A cof-based electrochemical sensor for detecting hhv-8, preparation method and application

Abstract

This invention belongs to the field of virus detection, and specifically relates to an electrochemical sensor for detecting HHV-8 based on COF, its preparation method, and its application. The preparation method includes the following steps: dissolving TMACl and KTPBCl in a solvent at a 1:1 equimolar ratio, stirring the reaction, centrifuging to obtain a precipitate, and drying to obtain TMATPBCl; mixing PDA, TAPB, and TMATPBCl, adding them to DCE and a solvent, and allowing the mixture to stand to obtain a mixed solution; removing the COF membrane, rinsing it, and ultrasonically dispersing it to obtain a COF dispersion material; cutting carbon paper into 1cm × 1cm dimensions for later use; ultrasonically immersing the clip electrode sequentially in water, anhydrous ethanol, and water; after the clip electrode dries, removing the carbon paper, coating the surface of the carbon paper electrode with the COF dispersion material obtained in step S2, storing and drying it, and then adding HHV8 antibody to obtain a COF electrochemical immunosensor for detecting HHV8.

Description

Technical Field

[0001] This invention belongs to the field of virus detection, and in particular relates to an electrochemical sensor for detecting HHV-8 based on COF, its preparation method, and its application. Background Technology

[0002] Human herpesvirus 8 (HHV-8) is a species of herpesvirus gamma subfamily. This virus is the pathogen that causes Kaposi's sarcoma (KS), a carcinogenic disease that has a significant impact on immunocompromised individuals. Furthermore, HHV-8 is closely associated with a wide range of clinical diseases, and recent research has revealed a potential link between it and multicentric Castellman's disease (MCD), a life-threatening virus-induced b-lymphoproliferative disorder. This undoubtedly broadens its relevance in clinical medicine. Therefore, the detection of HHV-8 infection is both crucial and urgent. Currently, real-time quantitative PCR has become the mainstream method for detecting HHV-8 infection. This technology can accurately quantify the levels of viral DNA and transcription in blood, other body fluids, and organs. However, it is undeniable that some detection procedures remain complex, time-consuming, and have limited sensitivity. During the latent infection phase of HHV-8, the virus stably expresses its antigens, a characteristic that makes it a hallmark biomarker for monitoring latent HHV-8 infection. This discovery provides strong support for the early diagnosis and scientific surveillance of HHV-8-related diseases. Therefore, developing a novel, highly sensitive, rapid, and cost-effective detection method specifically for detecting antigens in HHV-8 is particularly urgent and important. Summary of the Invention

[0003] To address the problems and shortcomings of existing technologies, this invention provides a method for preparing a COF-based electrochemical sensor for detecting HHV-8, comprising the following steps:

[0004] S1: Dissolve TMACl and KTPBCl in solvent at a 1:1 equimolar ratio, stir and react, centrifuge to obtain precipitate, and dry to obtain TMATPBCl.

[0005] S2: PDA, TAPB and TMATPBCl obtained from S1 are mixed, added to DCE and solvent, and allowed to stand to obtain a mixture; the COF membrane is taken out, rinsed, and ultrasonically dispersed to obtain COF dispersion material;

[0006] The molar ratio of PDA, TAPB, and TMATPBCl is 1:1.5:1;

[0007] S3: Cut the carbon paper into 1cm×1cm sizes for later use. Sonicate the clip electrode in water, anhydrous ethanol, and water in sequence. After the clip electrode dries, remove the carbon paper and coat the COF dispersion material obtained in S2 onto the surface of the carbon paper electrode. After preservation and drying, add HHV8 antibody to obtain the COF electrochemical immunosensor for detecting HHV8.

[0008] Furthermore, the solvent mentioned in S1 is a mixture of methanol and water, wherein the volume ratio of methanol to water is 2:1.

[0009] Furthermore, the settling time in S2 is 24 hours, the settling temperature is 2-8°C, and anhydrous ethanol is used in the ultrasonic dispersion process.

[0010] Furthermore, the storage time in S3 is 4 hours, and the storage temperature is 4–8°C.

[0011] An electrochemical sensor for detecting HHV-8 based on COF.

[0012] An application of a COF-based electrochemical sensor for detecting HHV-8, wherein the COF-based electrochemical sensor for detecting HHV-8 is used in the preparation of a device for detecting human herpesvirus HHV-8.

[0013] Beneficial effects

[0014] This invention provides a method for fabricating an electrochemical sensor for HHV-8 detection based on COF (carbon paper). The invention successfully utilizes a hydrophilic TAPB-PDA COF membrane with high mechanical strength and highly conductive carbon paper. The porous structure of COF provides a high specific surface area for antibody loading, while the rapid electron transfer of the carbon paper electrode amplifies the signal. Together, these two components achieve ultrasensitive detection of the HHV-8 viral antigen. This achievement is attributed to the effective binding of the hydrophilic amino groups in the material to the carboxyl groups of the HHV-8 antibody. Our designed biosensor exhibits excellent performance indicators: a broad detection range covering from 4.97 × 10⁻⁶. -16 g / mL to 3.68×10 -13 It exhibits a linear range of g / mL and possesses extremely high detection sensitivity, with a detection limit as low as 0.142 × 10⁻⁶ g / mL. -16 g / mL (signal-to-noise ratio S / N = 3), which is far lower than the commonly used fluorescence quantitative PCR method and also lower than the detection limit of most existing electrochemical sensors (≥10 g / mL). -13 (g / mL). Furthermore, the molar ratio of DA, TAPB, and TMATPBCl is 1:1.5:1, which also makes this electrochemical sensor perform better than existing technologies.

[0015] Existing technologies use Fe3O4@COF to enhance magnetic separation capabilities, but their detection limit is 0.84 copies / mL (approximately 10). - 15 The limit of detection (LOD) was 0.142 g / mL and was dependent on AuNP modification; however, this study achieved a lower detection limit (0.142 × 10⁻⁶ g / mL) without the use of precious metals by utilizing the inherent hydrophilicity of TAPB-PDA COF and the synergistic effect of carbon paper electrode. -16 g / mL).

[0016] This invention provides a COF-based electrochemical sensor for detecting HHV-8, which also performs excellently in practical applications, accurately detecting the antigen in real viral samples. Specifically, in real viral samples, with an RSD of 3.07%, the recovery rate remains at a high level of 101.30%.

[0017] Furthermore, this invention marks the first application of COF biosensors to the detection of antigens in the HHV-8 virus. This innovation not only broadens the application scope of COF materials in the field of biosensing but also provides a powerful tool for the trace analysis of HHV-8 antigens, helping researchers to more effectively prevent and control public health events caused by HHV-8 infection. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0019] Figure 1 This is a flowchart of the HHV-8 detection reaction using an immune sensor according to Example 1 of the present invention;

[0020] Figure 2 This is a CV comparison diagram of the carbon paper electrode of Embodiment 1 of the present invention and a commonly used glassy carbon electrode;

[0021] Figure 3 This is a comparison image of the carbon paper electrode of Embodiment 1 of the present invention and a commonly used glassy carbon electrode using EIS.

[0022] Figure 4 The image shows the conductivity characterization (CV) diagram of the sensor fabrication process in Example 1 of this invention.

[0023] Figure 5 EIS diagrams characterizing the conductivity of the sensor fabrication process in Example 1 of this invention;

[0024] Figure 6 Linear graph of the detection of human herpesvirus type 8 (HHV-8) using a self-made immune sensor under optimized conditions for Example 1 of the present invention. Detailed Implementation

[0025] The following will refer to Embodiment 1 of the present invention and the appendix. Figures 1-6 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] S1: Take TMACl and KTPBCl in an equimolar ratio of 1:1 in a beaker containing a mixture of methanol and water, with a volume ratio of methanol to water of 2:1, and place the beaker in a constant temperature stirrer to stir the mixture thoroughly. After the reaction is complete, centrifuge the resulting solution to obtain a precipitate, and dry the precipitate to obtain TMATPBCl.

[0028] S2: Mix 3.2 mg PDA, 5.5 mg TAPB, and 8.3 mg TMATPBCl, then add the mixture to a solution of 3.125 mL LDCE and 3.125 mL (water + 0.2 mg TMACl + 0.25 μL concentrated hydrochloric acid). Let the mixture stand at 2 degrees Celsius for 24 h. Remove the COF membrane and rinse it with anhydrous ethanol. After rinsing, add 2 mL of anhydrous ethanol and ultrasonically disperse the mixture to obtain the COF dispersion material.

[0029] S3: Cut the carbon paper to a size of 1cm × 1cm for later use. The introduction of carbon paper electrode (CPE) replaces the traditional glassy carbon electrode. Its porous structure synergistically enhances conductivity with COF and reduces background interference. Ultrasonically clean and dry the clip electrode. After the clip electrode is dry, clamp the cut carbon paper, take 50 μL of the obtained COF dispersion material, and drop it onto the surface of the carbon paper electrode. Place it in a 4°C refrigerator for about 4 hours to form a thin film. After the COF material dries, drop in 20 μL of HHV-8 antibody. The concentration of HHV-8 antibody is 1 × 10⁻⁶. -4 g / L, to obtain the COF detection HHV8 electrochemical immunosensor.

[0030] To apply the COF detection HHV8 (herpesvirus) electrochemical sensor obtained in Example 1, firstly, potassium dihydrogen phosphate was weighed into a beaker, and deionized water was added to the beaker to dissolve and bring the volume to prepare a 0.1 mol / L PBS buffer solution A. Secondly, potassium dihydrogen phosphate was weighed into another beaker, and deionized water was added to the beaker to dissolve and bring the volume to prepare a 0.1 mol / L PBS buffer solution B.

[0031] Mix solutions A and B to prepare PBS buffer solutions with pH = 6.50 and 7.00 (and add KCl, KCl concentration = 0.1 mol / L); transfer 10 μL of HHV-8 antigen (concentration = 1 g / L) into a beaker and make up to volume to prepare a concentration of 1 × 10⁻⁶. -4 A standard solution for detection was prepared by serially diluting a g / L HHV8 (herpesvirus) solution with ultrapure water. Potassium ferricyanide was dissolved in ultrapure water to prepare a 6 mM potassium ferricyanide solution. A three-electrode system was used as the base solution with a pH 6.50 PBS buffer solution and the potassium ferricyanide solution. The operating conditions were set as follows: initial voltage of CV detection was -0.8 V, peak voltage was 1.3 V, sampling interval was 0.001 V, scan rate was 0.1 V / s, and sensitivity was 10 μA / V. The three-electrode system consisted of a carbon paper electrode modified with COF and antibody sequentially as the working electrode, a platinum electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. 4 mL of PBS (pH 7.2) was transferred to the electrolytic cell, and 1 mL of potassium ferricyanide solution (6 mM) was added. The electrochemical properties of the electrode modification process were characterized by cyclic voltammetry.

[0032] 4 mL of PBS was transferred to the electrolytic cell, and 1 mL of potassium ferricyanide solution (6 mM) was added. The pH of the PBS was optimized using cyclic voltammetry, and the optimal pH was 6.5. 4 mL of PBS (pH = 6.5) was transferred to the electrolytic cell, and 1 mL of potassium ferricyanide solution (6 mM) was added. Under the optimal pH, the optimal settling time of the electrode material in the solution was optimized using cyclic voltammetry to be 10 min. 4 mL of PBS (pH = 6.5) was transferred to the electrolytic cell, and 1 mL of potassium ferricyanide solution was added. Under optimal pH and optimal settling time conditions, the ratio of the modified electrode material to HHV8Ab (herpes antibody) was optimized using cyclic voltammetry. The optimal ratio was COF:HHVAb = 5:2. 4 mL of PBS (pH = 6.5) was transferred to the electrolytic cell, and 1 mL of potassium ferricyanide solution (6 mM) was added. Under optimal pH, optimal settling time, and optimal material-to-antibody ratio conditions, the detection temperature was optimized using cyclic voltammetry. The optimal temperature was 30 °C.

[0033] Transfer 4 mL of PBS (pH = 6.5) to the electrolytic cell, add 1 mL of potassium ferricyanide solution (6 mM), and then sequentially add HHV8 (herpesvirus) standard solution (10 mM) starting from the lowest concentration. -16 g / mL and 10 -13 g / mL), each concentration was measured 4 times (except for the first 5 μL of each concentration, the remaining three were 10 μL each), and each measurement was accumulated to the bottom solution. The linearity was found by differential pulse voltammetry.

[0034] Combination Figure 1 The reaction flowchart for the HHV-8 detection of the immunosensor of the present invention can be obtained. Specifically, the prepared COF membrane is ultrasonically dispersed evenly in anhydrous ethanol to obtain COF material. An appropriate amount of material is dropped onto a carbon paper electrode. After the COF material dries and adheres to the carbon paper, an appropriate amount of HHV-8 antibody solution is dropped onto the modified COF carbon paper. After the antibody dries and adheres to the electrode, an HHV-8Ab / COF / CPE electrochemical immunosensor is formed.

[0035] pass Figures 2-3 It can be seen that by adding 4 mL of PBS (pH = 6.5) and 1 mL of 6 mM potassium ferricyanide solution to the electrolytic cell, and measuring the oxidation and reduction peak potentials of bare CPE and bare GCE by CV, the results can be obtained. Figure 2 We can see that the electrochemical response of CPE is much greater than that of GCE, proving that the conductivity of carbon paper electrode is much greater than that of glassy carbon electrode; 4 mL of PBS (pH = 6.5) and 1 mL of 6 mM potassium ferricyanide solution were added to the electrolytic cell, and the charge transfer resistance of bare CPE and bare GCE was measured by EIS. Figure 3 It can be seen that the resistance of carbon paper is lower than that of glassy carbon electrode. Characterization by CV and EIS shows that the conductivity of carbon paper is much higher than that of glassy carbon electrode.

[0036] pass Figures 4-5 It can be seen that adding 4 mL of PBS (pH=6.5) and 1 mL of 6 mM potassium ferricyanide solution to the electrolytic cell, and measuring the oxidation and reduction peak potentials of bare CPE, (COF / CPE), and (HHV-8Ab / COF / CPE) using colorimetry (CV), revealed a significant oxidation peak in the bare CPE. This is because the carbon paper itself has a large number of organic functional groups, resulting in a redox reaction. When COF was modified, the oxidation peak decreased significantly because the amino groups on COF bound to the functional groups on the carbon paper. Further modification with antibody resulted in a slight increase in current, because the antibody bound to the amino groups on COF, releasing a large number of functional groups from the carbon paper and enhancing the redox reaction. Adding 4 mL of PBS (pH=6.5) and 5 μL of 10 mM potassium ferricyanide solution to the electrolytic cell further enhanced the redox reaction. -4HHV-8 antigen solution and 1 mL of 6 mM potassium ferricyanide solution were added. The oxidation and reduction peak potentials of HHV-8Ab / COF / CPE were measured by CV. After the addition of the antigen solution, the antigen and antibody specifically bound, and a pair of distinct redox peaks appeared at other potentials. 4 mL of PBS (pH = 6.5) and 1 mL of 6 mM potassium ferricyanide solution were added to the electrolytic cell. The charge transfer resistance of bare CPE, (COF / CPE), and (HHV-8Ab / COF / CPE) was measured by EIS. -4 The charge transfer resistance of HHV-8Ab / COF / CPE was measured using an HHV-8 antigen solution and 1 mL of 6 mM potassium ferricyanide by electrochemical impedance spectroscopy (EIS). Electrochemical cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to measure the conductivity of the electrode during preparation. Both methods demonstrated that the immunosensor possesses excellent electrosensing detection performance.

[0037] pass Figure 6 It can be seen that 4 mL of PBS (pH=6.5) was transferred into the electrolytic cell, and 1 mL of potassium ferricyanide solution (6 mM) was added. Starting from the lowest concentration, HHV-8 virus antigen standard solution (10 mM) was then added sequentially. -16 g / mL and 10 -13 For each concentration (g / mL), 5 μL was taken sequentially, seven times, with each measurement taken and accumulated to the bottom solution. The oxidation peak current was detected using the DPV method. As shown in the figure, a good linear relationship was observed between the oxidation peak current and the concentration. At concentrations around 4.97 × 10⁻⁶ g / mL... -16 g / mL~3.68×10 -13 The linear regression equation for g / mL is y = 57.36894 + 1.99854x, and the correlation coefficient is R. 2 =0.99779. The limit of detection for the lowest concentration is 0.142 × 10⁻⁶. -16 g / mL.

[0038] The invention provides a method for preparing a COF-based electrochemical sensor for HHV-8 detection. The innovations include: controlling COF crystallinity and optimizing the pore structure by allowing the COF to stand at 2-8°C for 24 hours, thus significantly increasing antibody loading compared to traditional room-temperature synthesis; and using anhydrous ethanol for ultrasonic dispersion instead of other organic reagents and water as solvents to avoid COF pore blockage and ensure sufficient exposure of antibody modification sites. The introduction of a carbon paper electrode (CPE) replaces the traditional glassy carbon electrode; its porous structure synergistically enhances conductivity with COF and reduces background interference. Because the TAPB-PDA COF has hydrophilic amino groups that can effectively bind to the carboxyl groups on the antibody, the antibody is effectively immobilized on the COF membrane, significantly improving the sensor's specificity.

Claims

1. A method for preparing an electrochemical sensor for detecting HHV-8 based on COF, characterized in that, Includes the following steps: S1: Dissolve TMACl and KTPBCl in a solvent at a 1:1 equimolar ratio, stir and react, centrifuge to obtain a precipitate, and dry to obtain TMATPBCl; The solvent mentioned in S1 is a mixed solution of methanol and water, and the volume ratio of methanol to water is 2:

1. S2: PDA, TAPB and TMATPBCl obtained in S1 are mixed and added to DCE and solvent. The mixture is allowed to stand for 24 h at 2~8 ℃ to obtain a COF membrane. The COF membrane is taken out and rinsed with anhydrous ethanol. After rinsing, anhydrous ethanol is added and ultrasonically dispersed to obtain COF dispersion material. The solvent mentioned in S2 is a mixture of water, TMACl and concentrated hydrochloric acid. The molar ratio of PDA, TAPB, and TMATPBCl is 1:1.5:1; S3: Cut the carbon paper into 1 cm × 1 cm sizes for later use. Sonicate the clip electrode in water, anhydrous ethanol, and water in sequence. After the clip electrode dries, remove the carbon paper and coat the COF dispersion material obtained in S2 onto the surface of the carbon paper electrode. After preservation and drying, add HHV-8 antibody to obtain the COF detection HHV-8 electrochemical immunosensor.

2. The method for preparing a COF-based electrochemical sensor for detecting HHV-8 according to claim 1, characterized in that, The storage time in S3 is 4 hours, and the storage temperature is 4~8℃.

3. An electrochemical sensor for detecting HHV-8 based on COF, obtained by any one of the preparation methods according to claims 1 to 2.

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