prepared in Example 1 was used for detection: EtG solution and 5μg / mL EtG-BSA were mixed at a volume ratio of 1:1, and 10μL of the mixture was added dropwise to the electrode surface. The mixture was reacted at room temperature (RT, 25±2℃) for 45 minutes, and the corresponding impedance signal was measured. Detection results ( Figure 5 ) from a → h: 0.01 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, and 500 μg / mL. Through layer-by-layer assembly, the electrochemical impedance values ​​gradually changed, consistent with those observed by the CV method, further demonstrating the accuracy and reliability of the immunosensor for detecting ethyl glucuronide.

[0072] 2. Optimization experiments of different experimental conditions and experimental parameters

[0073] According to the preparation method of the EtG immunoelectrochemical biosensor in Example 1, a comparative experiment with different experimental conditions and experimental parameters was performed.

[0074] 1. Concentration of nanomaterials

[0075] The concentration of nanomaterials plays an important role in the detection performance of sensors. If the concentration of nanomaterials is too high, the conductivity will be lower and the corresponding signal will be higher. If the concentration of nanomaterials is too low, there will be less nanomaterials bound to the gold electrode and the conductivity will also be lower. Therefore, it is particularly necessary to study the concentration of nanomaterials.

[0076] This example compares different concentrations of nanomaterial Nb4C3Tx, and uses Nb4C3Tx with concentrations of 0.1, 0.5, 1, 5, and 10 mg / mL to prepare sensors. Figure 6 As shown in the figure, as the concentration of nanomaterials increases, the impedance signal decreases sharply. When the concentration is 1 mg / mL, the impedance is the lowest and the conductivity is the best. The conductivity decreases when the concentration exceeds or is lower than this concentration. Therefore, 1 mg / mL is the optimal concentration of nanomaterials.

[0077] 2. Antibody incubation time

[0078] The antibody incubation time in the step of "binding of antibody to his-PG / AuNPs-Nb4C3Tx" in Example 1 was compared, and the experiments were performed with incubation times of 1, 2, 3, and 4 hours, respectively.

[0079] The effect of antibody incubation time on signal generation is as follows Figure 7 As shown, from Figure 7 As can be seen from the figure, the incubation time shows an upward trend in the first 3 hours. When the incubation time reaches 3 hours, the signal reaches the maximum and is in a stable state. Therefore, we choose 3 hours as the optimal incubation time for the antibody.

[0080] 3. Concentration of EtG-BSA binding to EtG antibody

[0081] Different concentrations of EtG-BSA were incubated with the electrode coated with EtG antibody. The concentrations of EtG-BSA were: 5 ng / mL, 50 ng / mL, 500 ng / mL, 5 μg / mL, and 50 μg / mL.

[0082] The effect of the concentration of EtG-BSA combined with the antibody on the signal generation is as follows: Figure 8 As shown in the figure, the binding concentration is 5 ng / mL-50 μg / mL. As can be seen from the figure, the signal shows an upward trend in the binding concentration range of 5 ng / mL-5 μg / mL. When the binding concentration reaches 5 μg / mL, the signal reaches a maximum and is in a stable state. Therefore, we choose 5 μg / mL as the optimal binding concentration for EtG-BSA to bind to the antibody.

[0083] 4. Binding time of EtG-BSA and EtG antibody

[0084] 10 μL of 5 μg / mL EtG-BSA was added dropwise to the electrode surface coated with EtG antibody and reacted at room temperature (RT, 25±2°C). Different reaction times were set: 30 min, 45 min, 60 min, and 90 min.

[0085] The effect of the binding time of EtG-BSA and EtG antibody on the signal generation is shown in the attached figure. Figure 9 As shown. Figure 9 It can be seen that the incubation time shows an upward trend in the first 45 minutes. When the incubation time reaches 45 minutes, the signal reaches the maximum and is in a stable state. Therefore, we choose the optimal binding time of EtG-BSA and antibody as 45 minutes.

[0086] 5. Sensor specificity experiments, repeatability and stability

[0087] This example tests the specificity of the sensing strategy of the present invention, and the results are as follows Figure 10 As shown, from AE, they are: A (EtG+5μgEtG-BSA), B (pbs+5μgEtG-BSA), C (methylβ-D-glucuronide+5μgEtG-BSA), D (1-propylβ-Dglucuronide+5μgEtG-BSA), E (1-butylβ-D-glucuronide+5μgEtG-BSA), F (1-butylβ-D-glucuronide+5μgEtG-BSA), and G (tert-butylβ-D-glucuronide+5μgEtG-BSA). Compared with A (EtG+5μgEtG-BSA), the other groups showed higher impedance signals, and the impedance signals were relatively close, which indicates the excellent specificity of this sensing strategy.

[0088] The repeatability of the sensing strategy was tested as follows: three concentration levels of EtG samples (1 ng / L, 1 μg / L, 100 μg / L) were taken, each concentration was measured three times, and the relative standard deviation was calculated. Figure 11 As shown in the figure, the relative standard deviations of the EtG samples at the three concentration levels were 1.8%, 1.6%, and 1%, respectively. The results show that the sensing strategy has good repeatability.

[0089] The stability of the sensor was tested: the sensor prepared in Example 1 was placed at 4°C for different periods of time and then used for EtG detection. The assembled sensor was then incubated with the test solution-EtG-BSA mixture at room temperature for 45 minutes, and its electrochemical parameters were measured. Figure 12 As shown, the test results show that when stored at 4°C, the detection performance of the immunoelectrochemical biosensor of the present invention is still good after storage for 2 weeks.

[0090] 6. Sensor sensitivity

[0091] In order to evaluate the analytical performance of the sensing strategy of the present invention for the target, we studied the strategy using a series of different concentrations of EtG. The results of the detection of target EtG at different concentrations from 0.01 ng / mL to 100 μg / mL are as follows: Figure 13 As shown, from Figure 13 It can be seen that as the EtG concentration increases, the signal also increases. When the EtG concentration is in the range of 1 ng / mL to 100 μg / mL, the increase in the signal has a good linear relationship with the logarithm of the EtG concentration. The regression equation is Y=256.7152-47.20321logC(ng / mL), the correlation coefficient is 0.96234, the detection limit is 0.11 ng / mL, and the detection range is 1 ng / mL-100 μg / mL. Y represents the impedance value and logC represents the EtG concentration.

[0092] Example 3 Detection method

[0093] The sensor constructed in Example 1 was used to detect the sample to be tested, and the following steps were followed:

[0094] 1) Establishing a standard curve equation: EtG standard solutions with varying concentrations were prepared. Equal volumes of the EtG standard solutions and a 5 μg / mL EtG-BSA solution were mixed and then added dropwise to the electrode surface. The mixture was incubated at room temperature for 45 minutes to allow for full reaction. The corresponding impedance signal was measured using electrochemical impedance spectroscopy (EIS). A standard curve equation was established based on the relationship between EtG concentration and the impedance signal. The concentrations of the EtG standard solutions varied from 0.01 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, and 500 μg / mL. The regression equation for the standard curve was Y = 256.7152 - 47.20321 log C (ng / mL), with a correlation coefficient of 0.96234. The detection limit was 0.11 ng / mL, and the detection range was 1 ng / mL to 100 μg / mL. Y represents the impedance value, and log C represents the logarithm of the EtG concentration.

[0095] 2) Mix equal volumes of the sample solution and 5 μg / mL EtG-BSA solution, then add dropwise to the electrode surface. Incubate at room temperature for 45 minutes to allow for full reaction, and measure the impedance signal using electrochemical impedance spectroscopy.

[0096] 3) Substituting the impedance signal obtained in step 2) into the standard curve equation in step 1) to calculate the concentration of EtG in the sample solution to be tested.

Claims

1. A method for preparing an ethyl glucuronide electrochemical biosensor based on MXene nanomaterials, characterized in that: The AuNPs-Nb4C3Tx complex is fixed on the electrode surface via a bridging agent chitosan, and then histidine-tagged recombinant protein G is bound to the surface of the gold nanoparticles, and the EtG antibody is fixed on the electrode surface via the histidine-tagged recombinant protein G; the steps include: S1. Preparation of AuNPs-Nb4C3Tx / chitosan solution: Disperse the AuNPs-Nb4C3Tx composite material in chitosan solution and store for later use; S2. Fixation of AuNPs-Nb4C3Tx: coat the electrode with the AuNPs-Nb4C3Tx / chitosan solution prepared in step S1 and air dry; S3, Combination of his-PG and AuNPs-Nb4C3Tx: The his-PG solution was coated on the electrode modified with AuNPs-Nb4C3Tx; S4. Continue coating the electrode with EtG antibody, and then use blocking solution to block the non-specific binding sites on the electrode surface.

2. The preparation method according to claim 1, wherein: The preparation method of the AuNPs-Nb4C3Tx composite material is as follows: taking MXene nanomaterial Nb4C3Tx, adding it to deionized water, dispersing it evenly, then adding HAuCl4 solution, after sufficient reaction, centrifuging and washing, separating the supernatant and the precipitate, and the precipitate is the AuNPs-Nb4C3Tx composite material.

3. The preparation method according to claim 2, wherein: The concentration of Nb4C3Tx is 0.5-2 mg / mL.

4. The preparation method according to claim 1, wherein: The EtG antibody is a monoclonal antibody, the blocking solution is bovine serum albumin, and the electrode is a gold electrode or a glassy carbon electrode.

5. The preparation method according to claim 1, wherein: The concentration of the his-PG solution in step S3 is 15-25 μg / mL.

6. The preparation method according to claim 1, wherein: The antibody incubation time when coating with EtG antibody in step S4 is 2-4 hours; The concentration of EtG antibody is 15-25 μg / mL.

7. The ethyl glucuronide electrochemical biosensor prepared by the preparation method according to any one of claims 1 to 6.

8. A method for detecting ethyl glucuronide, characterized in that: The detection is performed using the ethyl glucuronide electrochemical biosensor according to claim 7.

9. The detection method according to claim 8, wherein: The detection is based on the competition between EtG and EtG-BSA for binding to EtG antibodies. Each sample containing EtG is mixed with an EtG-BSA solution of known concentration and then detected using the ethyl glucuronide electrochemical biosensor according to claim 7, comprising the following steps: 1) Establishing a standard curve equation: Prepare EtG standard solutions with different concentration gradients, mix the EtG standard solutions with EtG-BSA solution, and then add them dropwise to the electrode surface. Allow to react fully at room temperature, and measure the corresponding impedance signal using electrochemical impedance spectroscopy. Establish a standard curve equation based on the relationship between EtG concentration and impedance signal. 2) The sample solution to be tested was mixed with the EtG-BSA solution and then added dropwise to the electrode surface. The mixture was allowed to react fully at room temperature, and the impedance signal was detected by electrochemical impedance spectroscopy. 3) Substituting the impedance signal obtained in step 2) into the standard curve equation in step 1) to calculate the concentration of EtG in the sample solution to be tested.

10. The detection method according to claim 9, characterized in that: The concentration of the EtG standard solution in step 1) is 0.005 ng / mL to 520 μg / mL; In steps 1) and 2), the concentration of EtG-BSA is 3-7 μg / mL; the EtG standard solution or the test sample solution is mixed with the EtG-BSA solution in equal volumes; In steps 1) and 2), the incubation time of the EtG / EtG-BSA mixed solution and the antibody is 40 to 60 minutes; The regression equation of the standard curve is Y=256.7152-47.20321logC(ng / mL), the correlation coefficient is 0.96234, the detection limit is 0.11 ng / mL, the detection range is 1 ng / mL-100 μg / mL, Y represents the impedance value, and logC represents the logarithm of the EtG concentration.

11. The detection method according to claim 10, characterized in that: The different concentration gradients of the EtG standard solution in step 1) are 0.01 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, and 500 μg / mL.

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