Sensing electrode for diagnosing oral diseases as well as preparation method and application of sensing electrode

Abstract

The invention belongs to the field of detection and analysis, and particularly relates to a sensing electrode for diagnosing oral diseases, which comprises an electrode layer and a signal conversion layer positioned on one side of the electrode layer, a biological recognition probe is fixed on the surface and / or inside the side, far away from the electrode layer, of the signal conversion layer, and the side, far away from the electrode layer, of the signal conversion layer is further connected with a surfactant. The invention further relates to a preparation method and application of the sensing electrode. The sensing electrode is high in accuracy and reliability of detecting index components in a sample, good in repeatability and high in sensitivity.

Description

Technical Field

[0001] This application belongs to the field of detection and analysis, specifically relating to a sensing electrode for diagnosing oral diseases, as well as a method for preparing the sensing electrode and its medical applications. Background Technology

[0002] Saliva, as a non-invasive biological fluid, is rich in biomarkers such as amino acids, nucleic acids, enzymes, hormones, and inflammatory factors. Its compositional changes are closely related to oral diseases (periodontitis, dental caries, oral cancer, etc.) and overall health, making it an ideal sample for early disease screening and health monitoring.

[0003] The traditional gold standard for detecting oral disease biomarkers is enzyme-linked immunosorbent assay (ELISA). While it offers advantages such as pg-level sensitivity and low cost for batch testing, its complex procedure, poor batch-to-batch reproducibility, and inability to provide rapid, on-the-spot detection are drawbacks. Electrochemical sensors, with their high sensitivity, rapid response (minute-level), ease of operation, low cost, portability, and point-of-care testing (POCT), have become the preferred alternative to ELISA. However, current electrochemical sensors used for saliva detection suffer from several drawbacks:

[0004] (1) Saliva contains a large number of macromolecular impurities such as proteins, lipids, and food residues, which can easily contaminate the surface of the sensing electrode and occupy the target molecule recognition sites, affecting the detection sensitivity and accuracy.

[0005] (2) Single biomarker detection is difficult to simultaneously meet the clinical requirements of high sensitivity and high specificity. In addition, patients have large biological differences, and the risk of false negatives and false positives is high. However, existing electrochemical sensors for detecting saliva are prone to signal interference when detecting multiple biomarkers simultaneously, resulting in insufficient accuracy and stability.

[0006] Therefore, there is an urgent need for a sensing electrode and detection method that can separate target molecules from impurities and detect multiple markers, with high sensitivity, accuracy and stability. Summary of the Invention

[0007] One objective of this application is to provide a sensing electrode for diagnosing oral diseases, which exhibits high accuracy, reliability, repeatability, and sensitivity in detecting indicator components in test samples. Furthermore, another objective of this application is to provide a method for preparing this sensing electrode and its applications.

[0008] To achieve the above objectives, the first aspect of this application provides a sensing electrode for diagnosing oral diseases. The sensing electrode includes an electrode layer and a signal conversion layer located on one side of the electrode layer. A biometric probe (optionally a biometric probe for diagnosing oral diseases) is fixed on the surface and / or inside of the signal conversion layer on the side away from the electrode layer. Furthermore, a surfactant is attached to the side of the signal conversion layer away from the electrode layer.

[0009] When no voltage is applied to the sensing electrode, the modified surfactant is freely arranged and distributed, allowing the analyte in the sample to specifically bind to the biometric probe in the sensing electrode. When a voltage is applied, the modified surfactant is oriented under voltage-driven conditions, with the hydrophilic end facing the signal conversion layer surface and the hydrophobic end moving away from the signal conversion layer surface. This allows hydrophobic proteins and non-specifically adsorbed macromolecules to be kept away from the signal conversion layer surface, thereby achieving the separation of hydrophobic proteins and non-specifically adsorbed impurities from the sample and reducing their interference with sample detection.

[0010] In any embodiment of the first aspect of this application, the surfactant includes one or more of cationic surfactants and anionic surfactants.

[0011] In any embodiment of the first aspect of this application, the surfactant includes one or more of quaternary ammonium salt cationic surfactants, sulfate ester salt anionic surfactants, and sulfonate anionic surfactants.

[0012] In any embodiment of the first aspect of this application, the quaternary ammonium salt cationic surfactant includes one or more of hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, and tetradecyltrimethylammonium bromide.

[0013] In any embodiment of the first aspect of this application, the sulfate ester salt anionic surfactant includes one or more of sodium hexadecyl sulfate and sodium dodecyl sulfate.

[0014] In any embodiment of the first aspect of this application, the sulfonate anionic surfactant includes sodium dodecylbenzenesulfonate.

[0015] In any embodiment of the first aspect of this application, the biometric probe includes one or more of D-type amino acid oxidase, LTB4 receptor, L-type amino acid oxidase, lactate dehydrogenase, and matrix metalloproteinase.

[0016] In some embodiments of the first aspect of this application, the biometric probe includes a D-type amino acid oxidase and an LTB4 receptor.

[0017] Unconstrained by theoretical limitations, both D-amino acids and L-amino acids are microbial metabolites. Changes in D-amino acid levels reflect the balance of the oral microbiome and the state of inflammation, while changes in L-amino acid levels reflect the host's metabolic state. Measuring the ratio of these two amino acids can reduce the interference of amino acid level fluctuations and improve accuracy. The concentration of the inflammatory mediator LTB4 in the saliva of patients with oral inflammation is significantly elevated and can serve as an indicator component for periodontitis detection. Combined detection of the D-amino acid / L-amino acid ratio with LTB4 is beneficial for improving the accuracy of oral disease diagnosis.

[0018] D-type amino acid oxidase can specifically bind to D-type or L-type amino acids under different pH conditions, triggering changes in electrical signals. LTB4 receptors specifically bind to LTB4, producing an electrochemical steric hindrance effect, which causes changes in electrical signals. Therefore, the content of indicator components can be detected by detecting the electrical signal response value.

[0019] In any embodiment of the first aspect of this application, the enzyme activity of D-type amino acid oxidase in the sensing electrode per unit area is 0.001 U / mm². 2 -0.1 U / mm 2 For example, 0.003 U / mm 2 0.004 U / mm 2 0.005 U / mm 2 0.0057 U / mm 2 0.006 U / mm 2 0.007 U / mm 2 0.009 U / mm 2 0.01 U / mm 2 0.02 U / mm 2 0.0283U / mm 2 0.03 U / mm 2 0.04 U / mm 2 0.05 U / mm 2 0.06 U / mm 2 0.07 U / mm 2 0.08 U / mm 2 0.09 U / mm 2 0.1 U / mm 2 .

[0020] In any embodiment of the first aspect of this application, the mass of LTB4 receptors in the sensing electrode per unit area is 0.1 μg / mm². 2 -2 μg / mm 2 For example, 0.1 μg / mm 2 0.15μg / mm 20.2μg / mm 2 0.25μg / mm 2 0.283 μg / mm 2 0.3 μg / mm 2 0.4 μg / mm 2 0.5μg / mm 2 0.7 μg / mm 2 0.8 μg / mm 2 0.9μg / mm 2 1μg / mm 2 1.2 μg / mm 2 1.3 μg / mm 2 1.4 μg / mm 2 1.415 μg / mm 2 1.5μg / mm 2 1.6 μg / mm 2 1.7 μg / mm 2 1.8 μg / mm 2 1.9 μg / mm 2 2μg / mm 2 .

[0021] In any embodiment of the first aspect of this application, the signal conversion layer comprises undoped or nitrogen-doped carbon-based nanomaterials. Thus, nitrogen doping enhances the conductivity of the carbon-based nanomaterials, amplifying the sensing signal.

[0022] In any embodiment of the first aspect of this application, the mass content of the N element in the carbon-based nanomaterial is 1%-10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0023] In any embodiment of the first aspect of this application, the carbon-based nanomaterial includes one or more of carbon nanotubes, graphene, and transition metal carbide-transition metal nitride two-dimensional materials (MXene).

[0024] In any embodiment of the first aspect of this application, the carbon-based nanomaterial is further loaded with nanoparticles containing transition metal elements, wherein the nanoparticles containing transition metal elements are adapted to promote the decomposition of hydrogen peroxide.

[0025] In any embodiment of the first aspect of this application, the transition metal element includes one or more of Co, Fe, Ag, Cu, Au, and Ni, such as Co.

[0026] Since the D-amino acid oxidase in the biorecognition probe readily catalyzes the production of hydrogen peroxide from alanine in the sample, and hydrogen peroxide in turn inactivates the D-amino acid oxidase, thus losing its function as a biorecognition probe, nanoparticles containing transition metals can promote the decomposition of hydrogen peroxide to inhibit its destruction of the activity of the D-amino acid oxidase biorecognition probe.

[0027] In any embodiment of the first aspect of this application, the surface of the carbon-based nanomaterial is further modified with the compound 2-(3,4-dihydroxyphenyl)ethylamine.

[0028] Due to the strong hydrophobicity of carbon-based nanomaterials, they are prone to aggregation in aqueous solvents. Compound 2-(3,4-dihydroxyphenyl)ethylamine, with its weak negative charge and good hydrophilicity, can be used to modify the surface of carbon-based nanomaterials, thereby improving their hydrophilicity, stability, and dispersibility in aqueous solvents, and reducing aggregation. Furthermore, the amino groups in this compound can couple biometric probes through covalent bonds and other interactions, which is beneficial for the immobilization of biometric probes.

[0029] In any embodiment of the first aspect of this application, the thickness of the signal conversion layer is 1μm-2μm, for example 1.2μm or 1.4μm.

[0030] A second aspect of this application provides a method for preparing a sensing electrode, comprising: A signal conversion layer is disposed on one side of the electrode layer; A biometric probe (optionally a biometric probe for diagnosing oral diseases) is fixed on the surface and / or inside the signal conversion layer on the side away from the electrode layer. After fixing the biometric probe, a surfactant is modified on the side of the signal conversion layer away from the electrode layer to obtain a sensing electrode.

[0031] The signal conversion layer provided in this application is beneficial for improving electron conduction velocity, significantly amplifying electrical signals, and improving detection sensitivity and signal-to-noise ratio.

[0032] Because surfactants have electrostatic repulsion properties, they are not conducive to the fixation of biometric probes. Therefore, this application first fixes the biometric probes and then modifies the surfactants, reducing the aforementioned adverse effects, increasing the content of biometric probes in the signal conversion layer, and thus improving detection speed and sensitivity.

[0033] In any embodiment of the second aspect of this application, the surfactant is modified by immersing the side of the signal conversion layer away from the electrode layer in a surfactant solution.

[0034] In any embodiment of the second aspect of this application, the step of modifying the surfactant involves immersion at 20°C-30°C, for example, 25°C.

[0035] In any embodiment of the second aspect of this application, the immersion time in the step of modifying the surfactant is 20 minutes to 120 minutes, for example, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, or 120 minutes.

[0036] In any embodiment of the second aspect of this application, in the step of modifying the surfactant, the molar concentration of the surfactant solution is 0.1 mM-2 mM, for example 0.5 mM, 1 mM, or 1.5 mM.

[0037] In any embodiment of the second aspect of this application, the biometric probe is fixed by the following steps: at least one side of the signal conversion layer away from the electrode layer is immersed in a first solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloric acid and N-hydroxysuccinimide for reaction; The second solution containing the biometric probe is coated onto the surface of the signal conversion layer after the reaction, and then incubated at 20°C-35°C (e.g., 25°C, 30°C).

[0038] In any embodiment of the second aspect of this application, in the step of immobilizing the biometric probe, the molar concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in the first solution are each independently 5 mM-40 mM, for example 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM.

[0039] In any embodiment of the second aspect of this application, the biometric probe includes one or more of D-type amino acid oxidase, LTB4 receptor, L-type amino acid oxidase, lactate dehydrogenase, and matrix metalloproteinase.

[0040] In some embodiments of the second aspect of this application, the biometric probe includes a D-type amino acid oxidase and an LTB4 receptor.

[0041] In any embodiment of the second aspect of this application, the concentration of D-type amino acid oxidase in the second solution is 10 U / mL-30 U / mL, for example 15 U / mL, 20 U / mL, or 25 U / mL.

[0042] In any embodiment of the second aspect of this application, the concentration of the LTB4 receptor in the second solution is 0.05 mg / mL to 0.4 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, or 0.4 mg / mL.

[0043] In any embodiment of the second aspect of this application, the reaction temperature is 20°C-35°C, for example 25°C or 30°C.

[0044] In any embodiment of the second aspect of this application, the reaction time is 20-60 minutes, for example, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes.

[0045] In any embodiment of the second aspect of this application, the incubation time is 5-30 minutes, for example 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0046] In any embodiment of the second aspect of this application, the signal conversion layer is set by the following steps: coating a dispersion of undoped or N-doped carbon-based nanomaterials onto one side of the electrode layer and drying it.

[0047] In any embodiment of the second aspect of this application, in the step of setting the signal conversion layer, the concentration of the carbon-based nanomaterial dispersion is 0.1 mg / mL-5 mg / mL, for example 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL.

[0048] In any embodiment of the second aspect of this application, during the step of setting the signal conversion layer, the carbon-based nanomaterial is further loaded with nanoparticles containing transition metal elements, wherein the nanoparticles containing transition metal elements are suitable for promoting the decomposition of hydrogen peroxide.

[0049] In any embodiment of the second aspect of this application, during the step of setting the signal conversion layer, the surface of the carbon-based nanomaterial is further modified with the compound 2-(3,4-dihydroxyphenyl)ethylamine.

[0050] In any embodiment of the second aspect of this application, the dispersion of the carbon-based nanomaterial is prepared by the following steps: Carbon-based nanomaterials, whether undoped or doped with nitrogen, are dispersed in a first solvent. Optionally, the pH of the dispersion is adjusted to 9-11 (e.g., 10), and the dispersion is mixed with a transition metal salt solution. Solid-liquid separation is performed, the solid phase is collected, dried, and calcined. The calcined material was dispersed in a second solvent to obtain a dispersion of carbon-based nanomaterials.

[0051] In any embodiment of the second aspect of this application, during the step of preparing a dispersion of carbon-based nanomaterials, a compound (e.g., 2-methylimidazole) that coordinates with transition metal ions is added during the mixing process. This facilitates the uniform dispersion of transition metal ions in the carbon-based nanomaterials.

[0052] In any embodiment of the second aspect of this application, in the step of preparing a dispersion of carbon-based nanomaterials, the ratio of undoped or N-doped carbon-based nanomaterials to the first solvent is 1:1 mg / mL to 7:1 mg / mL, for example, 2:1 mg / mL or 4:1 mg / mL.

[0053] In any embodiment of the second aspect of this application, in the step of preparing a dispersion of carbon-based nanomaterials, the first solvent is an alcohol solvent, such as ethylene glycol.

[0054] In any embodiment of the second aspect of this application, in the step of preparing a dispersion of carbon-based nanomaterials, the transition metal salt solution is selected from one or more of the transition metal nitrate solution, chloride solution, and carbonate solution.

[0055] In any embodiment of the second aspect of this application, in the step of preparing a dispersion of carbon-based nanomaterials, the molar concentration of the transition metal salt solution is 30-80 mM, for example 40 mM, 50 mM, 60 mM, or 70 mM.

[0056] In any embodiment of the second aspect of this application, in the step of preparing the dispersion of carbon-based nanomaterials, the mixing temperature is 20°C-30°C (e.g., 25°C), and the mixing time is 30 minutes-90 minutes (e.g., 60 minutes).

[0057] In any embodiment of the second aspect of this application, in the step of preparing the dispersion of carbon-based nanomaterials, the drying temperature is 20°C-30°C (e.g., 25°C), and the drying time is 18-48 hours (e.g., 24 hours).

[0058] In any embodiment of the second aspect of this application, in the step of preparing the dispersion of carbon-based nanomaterials, the calcination temperature is 400℃-700℃ (e.g., 500℃, 600℃), and the calcination time is 1-5 hours (e.g., 2 hours, 3 hours, 4 hours). Optionally, the calcination heating rate is 1℃ / min-3℃ / min, for example, 1℃ / min.

[0059] In any embodiment of the second aspect of this application, in the step of preparing the dispersion of carbon-based nanomaterials, the calcined material is mixed with a 2-(3,4-dihydroxyphenyl)ethylamine solution, the solid and liquid phases are separated, and the resulting solid phase is dried and dispersed in a solvent. Optionally, the molar concentration of the 2-(3,4-dihydroxyphenyl)ethylamine solution is 0.05-0.3 mM, for example, 0.1 mM. Optionally, the ratio of undoped or nitrogen-doped carbon-based nanomaterials to 2-(3,4-dihydroxyphenyl)ethylamine is (3000-5000):1 mg / mmol, for example, 4000:1 mg / mmol. Optionally, the mixing temperature is 20℃-30℃ (for example, 25℃), and the mixing time is 1-5 hours (for example, 2 hours).

[0060] In any embodiment of the second aspect of this application, in the step of preparing a dispersion of carbon-based nanomaterials, the second solvent is water, such as deionized water, high-purity water, etc.

[0061] In any embodiment of the second aspect of this application, the surfactant includes one or more of cationic surfactants and anionic surfactants.

[0062] In any embodiment of the second aspect of this application, the carbon-based nanomaterial includes one or more of carbon nanotubes, graphene, and transition metal carbide-transition metal nitride two-dimensional materials (MXene).

[0063] In any embodiment of the second aspect of this application, the mass content of the N element in the carbon-based nanomaterial is 1%-10%.

[0064] In any embodiment of the second aspect of this application, the sensing electrode is as described in the first aspect of this application.

[0065] In some embodiments, the transition metal carbide-transition metal nitride two-dimensional material is Ti3C2T. x In this application, Ti3C2T x The "T" in x "x" is an abbreviation for the chemical symbol representing various functional groups attached to the surface of the two-dimensional material Ti3C2. These functional groups are not inherent to the material itself, but are introduced during the synthesis process. T includes F, O, and hydroxyl groups, and x represents a non-stoichiometric variable coverage. This material is obtained by etching away Al from the precursor material Ti3AlC2. During the etching process, ions in the etching solution (such as fluoride ions, oxygen ions, and hydroxide ions) combine with titanium atoms to form the aforementioned functional groups.

[0066] The third aspect of this application provides the application of the sensing electrode of the first aspect of this application in the manufacture of a device for diagnosing oral diseases.

[0067] The fourth aspect of this application provides a method for diagnosing oral diseases, including: Using the sensing electrode of the first aspect of this application as the working electrode, an electrochemical workstation is used to detect the sample to be tested (such as saliva or artificial saliva). During the detection process, a voltage (such as a positive voltage or a negative voltage) is applied to the sensing electrode to obtain a cyclic voltammetry curve. The content of the index component in the sample to be tested is calculated based on the peak current corresponding to the oxidation peak or reduction peak in the cyclic voltammetry curve. Oral diseases are diagnosed based on the content of indicator components in the sample to be tested.

[0068] In any embodiment of the fourth aspect of this application, the method involves using an electrochemical workstation to detect the sample to be tested (e.g., saliva or artificial saliva) under conditions where the pH value is 7-8 (e.g., 7.4, 7.5) and different scanning ranges of the electrochemical workstation, and obtaining a first cyclic voltammetry curve and a second cyclic voltammetry curve respectively. The sample was tested using an electrochemical workstation at a pH of 9-10 (e.g., 9.8) to obtain the third cyclic voltammetric curve. The contents of D-alanine and BLT4 in the sample were calculated based on the peak current corresponding to the -0.54V reduction peak in the first cyclic voltammetry curve and the peak current corresponding to the 0.59V oxidation peak in the second cyclic voltammetry curve. The contents of L-alanine in the sample were calculated based on the peak current corresponding to the -0.54V reduction peak in the third cyclic voltammetry curve.

[0069] In any embodiment of the fourth aspect of this application, the range of the applied positive voltage is 0.5-10V, for example 0.5V, 1V, 2V, 3V, 4V, 5V, 6V, 7V, 8V, 9V, 10V.

[0070] In any embodiment of the fourth aspect of this application, the range of the applied negative voltage is -0.5 to -10V, for example -0.5V, -1V, -2V, -3V, -4V, -5V, -6V, -7V, -8V, -9V, -10V.

[0071] In any embodiment of the fourth aspect of this application, the external standard analysis method is used for calculation.

[0072] In any embodiment of the fourth aspect of this application, the operating conditions of the electrochemical workstation for testing the first and third cyclic voltammetry curves include one or more of the following: Scan range: 0 to -0.8 V; Initial potential 0 V; Scan speed: 0.1 V / s.

[0073] In any embodiment of the fourth aspect of this application, the operating conditions of the electrochemical workstation for testing the second cyclic voltammetry curve include one or more of the following: Scanning range: 0~0.8 V; Initial potential 0 V; Scan speed: 0.1 V / s.

[0074] The fifth aspect of this application provides a method for diagnosing oral diseases, including: The sample to be tested (such as saliva or artificial saliva) is placed on the surface of the sensing electrode of the first aspect of this application and incubated for a period of time under a pH value of 7-8 (such as 7.4 or 7.5). A voltage (such as positive or negative voltage) is applied to the sensing electrode, and then the sample to be tested on the surface of the sensing electrode is removed. The area where the sample to be tested is placed on the surface of the sensing electrode is detected by a terahertz spectrometer to obtain a spectrum. The time-domain signal of the spectrum is subjected to Fourier transform to obtain the terahertz frequency domain signal map. The presence of D-amino acids in the sample to be tested can be determined from the terahertz frequency domain signal map.

[0075] In any embodiment of the fifth aspect of this application, the range of the applied positive voltage is 0.5-10V, for example 0.5V, 1V, 2V, 3V, 4V, 5V, 6V, 7V, 8V, 9V, 10V.

[0076] In any embodiment of the fifth aspect of this application, the range of the applied negative voltage is -0.5 to -10V, for example -0.5V, -1V, -2V, -3V, -4V, -5V, -6V, -7V, -8V, -9V, -10V.

[0077] In any embodiment of the fifth aspect of this application, the incubation temperature is 20°C-35°C, for example 25°C or 30°C.

[0078] In any embodiment of the fifth aspect of this application, the incubation time is 2 minutes to 20 minutes, for example, 5 minutes, 10 minutes, 15 minutes, or 20 minutes.

[0079] In any embodiment of the fifth aspect of this application, the sample to be tested is placed by dripping or coating.

[0080] In any embodiment of the fifth aspect of this application, the operating conditions of the terahertz spectrometer include one or more of the following: The test temperature was 21±3℃; The tested humidity was 30.3% RH%. The scan window is 160 ps.

[0081] The sensing electrode of the first aspect of this application is used for diagnosing oral diseases.

[0082] In any embodiment of this application, oral diseases include one or more of periodontitis, dental caries, and oral cancer.

[0083] This application has achieved at least one of the following beneficial effects:

[0084] (1) The sensing electrode of this application has high accuracy and reliability, good repeatability and high sensitivity in detecting the index components in the sample to be tested.

[0085] (2) After the voltage is applied to the sensing electrode of this application, the hydrophobic ends of the surfactant modified by the signal conversion layer are distributed far away from the signal conversion layer. These hydrophobic ends can bind to the hydrophobic regions of macromolecules such as lipids and proteins, thereby realizing the separation of macromolecules in the sample, reducing the interference of macromolecules on sample detection, and the sample does not need to be pretreated.

[0086] (3) The signal conversion layer of this application improves the electronic conduction rate of the sensing electrode, amplifies the electrical signal, and improves the detection sensitivity and signal-to-noise ratio.

[0087] (4) The sensing electrode of this application includes a variety of biometric probes, which can simultaneously detect a variety of indicator components to diagnose oral diseases. Attached Figure Description

[0089] To make the content of this application easier to understand, the following detailed description is provided based on specific embodiments and accompanying drawings, wherein:

[0090] Figure 1 This is a flowchart illustrating the fabrication process of the sensing electrode in Embodiment 1 of this application.

[0091] Figure 2 This is a SEM image of the nanocomposite material in Example 1 of this application.

[0092] Figure 3 This is a SEM image of the nanocomposite material in Example 3 of this application.

[0093] Figure 4 Electrochemical impedance spectroscopy (EIS) of the bare electrode and the sensing electrodes of Examples 1 and 3 of this application.

[0094] Figure 5 Cyclic voltammetry curves of the sensing electrodes used as working electrodes in phosphate buffer solution for Examples 1 and 1 of this application are shown.

[0095] Figure 6a This is a schematic diagram of the surface contact angle of the sensing electrode before voltage is applied, as shown in Comparative Example 2.

[0096] Figure 6bThis is a schematic diagram of the surface contact angle of the sensing electrode after applying voltage to it, as shown in Comparative Example 2.

[0097] Figure 7a This is a schematic diagram of the surface contact angle of the sensing electrode before voltage is applied in Embodiment 1 of this application.

[0098] Figure 7b This is a schematic diagram of the surface contact angle of the sensing electrode after a voltage is applied in Embodiment 1 of this application.

[0099] Figure 8a Cyclic voltammetry curves of the sensing electrode used as the working electrode in Example 1 of this application were tested in phosphate buffer, L-alanine solution and D-alanine solution at pH 7.4.

[0100] Figure 8b Cyclic voltammetry curves of the sensing electrode used as the working electrode in Example 1 of this application were tested in phosphate buffer, L-alanine solution and D-alanine solution with a pH of 9.8.

[0101] Figure 9a The cyclic voltammetry curves for Example 1 of this application, where the sensing electrode is the working electrode, were tested in a series of L-alanine solutions with a pH of 9.8.

[0102] Figure 9b A standard curve for quantitatively calculating L-alanine content.

[0103] Figure 10a The cyclic voltammetry curves for Example 1 of this application, where the sensing electrode is the working electrode, were tested in a series of D-alanine solutions with a pH of 7.4.

[0104] Figure 10b A standard curve for quantitatively calculating D-alanine content.

[0105] Figure 11a The cyclic voltammetry curves for Example 1 of this application, where the sensing electrode is the working electrode, were tested in a series of LTB4 solutions with a pH of 7.4.

[0106] Figure 11b A standard curve for quantitatively calculating LTB4 content.

[0107] Figure 12 shows the terahertz frequency domain signals of phosphate buffer, L-alanine solution and D-alanine solution with a pH of 7.4 tested by the sensing electrode of Example 1 of this application. Detailed Implementation

[0109] The embodiments of this application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this application or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0110] Example 1: Fabrication of Sensing Electrode 1

[0111] Figure 1 This is a flowchart of the fabrication process for the sensing electrode.

[0112] (I) Synthesis of Nanocomposite Materials Take 40 mg of nitrogen-doped graphene (nitrogen content of 4%) and disperse it in 10 mL of ethylene glycol. Sonicate the resulting dispersion for 30 minutes. Add 0.1 M sodium hydroxide aqueous solution dropwise to the resulting dispersion to adjust the pH value to 10.

[0113] Add 10 mL of 60 mmol / L cobalt nitrate hexahydrate aqueous solution dropwise to the pH-adjusted dispersion, mix well to obtain a mixture; quickly inject 70 mL of an aqueous solution containing 4.54 g of 2-methylimidazole (which coordinates with cobalt ions to improve the uniformity of cobalt dispersion and the loading of cobalt) into the mixture, stir at 25 °C for 1 hour, centrifuge to collect the precipitate, wash the precipitate three times with ethanol, and dry at 25 °C for 24 hours.

[0114] The dried material is placed in a tube furnace and heated to 600°C at a rate of 1°C / min in a nitrogen atmosphere. The temperature is maintained at 600°C for 2 hours to achieve carbonization, thus obtaining carbonized material.

[0115] The carbonized material was dispersed in 100 mL of 0.1 mM dopamine (chemical name: 2-(3,4-dihydroxyphenyl)ethylamine) aqueous solution. The resulting dispersion was incubated at 25 °C for 2 hours to enhance surface hydrophilicity. The precipitate was collected by centrifugation and dried to obtain Co NPs / nitrogen-doped graphene (NGO) nanocomposite material with a Co element mass content of 2.1%.

[0116] (II) Preparation of sensing electrode 1 Two mg of Co NPs / nitrogen-doped graphene (NGO) nanocomposite material was dispersed in 1 ml of ultrapure water to obtain a nanocomposite dispersion with a concentration of 2 mg / ml. A glassy carbon electrode (7.065 mm²) was then used. 2 After cleaning, the above-mentioned nanocomposite dispersion is drop-coated and dried at 25°C to form a signal conversion layer with a thickness of 1.4 μm.

[0117] Preparation of EDC / NHS solvent: Weigh 1.952 g of 2-(N-morpholino)ethanesulfonic acid (MES) powder and add it to 80 mL of deionized water. Stir to dissolve, and adjust the pH to 5.5 dropwise with 1 M NaOH (approximately 15-20 mL). Transfer the resulting solution to a 100 mL volumetric flask and dilute to volume with ultrapure water to obtain the MES solution. Dilute 191.7 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution in 50 mL of MES solution to obtain an EDC solution with a molar concentration of 20 mM. Dissolve 287.5 mg of N-hydroxysuccinimide (NHS) in 50 mL of MES solution to obtain an NHS solution with a molar concentration of 50 mM. Mix 1 mL of EDC solution and 1 mL of NHS solution to obtain the EDC / NHS solvent.

[0118] The electrode forming the signal conversion layer was immersed in EDC / NHS solvent and reacted at 25°C for 30 minutes, followed by rinsing three times with MES solution. Then, 20 μL of MES solution containing 20 U / mL D-amino acid oxidase (DAAO, purchased from Sigma-Aldrich) and 0.2 mg / mL BLT4 receptor (BLTR, purchased from Sigma-Aldrich) was drop-coated onto the surface of the EDC / NHS-modified signal conversion layer. After incubation at 25°C for 15 minutes, the electrode was rinsed three times with MES solution. The resulting signal conversion layer surface was immobilized with DAAO (0.2 U) and BLTR (2 μg), with a DAAO immobilization amount of 0.0283 U / mm². 2 The LBLTR fixation amount was 0.283 μg / mm. 2 .

[0119] The electrode with the biometric probe fixed was immersed in a 0.5 mM hexadecyltrimethylammonium bromide (CTAB) aqueous solution and left to stand at 25 °C for 30 minutes to allow the amphiphilic surfactant to self-assemble on the surface of the signal conversion layer. The loosely adsorbed surfactant was removed by slowly rinsing with ultrapure water for 5 seconds to obtain sensing electrode 1.

[0120] Example 2: Fabrication of Sensing Electrode 2

[0121] In the synthesis of the nanocomposite material, 120 mg of nitrogen-doped graphene (nitrogen content of 4%) was used, and the remaining operations were the same as in Example 1 to obtain Co NPs / nitrogen-doped graphene (NGO) nanocomposite material with a Co element content of 0.7%.

[0122] During the fabrication of the sensing electrode, when immobilizing the biorecognition probe, 20 μL of MES solution containing DAAO (4 U / ml) and BLTR (1 mg / ml) was drop-coated onto the surface of the signal conversion layer modified with EDC / NHS. After incubation at 25°C for 15 minutes, the electrode was rinsed three times with MES solution. The resulting signal conversion layer surface was immobilized with DAAO (0.04 U) and BLTR (10 μg), and the DAAO immobilization amount in the signal conversion layer was 0.0057 U / mm. 2 The BLTR fixation amount was 1.415 μg / mm. 2 .

[0123] The electrode with the fixed biometric probe was immersed in a 1 mM hexadecyltrimethylammonium bromide (CTAB) aqueous solution and left to stand at 25 °C for 30 minutes to allow the amphiphilic surfactant to self-assemble on the surface of the signal conversion layer. The remaining operations were the same as in Example 1 to obtain sensing electrode 2.

[0124] Example 3: Fabrication of sensing electrode 3

[0125] In the synthesis step of the nanocomposite material, 120 mg of Ti3C2T was used. x (Purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.) To replace graphene, the remaining operations were the same as in Example 1, and Co NPs / MXene nanocomposite material was obtained, with the same Co element mass content as in Example 2.

[0126] The fabrication steps for the sensing electrode are the same as in Example 2, resulting in sensing electrode 3. The DAAO fixation amount in the signal conversion layer is 0.0057 U / mm. 2 BLTR fixation amount 1.415 μg / mm 2 .

[0127] Comparative Example 1: Preparation of Sensing Electrode A

[0128] After cleaning the glassy carbon electrode, DAAO (0.2 U) and BLTR (2 μg) were fixed on the surface of the glassy carbon electrode according to the procedure in Example 1.

[0129] The electrode with the biometric probe fixed was immersed in a 0.5 mM hexadecyltrimethylammonium bromide (CTAB) aqueous solution and left to stand at 25°C for 30 minutes to allow the amphiphilic surfactant to self-assemble on the electrode surface. The loosely adsorbed surfactant was removed by slowly rinsing with ultrapure water for 5 seconds to obtain sensing electrode A.

[0130] Comparative Example 2: Preparation of Sensing Electrode B

[0131] Based on Example 1, the operation step of self-assembling amphiphilic surfactant was omitted, and the rest was the same as in Example 1, to obtain sensing electrode B.

[0132] Experimental Example 1: Microstructure of Nanocomposite Materials

[0133] The nanocomposites of Examples 1 and 3 were observed using scanning electron microscopy (SEM), as shown below. Figure 2-3 As shown.

[0134] The images show that Co nanoparticles are embedded in a carbon framework and loaded into sheet structures of graphene or MXene, respectively, to form a nanocomposite structure.

[0135] Experimental Example 2: Electrochemical Impedance Spectroscopy of Sensing Electrode

[0136] Test method: A three-electrode system was formed using the electrode under test as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. 10 mL of electrolyte was added, ensuring the three electrodes were completely submerged. The system was allowed to stand for 5-10 minutes to allow the electrode / solution interface to stabilize. The electrolyte preparation steps were as follows: 164.6 mg of potassium ferricyanide and 211.5 mg of potassium ferrocyanide trihydrate were weighed, and 3.73 g of potassium chloride was added. The solution was dissolved in ultrapure water and diluted to a final volume of 500 mL.

[0137] The open circuit potential (OCP) was tested using an electrochemical workstation, and the stabilization time was 400 s. The OCP value after stabilization was recorded as the DC bias potential for electrochemical impedance spectroscopy.

[0138] Electrochemical impedance spectroscopy was tested using an electrochemical workstation. The measured open-circuit potential was used as the initial voltage. The scanning frequency range was 100 kHz to 0.1 Hz, the AC amplitude was 5 mV, and the resting time before each frequency point was 2 seconds.

[0139] Figure 4 Electrochemical impedance spectroscopy spectra of the bare electrode (glassy carbon electrode), Example 1 (modified Co NPs / NGO), and Example 3 (modified Co NPs / MXene) sensing electrodes are shown.

[0140] Depend on Figure 4 It can be seen that after modifying the electrodes with nanocomposite materials Co NPs / nitrogen-doped graphene (NGO) and Co NPs / MXene in Examples 1 and 3 respectively, the resistance of the electrode surface was significantly reduced, indicating that nanocomposite materials are beneficial to accelerating electron transfer on the electrode surface and improving conductivity.

[0141] Experimental Example 3: Cyclic Volt-Ampere Curves of Sensing Electrodes

[0142] A three-electrode system was used (the working electrodes were the sensing electrodes of Example 1 and Comparative Example 1, respectively; Ag / AgCl was the reference electrode; and platinum wire was the counter electrode). The three-electrode system was placed in 10 mL of phosphate buffer (50 mM, pH 7.4), and cyclic voltammetry was tested using an electrochemical workstation (CHI660E). The scan range was 0 to -0.8 V, the initial potential was 0 V, and the scan rate was 0.1 V / s. The phosphate buffer was prepared as follows: 3.90 g of sodium dihydrogen phosphate dihydrate was weighed and dissolved in 500 mL of deionized water, and stirred thoroughly to dissolve (solution A). 8.95 g of disodium hydrogen phosphate dodecahydrate was weighed and dissolved in 500 mL of deionized water, and stirred thoroughly to dissolve (solution B). Approximately 100 mL of solution A was placed in a beaker, and approximately 400 mL of solution B was added, with pH monitored while stirring. If the pH is too acidic, continue adding solution B; if it is too alkaline, add solution A. Finely adjust to pH 7.4, then bring the volume to 1000 mL with deionized water. The cyclic voltammetry curve is shown below. Figure 5 As shown.

[0143] Depend on Figure 5 It can be seen that the sensing electrode prepared in Example 1 exhibits enhanced electrochemical redox peaks, while the electrode prepared in Comparative Example 1 only exhibits weak redox peaks. This indicates that the nanocomposite material can significantly enhance the electron transfer of the recognition probe on the surface of the sensing electrode, which helps to improve the sensitivity of the sensing electrode.

[0144] Experimental Example 4: Contact Angle of Sensing Electrode Surface

[0145] The hydrophilicity / hydrophobicity of the sensor electrode surfaces prepared in Example 1 and Comparative Example 2 were tested. The sensor electrode was placed on the stage of a surface analyzer. Using a microsyringe, 2 to 5 microliters of deionized water were drawn, air bubbles were expelled, and the syringe needle was brought close to the sample surface (approximately 1 to 2 millimeters away). The droplet was slowly ejected and allowed to drip naturally onto the sensor electrode surface. The contact angle of the water droplet spreading on the sensor electrode surface was photographed and analyzed using a surface analyzer (Ningbo New Boundary Scientific Instruments Co., Ltd., OSA200). The test results are as follows: Figure 6a , 6b As shown in 7a and 7b. Among them, Figure 6a and 6b The surface contact angle of the sensing electrode before and after applying voltage (applied voltage - 1 V) is shown in Comparative Example 2. Figure 7a and 7b The surface contact angle of the sensing electrode in Example 1 before and after applying voltage (applied voltage -1V).

[0146] Depend on Figure 6a , 6bAs shown in 7a and 7b, the surface contact angle of the sensing electrode in Comparative Example 2 remains almost unchanged before and after the voltage is applied, and the hydrophilicity or hydrophobicity of the sensing electrode surface cannot be controlled electronically. After the voltage is applied, the surface contact angle of the sensing electrode in Example 1 increases significantly compared with that before the voltage is applied, and the surface changes from a hydrophilic surface to a hydrophobic surface. This indicates that after the voltage is applied to the sensing electrode, the hydrophilic end of the modified surfactant faces the surface of the signal conversion layer, and the hydrophobic end is away from the signal conversion layer. This creates a selective molecular transport channel on the surface of the signal conversion layer, which can repel hydrophobic polar proteins and non-specifically adsorbed macromolecules, keeping them away from the surface of the signal conversion layer. This achieves the separation of hydrophobic polar proteins, non-specifically adsorbed impurities and other macromolecules in the sample, reducing their interference with sample detection.

[0147] Experimental Example 5

[0148] (1) Specificity of the sensor for the detection of D and L alanine under different pH conditions

[0149] A three-electrode system was used (the working electrode was the sensing electrode prepared in Example 1, Ag / AgCl was the reference electrode, and platinum wire was the counter electrode).

[0150] The three-electrode system was placed in 5 μM L-alanine solution, 5 μM D-alanine solution, and phosphate buffer solution with a pH of 7.4 (composition: 61.2 ml, 50 mM disodium hydrogen phosphate dodecahydrate; 38.8 ml, 50 mM sodium dihydrogen phosphate dihydrate). Both solutions were prepared using the aforementioned phosphate buffer solution with a pH of 7.4. Cyclic voltammetry curves were measured using an electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., CHI660E), with a scan range of 0 to -0.8 V, an initial potential of 0 V, and a scan rate of 0.1 V / s. The results are as follows: Figure 8a As shown.

[0151] The three-electrode system was placed in 5 μM L-alanine solution, 5 μM D-alanine solution, and phosphate buffer solution with a pH of 9.8 (composition: 50 mM disodium hydrogen phosphate dodecahydrate solution, adjusted to pH 9.8 with 1 M NaOH). Both solutions were prepared using the aforementioned phosphate buffer solution with a pH of 9.8. Cyclic voltammetry curves were tested using an electrochemical workstation (CHI660E) under the same operating conditions as above. The results are as follows. Figure 8b As shown.

[0152] Depend on Figure 8a , 8bAs can be seen, in the buffer solution with a pH of 7.4, the peak current of the L-alanine solution remained unchanged, while the peak current of the D-alanine solution significantly increased. This indicates that the sensing electrode of this application can achieve rapid and sensitive detection of D-alanine at a pH of 7.4. Furthermore, the sensing electrode of this application exhibits an electrochemical reduction peak at -0.545 V. Under the buffer solution condition with a pH of 9.8, the peak current of the D-alanine solution remained essentially unchanged, while the peak current of the L-alanine solution significantly increased. This indicates that the sensing electrode of this application can achieve rapid and sensitive detection of L-alanine at a pH of 9.8.

[0153] (2) Establishment of standard curve and quantitative testing

[0154] A three-electrode system was used (the working electrode was the sensing electrode prepared in Example 1, Ag / AgCl was the reference electrode, and platinum wire was the counter electrode).

[0155] Chiral amino acid standard curve: The three-electrode system was placed in a series of L-alanine solutions (5 μM, 10 μM, 30 μM, 60 μM, 150 μM, 400 μM) at pH 9.8 and a series of D-alanine solutions (10 nM, 40 nM, 300 nM, 600 nM, 1000 nM, 5000 nM) at pH 7.4. The L-alanine solution was prepared using phosphate buffer at pH 9.8 (composition: 50 mM disodium hydrogen phosphate dodecahydrate solution, adjusted to pH 9.8 with 1 M NaOH). The D-alanine solution was prepared using phosphate buffer at pH 7.4 (composition: 61.2 ml, 50 mM disodium hydrogen phosphate dodecahydrate; 38.8 ml, 50 mM disodium hydrogen phosphate dodecahydrate solution). Sodium dihydrogen phosphate dihydrate was prepared, and cyclic voltammetry curves were tested using an electrochemical workstation (CHI660E). During testing, a voltage of -1V was applied to the working electrode. The operating conditions of the electrochemical workstation were: scan range 0~-0.8V, initial potential 0V, scan rate 0.1V / s. The results are as follows: Figure 9a , 10a As shown. Record. Figure 9a , 10a The reduction peak current at -0.54 V was used to plot a standard curve with concentration on the x-axis and peak current on the y-axis, as shown below. Figure 9b , 10b As shown, the standard curves for the detection of L-alanine and D-alanine were established as y=-0.1593x-0.9817 and y=-0.3532x-2.0124, respectively, with sensitivities of 0.1593 mA / μM and 0.3532 mA / μM, respectively.

[0156] LTB4 Standard Curve: The three-electrode system was placed in a series of LTB4 solutions (0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 250 μM, 750 μM) at pH 7.4. The LTB4 solutions were prepared using a phosphate buffer solution at pH 7.4 (composition: 61.2 ml, 50 mM disodium hydrogen phosphate dodecahydrate; 38.8 ml, 50 mM sodium dihydrogen phosphate dihydrate). Luminol was added to a concentration of 0.1 mM as an indicator molecule. Cyclic voltammetry was tested using an electrochemical workstation (CHI660E). A voltage of -1 V was applied to the working electrode during testing. The operating conditions of the electrochemical workstation were: scan range 0–0.8 V, initial potential 0 V, scan rate 0.1 V / s. The results are as follows: Figure 11a Record the oxidation peak current at 0.59 V, and plot a standard curve with concentration on the x-axis and peak current on the y-axis, as shown below. Figure 11b As shown, the standard curve for LBT4 detection was established as y = -2.21023x + 28.5976, with a sensitivity of 2.2102 mA / μM.

[0157] Artificial saliva with pH values ​​of 7.4 and 9.8 was prepared. The artificial saliva with pH value of 7.4 consisted of the following per 1000 mL of ultrapure water: NaCl 0.40 g, KCl 0.40 g, NaH2PO4·2H2O 0.69 g, NaHCO3 0.20 g, urea 1.00 g, mucin (MUC5B, purchased from Sigma-Aldrich) at a concentration of 1.0 mg / mL, alkaline stable α-amylase (purchased from Sigma-Aldrich) at a concentration of 2.0 mg / mL, lysozyme (purchased from Sigma-Aldrich) at a concentration of 0.1 mg / mL, and bovine serum albumin (BSA, purchased from Sigma-Aldrich) at a concentration of 0.1 mg / mL. The pH value was adjusted using 0.1 mol / L hydrochloric acid solution or 0.1 mol / L sodium hydroxide solution. The composition of artificial saliva with a pH of 9.8 is as follows: per 1000 mL of ultrapure water, there are 0.40 g of NaCl, 0.40 g of KCl, 0.69 g of NaH2PO4·2H2O, 0.20 g of NaHCO3, 1.00 g of urea, 1.0 mg / mL of mucin (MUC5B), 2.0 mg / mL of alkaline stable α-amylase, 0.1 mg / mL of lysozyme, and 0.1 mg / mL of bovine serum albumin (BSA). The pH is adjusted using 0.1 mol / L sodium hydroxide solution.

[0158] A solution containing 10 μM D-alanine and 5 μM LBT4 was prepared using artificial saliva at pH 7.4 as test sample 1. A solution containing 10 μM L-alanine was prepared using artificial saliva at pH 9.8 as test sample 2.

[0159] Samples 1 and 2 were taken. Using the sensing electrode from Example 1 of this application as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode, the three-electrode system was placed in sample 1 and incubated for 1 minute to ensure sufficient contact between the analyte molecules and the sensing electrode. Then, an electrochemical workstation (CHI660E) was used to apply a -1V voltage to the working electrode, driving the hydrophobic long alkyl chain of the surfactant away from the signal conversion layer, thereby separating the hydrophobic polar protein and the large-molecule non-specifically adsorbed protein. Cyclic voltammetry curves were then measured using the electrochemical workstation (CHI660E). The operating conditions of the electrochemical workstation were: scan range 0 to -0.8 V, initial potential 0 V, scan rate 0.1 V / s. Voltammetric curves A and B were obtained.

[0160] Take sample 1 and perform the test according to the above procedure, except that the scanning range of the electrochemical workstation is 0~0.8 V, and the other operations remain unchanged. Obtain the voltammetric curve C.

[0161] The peak current value of voltammetry curve A at -0.54 V was recorded, yielding a peak current value of -2.364 mA for D-alanine. The peak current value of voltammetry curve B at -0.54 V was recorded, yielding a peak current value of -1.139 mA for L-alanine. The peak current value of voltammetry curve C at 0.59 V was recorded, yielding a peak current value of 27.066 mA for BLT4.

[0162] Substituting the measured peak current values ​​into the aforementioned standard curve, the molar concentrations of L-alanine, D-alanine, and LBT4 in the sample were calculated to be 9.71 μM, 9.87 μM, and 4.96 μM, respectively.

[0163] The same sample was measured five times using the above method. The relative standard deviations of the molar concentrations of L-alanine, D-alanine, and BLT4 were 3.76%, 3.42%, and 2.78%, respectively, indicating that the repeatability of the detection using the sensing electrode of this application is good.

[0164] (3) Average spiked recovery test

[0165] Add 0.1 μmol D-alanine and 0.05 μmol BLT4 to 10 mL of sample 1 to obtain spiked sample 1. Add 0.1 μmol L-alanine to 10 mL of sample 2 to obtain spiked sample 2.

[0166] The molar concentrations of D-alanine and BLT4 in spiked sample 1 and the molar concentration of L-alanine in spiked sample 2 were determined using the above method. The spike recovery rate was then calculated. The results were obtained by taking the average value of three parallel determinations. The results are shown in Table 1.

[0167] Table 1. Average Spike Recovery Results

[0168]

[0169] As shown in the table above, the average spiked recoveries of L-alanine, D-alanine and BLT4 detected by the sensing electrode of this application were 98.28%, 98.77% and 98.66%, respectively, indicating that the quantitative detection of L-alanine, D-alanine and BLT4 by the sensing electrode of this application has high accuracy and good reliability.

[0170] Experimental Example 6

[0171] Terahertz spectroscopy was acquired on the surface of the sensing electrode to qualitatively determine L-alanine and D-alanine in the sample.

[0172] Prepare 100 μL of phosphate buffer solution with pH 7.4 (composition: 61.2 ml, 50 mM disodium hydrogen phosphate dodecahydrate; 38.8 ml, 50 mM sodium dihydrogen phosphate dihydrate), 5 μL of 5 μM L-alanine solution, and 5 μL of 5 μM D-alanine solution, both prepared using the aforementioned phosphate buffer solution with pH 7.4. Add the three solutions dropwise to the surface of the sensing electrode prepared in Example 1, incubate at 25°C for 5 minutes to allow the target molecules to adsorb onto the sensing area, then apply a -1 V voltage for 1 minute. Afterward, remove the test solution with filter paper and detect using a terahertz spectrometer (Beijing Yuanda Hengtong Technology Co., Ltd., instrument model TA-KYLIN). Operating conditions include: test temperature 21±3°C; test humidity 30.3 RH%; scan window 160 ps. Perform Fourier transform on the time-domain signal to obtain the terahertz frequency domain signal graph, with the horizontal axis representing frequency (in Hz) and the vertical axis representing amplitude, as shown below. Figure 12 As shown.

[0173] like Figure 12 As shown, the sensor electrode surface tested with phosphate buffer solution generates a characteristic frequency domain signal of DAAO. When testing the sensor electrode surface with L-alanine solution, the DAAO frequency domain signal remains unchanged, indicating that the sensor electrode of this application does not respond to L-alanine at pH 7.4. When testing the sensor electrode surface with D-alanine solution, the DAAO frequency domain signal decreases overall, indicating that the sensor electrode of this application can achieve a specific response to D-alanine at pH 7.4.

[0174] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A sensing electrode for diagnosing oral diseases, characterized in that, The sensing electrode includes an electrode layer and a signal conversion layer located on one side of the electrode layer; a biometric probe is fixed on the surface and / or inside of the signal conversion layer on the side away from the electrode layer, and a surfactant is also attached to the side of the signal conversion layer away from the electrode layer.

2. The sensing electrode according to claim 1, characterized in that, The surfactant includes one or more of cationic and anionic surfactants; or... The surfactant includes one or more of the following: quaternary ammonium salt cationic surfactants, sulfate ester salt anionic surfactants, and sulfonate anionic surfactants.

3. The sensing electrode according to claim 2, characterized in that, The quaternary ammonium salt cationic surfactant includes one or more of hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, and tetradecyltrimethylammonium bromide; and / or, The sulfate ester salt anionic surfactant includes one or more of sodium hexadecyl sulfate and sodium dodecyl sulfate; and / or, The sulfonate anionic surfactants include sodium dodecylbenzenesulfonate.

4. The sensing electrode according to claim 1, characterized in that, The biometric probes include one or more of D-type amino acid oxidase, LTB4 receptor, L-type amino acid oxidase, lactate dehydrogenase, and matrix metalloproteinase.

5. The sensing electrode according to claim 4, characterized in that, The biorecognition probe includes a D-type amino acid oxidase and an LTB4 receptor, wherein, The enzyme activity of D-type amino acid oxidase in the sensing electrode per unit area is 0.001 U / mm². 2 -0.1 U / mm 2 ; and / or, The mass of LTB4 receptors per unit area of ​​the sensing electrode is 0.1 μg / mm². 2 -2 μg / mm 2 .

6. The sensing electrode according to claim 1, characterized in that, The signal conversion layer comprises undoped or nitrogen-doped carbon-based nanomaterials.

7. The sensing electrode according to claim 6, characterized in that, The mass content of nitrogen element in the carbon-based nanomaterial is 1%-10%; and / or, The carbon-based nanomaterials include one or more of carbon nanotubes, graphene, and transition metal carbide-transition metal nitride two-dimensional materials; and / or, The carbon-based nanomaterial is further loaded with nanoparticles containing transition metal elements, wherein the nanoparticles containing transition metal elements are suitable for promoting the decomposition of hydrogen peroxide.

8. The sensing electrode according to claim 7, characterized in that, The transition metal element includes one or more of Co, Fe, Ag, Cu, Au, and Ni.

9. The sensing electrode according to any one of claims 1 to 8, characterized in that, The thickness of the signal conversion layer is 1μm-2μm.

10. A method for preparing a sensing electrode, characterized in that, The method includes: A signal conversion layer is disposed on one side of the electrode layer; A biometric probe is fixed on the surface and / or inside the signal conversion layer on the side away from the electrode layer; After fixing the biometric probe, a surfactant is modified on the side of the signal conversion layer away from the electrode layer to obtain a sensing electrode.

11. The method according to claim 10, characterized in that, The surfactant is modified by immersing the side of the signal conversion layer away from the electrode layer in a surfactant solution.

12. The method according to claim 11, characterized in that, In the step of modifying the surfactant, immersion at 20°C-30°C; and / or, Immersion time is 20-120 minutes; and / or, The molar concentration of the surfactant solution is 0.1 mM-2 mM.

13. The method according to claim 10, characterized in that, Immobilize the biometric probe using the following steps: At least one side of the signal conversion layer away from the electrode layer is immersed in a first solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloric acid and N-hydroxysuccinimide for reaction; The second solution containing the biometric probe is coated onto the surface of the signal conversion layer after the reaction, and then incubated at 20°C-35°C.

14. The method according to claim 13, characterized in that, In the step of immobilizing the biometric probe, the molar concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in the first solution are each independently 5 mM-40 mM; and / or, The biometric probe includes one or more of D-type amino acid oxidase, LTB4 receptor, L-type amino acid oxidase, lactate dehydrogenase, and matrix metalloproteinase; and / or The reaction temperature is 20℃-35℃; and / or, The reaction time is 20-60 minutes; and / or, The incubation time is 5-30 minutes.

15. The method according to claim 10, characterized in that, The signal conversion layer is set up by the following steps: a dispersion of undoped or nitrogen-doped carbon-based nanomaterials is coated on one side of the electrode layer and then dried.

16. The method according to claim 15, characterized in that, In the step of setting the signal conversion layer, the concentration of the carbon-based nanomaterial dispersion is 0.1 mg / mL-5 mg / mL; and / or, The carbon-based nanomaterial further comprises nanoparticles containing transition metal elements, wherein the nanoparticles containing transition metal elements are suitable for promoting the decomposition of hydrogen peroxide; and / or, The carbon-based nanomaterials include one or more of carbon nanotubes, graphene, and transition metal carbide-transition metal nitride two-dimensional materials; and / or, The mass content of nitrogen element in the carbon-based nanomaterial is 1%-10%.

17. The method according to any one of claims 10 to 16, characterized in that... One or more of the following: The surfactant includes one or more of cationic surfactants and anionic surfactants; The sensing electrode is as described in any one of claims 1 to 9.

18. The use of the sensing electrode according to any one of claims 1 to 9 in the manufacture of a device for diagnosing oral diseases.

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