A method for detecting hepatitis b virus based on a magnetic covalent organic framework material to construct a biosensor

Abstract

The present application relates to a kind of based on magnetic covalent organic framework material construction biological sensor method for detecting hepatitis B virus, the method includes Au@ZnFe2O4@COF Nanocomposite and intermediate product material: ZnFe2O4@COF.The outstanding creativity of the present application: 1.firstly using magnetic adsorption biological sensor detects HBV.2.design capture probe, signal probe and can specifically recognize the characteristic of hepatitis B virus DNA instead of using antibody to capture hepatitis B virus and utilize CuO laccase activity determination virus concentration sandwich type photochemical immunosensor.

Description

Technical Field

[0001] This invention belongs to the field of functional materials and biosensing technology, specifically relating to a method for preparing a magnetic adsorption colorimetric immunoassay biosensor and detecting hepatitis B virus. Background Technology

[0002] Hepatitis B virus (HBV) is one of the known viruses that can cause viral hepatitis. There are over 20 million people infected with HBV worldwide, of whom 350 million are chronic HBV carriers. Chronic carriers have a higher risk of complications, including chronic hepatitis and cirrhosis. Serological markers are commonly used as diagnostic or prognostic indicators for acute or chronic HBV infection. Studies have reported that serum HBV DNA testing has prognostic value for both acute and chronic HBV infection.

[0003] Traditional detection methods include enzyme-linked immunosorbent assay (ELISA), real-time RT-PCR, and microparticle chemiluminescence immunoassay. ELISA directly uses antibodies or antigens to capture hepatitis B surface antigen and surface antibody, e antigen and e antibody, and core antigen and antibody, and then performs colorimetric detection using antibodies carrying probes. Real-time RT-PCR involves extracting viral RNA, designing and synthesizing primers and probes, and performing quantitative PCR using fluorescence quantification. ELISA is characterized by its simplicity, high sensitivity, and rapid detection; however, it suffers from drawbacks such as significant differences in sensitivity and specificity among various reagents, high cost, and complex reagent preparation. Real-time RT-PCR allows for targeted detection of hepatitis B virus, but its detection system requires strict technical specifications and is also costly. Summary of the Invention

[0004] This invention aims to address the aforementioned problems and deficiencies by providing a method for detecting hepatitis B virus (HBV) using a biosensor constructed based on a magnetic covalent organic framework material. The objective of this invention is to assemble HBV-specific recognition probes onto gold nanoparticles and CuO, forming a capture probe and signal probe complex capable of capturing and immobilizing HBV particles in a sandwich structure. The magnetic composite covalent organic framework nanomaterial loaded with the capture probes is then used to identify the virus in the system. Simultaneously, the laccase activity of the CuO nanomaterial causes 4-AP and 2,4-DP to turn red, exhibiting characteristic peaks at 510 nm. Finally, a colorimetric reaction is performed in 400 μL of a 50 mmol / L, pH 6.8 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, resulting in a biosensor capable of rapidly and effectively detecting the concentration of HBV in a sample.

[0005] In this invention, the magnetic composite covalent organic framework nanomaterial (AuNPs@ZnFe2O4@COF), as a new generation of composite nanomaterials, possesses excellent adsorption properties, can be highly dispersed in water, and can bind biomolecules with recognition functions such as probes, antibodies, aptamers, and peptides. Simultaneously, the CuO nanomaterial exhibits good laccase activity, enabling the signal molecules 4-aminopyridine (4-AP) and 2-(2,4-dichlorophenoxy)propionic acid (2,4-DP) to exhibit a red color with a characteristic peak at 510 nm.

[0006] Based on the whole genome analysis of hepatitis B virus (HBV), we designed a signal probe targeting the HBV core region and modified it with thiol groups at the 3' end. The thiol groups on the probe can self-assemble with gold nanoparticles to form gold-sulfur bonds (Au-S), or they can bond with CuO. Combined with the probe's specific ability to recognize HBV, and through the synergistic effect of magnetic composite covalent organic framework nanomaterials, we constructed an HBV biosensor with advantages such as high sensitivity, a wide detection range, fast detection speed, low detection limit, and ease of operation.

[0007] The present invention is implemented using the following technical solution.

[0008] 1. ZnFe2O4@COF was synthesized and characterized according to previous literature reports.

[0009] (1) To synthesize ZnFe₂O₄, ferric chloride hexahydrate and anhydrous zinc chloride were dissolved in ethylene glycol to form a transparent solution. Sodium acetate and polyethylene glycol 20000 were then added. The mixture was stirred vigorously for 0.5–2 h and then transferred to a stainless steel autoclave (50 mL capacity). The reaction was carried out at 200 °C for 6–10 h, and then allowed to cool naturally to room temperature after the reaction was terminated. The product was washed several times with ethanol and water, and the black product was dried under vacuum at 60 °C for 4–8 h. Samples were collected for subsequent experiments and characterization.

[0010] (2) Further synthesis of ZnFe2O4@COF: 1,3,5-tris(4-aminophenyl)benzene (TAPB), terephthalaldehyde (TPA), and ZnFe2O4 were dissolved in dimethyl sulfoxide. The mixture was ultrasonically dispersed, and anhydrous acetic acid was added under ultrasonication. The reaction was carried out at room temperature. ZnFe2O4@COF was collected using a magnet, washed three times with tetrahydrofuran and methanol, and dried under vacuum at 60 °C. The collected samples were used for subsequent experiments and characterization.

[0011] Characterization methods include: infrared spectroscopy, scanning electron microscopy (SEM) images, transmission electron microscopy (TEM) images, EDS elemental analysis, zeta potential, XRD powder X-ray diffraction patterns, and XPS photoelectron spectroscopy.

[0012] 2. Synthesize and characterize Au@ZnFe2O4@COF nanocomposites.

[0013] (1) To synthesize AuNPs, add 0.5-2 mL of HAuCl4 solution with a concentration of 10-30 mmol / L to 50-200 mL of deionized water, heat and stir at 80-120℃ for 2-10 min, add 5-20 mL of sodium citrate solution with a concentration of 10-20 mmol / L, reflux for 20-40 min until the solution turns wine red, cool naturally to room temperature, and store at 4℃.

[0014] (2) The ZnFe2O4@COF powder sample was prepared into a ZnFe2O4@COF solution with a concentration of 1-5 mg / mL by adding water. 1-5 mL of ZnFe2O4@COF solution was added dropwise to 2-20 mL of gold nanoparticle (AuNPs) solution with a concentration of 0.0001-0.0005 mol / L. The mixture was stirred at 4℃ for 12 h, centrifuged and washed to obtain Au@ZnFe2O4@COF nanocomposite material.

[0015] Characterization methods include: transmission electron microscopy (TEM) images, EDS elemental analysis, zeta potential, XRD powder X-ray diffraction patterns, and XPS photoelectron spectroscopy.

[0016] 3. Synthesis of CP@Au@ZnFe2O4@COF nanocomposites.

[0017] The hepatitis B virus capture probe (CP) was added to the Au@ZnFe2O4@COF nanocomposite solution, shaken at 4℃ for 12h, blocked with hexamethylenetetramine (HT), and washed with magnetic adsorption for later use.

[0018] 4. Synthesis of CuO nanomaterials.

[0019] Polyvinylpyrrolidone (PVP) and CuCl2·H2O were fully dissolved in deionized water, then NaOH was added to the solution, and finally H2O2 was added. The mixture was stirred for 30 min, washed by centrifugation with deionized water, and dried under vacuum at 60 °C.

[0020] 5. Synthesis of LP@CuO

[0021] The hepatitis B virus signal probe (LP) was added to a CuO nanomaterial solution, shaken at room temperature for 8 hours, blocked with hexamethylenetetramine, and then centrifuged and washed for later use.

[0022] 6. Investigate the conditions for the colorimetric reaction of the system.

[0023] (1) Optimal reaction pH value

[0024] Prepare a 50 mmol / L MES solution with pH = 3.0-9.0. Add CP@Au@ZnFe2O4@COF to a 2 mL centrifuge tube, and add 10-10 HBV DNA target compound was added at mol / L, incubated at room temperature, and washed three times with magnetic adsorption. LP@CuO was added, and incubation was repeated for 15 min at room temperature, followed by three more magnetic adsorption washes. Finally, 400 μL of 50 mmol / L MES buffer solution at different pH values ​​was added, along with 4-AP and 2,4-DP, and the mixture was incubated at 60 °C for 15 min. The supernatant was then aspirated, and the absorption peak at 510 nm was measured. The results showed that the MES buffer solution at pH 6.8 exhibited the highest absorption peak, indicating that these were the optimal conditions.

[0025] (2) Optimal reaction temperature

[0026] Add CP@Au@ZnFe2O4@COF to a 2mL centrifuge tube, then add 10 -10 mol / L HBV DNA target was incubated at room temperature for 15 min, followed by three magnetic washes. LP@CuO was then added, and the mixture was incubated at room temperature for 15 min, followed by three magnetic washes. Finally, MES buffer solutions of different pH values ​​were added, along with 4-AP and 2,4-DP, and the mixture was incubated at 30℃-70℃ for 15 min each. The supernatant was then measured to determine the absorption peak at 510 nm. The results showed that the highest absorption peak did not change significantly after 60℃, therefore 60℃ is the optimal temperature.

[0027] (3) Optimal reaction time of CP

[0028] Add CP@Au@ZnFe2O4@COF to a 2mL centrifuge tube, then add 10 -10 mol / L HBV DNA target compound was incubated at room temperature for 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, followed by three magnetic washes. LP@CuO was then added, and incubation at room temperature was repeated for 15 min, followed by three magnetic washes. Finally, MES buffer, along with 4-AP and 2,4-DP, was added, and the mixture was incubated at 60℃ for 15 min. The results showed that the highest absorption peak was observed after 15 min of incubation at room temperature; therefore, 15 min of incubation with CP@Au@ZnFe2O4@COF is the optimal incubation time.

[0029] (4) LP Optimal Reaction Time

[0030] Add CP@Au@ZnFe2O4@COF to a 2mL centrifuge tube, then add 10 -10mol / L HBV DNA target was incubated at room temperature for 15 min, followed by three magnetic washes. LP@CuO was then added, and incubation times were increased to 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min at room temperature, with three magnetic washes. Finally, MES buffer, along with 4-AP and 2,4-DP, was added, and the mixture was incubated at 60 °C for 15 min. The detection results showed that the highest absorption peak was observed after 15 min of incubation at room temperature, therefore, 15 min of LP@CuO incubation is the optimal incubation time.

[0031] 7. Procedure for detecting hepatitis B virus.

[0032] (1) Add 50-200 μL of 1 μmol / L CP@Au@ZnFe2O4@COF to a 2 mL centrifuge tube, and add 50-200 μL of 10 -10 Incubate 1 mol / L HBV DNA target at room temperature for 15 min. Wash three times with PBST solution using magnetic adsorption.

[0033] (2) Add 50-200 μL of 1 μmol / L LP@CuO and incubate at room temperature for 15 min. Wash three times with PBST magnetic adsorption.

[0034] (3) Add 200-800 μL of 50 mmol / L pH 6.8 MES buffer solution, and simultaneously add 50-200 μL of 1 mg / mL 4-AP and 50-200 μL of 1 mg / mL 2,4-DP. Incubate at 60℃ for 15 min.

[0035] (4) Take the supernatant and use a UV spectrophotometer to measure the absorption peak at 400-700 nm, and record the highest absorption peak at 510 nm. Record and plot the working curve.

[0036] In addition to the above-mentioned technical solutions, the molecules used in this invention to identify hepatitis B virus can also be other biomolecules, such as hepatitis B virus antibodies and aptamers. The ZnFe2O4@COF in this invention can also be replaced by other covalent organic framework materials. The CuO in this invention can also be replaced by other nanomaterials with laccase activity.

[0037] The beneficial effects of this invention are as follows: 1. Transmission electron microscopy reveals that ZnFe2O4 exhibits uniform and dispersed spherical shapes with a size of approximately 100-200 nm. After ZnFe2O4 is composited with COF, a thin COF shell, approximately 30 nm thick, is clearly observed on the ZnFe2O4 surface, surrounding the ZnFe2O4 magnetic core. These results indicate that COF has been successfully composited onto the ZnFe2O4 surface; AuNPs particles are uniformly and evenly distributed on ZnFe2O4@COF. These results prove that the Au@ZnFe2O4@COF material has been successfully synthesized.

[0038] 2. X-ray diffraction (XRD) results showed that the prepared ZnFe2O4 exhibited a distinct spinel structure at 62.5°, 56.8°, 53.2°, 43.0°, 35.4°, and 30.0°. o The presence of diffraction peaks, and the fact that these peaks did not shift significantly after being loaded with COF, indicates that the polymerization of COF on the ZnFe2O4 surface did not change the crystal structure of ZnFe2O4, and also demonstrates the successful synthesis of ZnFe2O4@COF.

[0039] 3. Infrared spectrum displayed at 3430cm -1 Each peak exhibits a relatively large stretching vibration peak, which is attributed to the stretching vibration of the hydroxyl group (-OH) in the residual water. 2920 cm⁻¹ -1 and 2850cm -1 The absorption peak at 1630 cm⁻¹ is related to the CH bond; -1 and 1402cm -1 The absorption peaks at 550 cm⁻¹ are due to the stretching vibrations of C=O and CO. These peaks reveal the infrared absorption characteristics of the carboxyl groups on the ZnFe₂O₄MNPs surface. -1 The absorption peaks around the left and right are attributed to the Zn-O-Fe vibration. Compared to ZnFe2O4, the infrared spectrum of ZnFe2O4@COF shows two absorption peaks unique to COF, located at 1700 cm⁻¹. -1 and 1489cm -1 The positions correspond to the CN bond deformation vibration of the amide bond and the C=C stretching vibration of the benzene ring, respectively, which indicates the formation of the COF shell in the acetal reaction.

[0040] 4. The Zeta potential test results show that the average Zeta potential of ZnFe2O4 is 23.83 mV, the average Zeta potential of COF is 13.21 mV, and the Zeta potential of AuNPs particles is known to be negative. The average Zeta potential of Au@ZnFe2O4@COF is -32.78 mV, indicating that AuNPs have been successfully loaded onto ZnFe2O4@COF.

[0041] 5. The photoelectron spectroscopy (XPS) results also clearly show the Zn and Fe elements in ZnFe2O4 and the C and N elements in ZnFe2O4@COF, further proving the successful preparation of ZnFe2O4 and ZnFe2O4@COF.

[0042] 6. The TEM image shows that CuO consists of spherical particles with a diameter of about 10 nm and good dispersion.

[0043] 7. As the concentration of hepatitis B virus increases, the optical signal detected by the ultraviolet spectrophotometer strengthens, especially at 10... -18 -10 -8 A good linear relationship was observed within the hepatitis B virus concentration range of mol / mL. Based on the absorption peak change at 510 nm using a UV spectrophotometer, the absorption peak value for each concentration was taken for data analysis. A standard curve was constructed with the logarithm of virus concentration as the X-axis and absorbance value as the Y-axis. The equation of the standard curve was Y = 0.0461log[C] + 1.1769(R). 2 =0.9931)

[0044] 8. Without the addition of ASFV, the sensor detected bovine serum albumin (BSA), lysozyme, Escherichia coli, Staphylococcus aureus, and Na+. + Mg 2+ K + Ca 2+ Zn 2+ Neither HCV nor DNA showed a signal response. However, when HBV standard was added to the mixture (HBV+Mix), the sensor showed a significant signal response, indicating that the sensor can specifically detect HBV.

[0045] This paper successfully prepared Au@ZnFe2O4@COF nanocomposites and CuO nanomaterials. The Au@ZnFe2O4@COF nanocomposites can effectively bind to a hepatitis B virus (HBV) capture probe and specifically recognize HBV DNA. With the assistance of LP@CuO, the signal is enriched and amplified. Finally, the highest absorption peak was measured at 510 nm using the laccase activity of CuO. Simultaneously, CP and LP can specifically recognize HBV and form a sandwich structure with the target. The constructed biosensor exhibits specific recognition ability for HBV, and the linear detection range of the prepared sensor is 10 nm. -18 -10 -8 mol / L.

[0046] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0047] Figure 1 This is a schematic diagram illustrating the principle of the biosensor for detecting hepatitis B virus in this invention.

[0048] Figure 2 TEM images of the substances related to this invention:

[0049] (A)ZnFe2O 4； TEM images of (B) ZnFe2O4@COF; (C) Au@ZnFe2O4@COF.

[0050] Figure 3 The images shown are (A) XRD patterns and (B) FT-IR patterns of some of the materials in this invention.

[0051] Figure 4 The following is a graph showing the Zeta potential results of Au@ZnFe2O4@COF in this invention (A):

[0052] (a) ZnFe2O4; (b) COF; (c) Au@ZnFe2O4@COF;

[0053] (B) XPS plot of ZnFe2O4; (C) XPS plot of ZnFe2O4@COF; (D) XPS plot of N element in ZnFe2O4@COF.

[0054] Figure 5 This is a TEM image of the CuO produced in this invention.

[0055] Figure 6 This is a diagram showing the results of the condition optimization for this invention.

[0056] (A) Results of optimal incubation time for CP; (B) Results of optimal incubation time for LP;

[0057] (C) Result of the optimal pH value of the color development reaction buffer; (D) Result of the optimal color development time.

[0058] Figure 7 The linear detection range diagram of the sensor of the present invention: (A) the relationship between ultraviolet absorption light intensity and target concentration; (B) the linear relationship between ultraviolet absorption light change value and target concentration.

[0059] Figure 8 The specific detection results of the sensor of this invention are shown in the figure. Detailed Implementation

[0060] To further disclose, and not limit, the present invention, the following detailed description is provided in conjunction with embodiments.

[0061] All chemical reagents and solvents used in the examples were of analytical grade; all probes used were obtained by commercial methods; and the instrument used for photochemical detection was an ultraviolet spectrophotometer.

[0062] Experimental Example 1: The method for preparing and characterizing ZnFe2O4@COF is as follows:

[0063] (1) Preparation of ZnFe2O4 nanoclusters: Ferric chloride hexahydrate (1.35 g) and anhydrous zinc chloride (0.34 g) were dissolved in ethylene glycol (40 mL) to form a transparent solution. Sodium acetate (3.6 g) and polyethylene glycol 20000 (1.0 g) were then added. After vigorous stirring for 0.5 h, the mixture was transferred to a stainless steel autoclave (50 mL capacity) and reacted at 200 °C for 8 h. After the reaction was terminated, the mixture was allowed to cool naturally to room temperature. The product was washed several times with ethanol and water, and the black product was dried under vacuum at 60 °C for 6 h. Samples were collected for subsequent experiments and characterization.

[0064] (2) Further synthesis of ZnFe2O4@COF: 1,3,5-tris(4-aminophenyl)benzene (TAPB, 0.106 g), terephthalaldehyde (TPA, 0.06 g), and ZnFe2O4 (0.15 g) were dissolved in 50 mL of dimethyl sulfoxide. The mixture was ultrasonically dispersed for 5 min, and then 1.5 mL of anhydrous acetic acid was added under ultrasonication. The reaction was carried out at room temperature for 15 min. ZnFe2O4@COF was collected using a magnet, washed three times with tetrahydrofuran and methanol, and dried under vacuum at 60 °C. The collected samples were used for subsequent experiments and characterization.

[0065] Experimental Example 2: Methods for preparing and characterizing Au@ZnFe2O4@COF nanocomposites:

[0066] (1) Preparation of AuNPs: Under stirring conditions, 1.07 mL of HAuCl4 solution with a concentration of 23.46 mmol / L was added to 100 mL of boiling deionized water, heated and stirred at 110 °C for 5 min, and 10 mL of sodium citrate solution with a concentration of 14.53 mmol / L was slowly added dropwise. The solution was refluxed for 30 min until it turned wine red. The solution was stirred at room temperature for 15 min and stored at 4 °C.

[0067] (2) ZnFe2O4@COF powder was dispersed in deionized water and sonicated for 2 hours. Then, a suspension of ZnFe2O4@COF was obtained by centrifugation to remove larger particles. After centrifugation, the suspension was freeze-dried for 12 hours and stored at 4°C for later use.

[0068] (3) The ZnFe2O4@COF obtained was prepared into a ZnFe2O4@COF solution with a concentration of 1 mg / mL by adding water. 1 mL of ZnFe2O4@COF solution was added dropwise to 10 mL of gold nanoparticle solution with a concentration of 0.0002 mol / L. The mixture was stirred at 4℃ for 12 h, centrifuged and washed to obtain Au@ZnFe2O4@COF nanocomposite material.

[0069] Experimental Example 3: Synthesis of CP@Au@ZnFe2O4@COF nanocomposite material.

[0070] 100 μL of 1 μmol / L hepatitis B virus capture probe (CP) was added to 1 mL of 1 mg / mL Au@ZnFe2O4@COF nanocomposite solution, shaken at room temperature for 12 h, then 100 μL of 50 mmol / L hexamethylenetetramine was added for blocking for 1 h, and the mixture was washed with magnetic adsorption for later use.

[0071] Experimental Example 4: Synthesis of CuO nanomaterials.

[0072] Dissolve 0.5 g of polyvinylpyrrolidone (PVP) and 0.01 g of CuCl2·H2O in 5 mL of deionized water. Then add 5 mL of 0.02 M NaOH to the solution. Finally, add 0.1 mL of 30% H2O2, stir for 30 min, wash with deionized water by centrifugation, and dry under vacuum at 60 °C.

[0073] Experimental Example 5: Synthesis of LP@CuO

[0074] Add 100 μL of 1 μmol / L hepatitis B virus signal probe (LP) to 1 mL of 1 mg / mL CuO nanomaterial solution, shake at room temperature for 8 h, then add another 100 μL, shake at room temperature for 8 h, add 100 μL of 50 mmol / L hexamethylenetetramine to block for 1 h, centrifuge and wash for later use.

[0075] Experiment Example 6: Investigating the conditions for the colorimetric reaction of the system.

[0076] (1) Optimal reaction pH value

[0077] Prepare a 50 mmol / L MES solution with pH = 3.0-9.0. Add 100 μL of 1 μmol / L CP@Au@ZnFe2O4@COF to a 2 mL centrifuge tube, then add 50 μL of 10... -101 mol / L HBV DNA target was added and incubated at room temperature for 15 min, followed by three magnetic washes. Then, 100 μL of 1 μmol / L LP@CuO was added, and the mixture was incubated at room temperature for 15 min, followed by three magnetic washes. Finally, 400 μL of 50 mmol / L MES buffer solution at different pH values ​​was added, along with 50 μL of 1 mg / mL 4-AP and 50 μL of 1 mg / mL 2,4-DP. The mixture was incubated at 60 °C for 15 min. The supernatant was then measured to determine the absorption peak at 510 nm. The results showed that the MES buffer solution at pH 6.8 exhibited the highest absorption peak, indicating that these were the optimal conditions.

[0078] (2) Temperature

[0079] Add 100 μl of 1 μmol / L CP@Au@ZnFe2O4@COF to a 2 mL centrifuge tube, then add 50 μl of 10 -10 1 mol / L HBV DNA target compound was incubated at room temperature for 15 min, followed by three magnetic washes. Then, 100 μL of 1 μmol / L LP@CuO was added, and the mixture was incubated at room temperature for 15 min, followed by three magnetic washes. Finally, 400 μL of 50 mmol / L MES buffer solution at different pH values ​​was added, along with 50 μL of 1 mg / mL 4-AP and 50 μL of 1 mg / mL 2,4-DP. The mixture was then incubated at 30℃-70℃ for 15 min. The supernatant was then measured to determine the absorption peak at 510 nm. The results showed that the highest absorption peak did not change significantly after 60℃, therefore 60℃ is the optimal temperature.

[0080] (3) Optimal reaction time of CP

[0081] Add 100 μL of 1 μmol / L CP@Au@ZnFe2O4@COF to a 2 mL centrifuge tube, then add 50 μL of 10 -10 1 mol / L HBV DNA target compound was incubated at room temperature for 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, followed by three magnetic washes. Then, 100 μL of 1 μmol / L LP@CuO was added, and incubation was repeated at room temperature for 15 min, followed by three magnetic washes. Finally, 400 μL of 50 mmol / L MES buffer (pH 6.8) was added, along with 50 μL of 1 mg / mL 4-AP and 50 μL of 1 mg / mL 2,4-DP, and the mixture was incubated at 60 °C for 15 min. The results showed that the highest absorption peak was observed after 15 min of incubation at room temperature. Therefore, 15 min of incubation with CP@Au@ZnFe2O4@COF is the optimal incubation time.

[0082] (4) LP Optimal Reaction Time

[0083] Add 100 μL of 1 μmol / L CP@Au@ZnFe2O4@COF to a 2 mL centrifuge tube, then add 50 μL of 10 -10 1 mol / L HBV DNA target was incubated at room temperature for 15 min, followed by three magnetic washes. Then, 100 μL of 1 μmol / L LP@CuO was added, and incubation was continued at room temperature for 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, followed by three magnetic washes. Finally, 400 μL of 50 mmol / L MES buffer (pH 6.8) was added, along with 50 μL of 1 mg / mL 4-AP and 50 μL of 1 mg / mL 2,4-DP. The mixture was then incubated at 60 °C for 15 min. The results showed that the highest absorption peak was observed after 45 min of incubation at room temperature, indicating that 15 min of LP@CuO incubation was the optimal incubation time.

[0084]

[0085] The outstanding inventiveness of this invention:

[0086] 1. First time using a magnetic adsorption biosensor to detect HBV.

[0087] 2. A sandwich photochemical immunosensor was designed with capture probes and signal probes that can specifically recognize hepatitis B virus DNA, replacing the method of using antibodies to capture hepatitis B virus and using CuO laccase activity to determine virus concentration.

[0088] The above descriptions are merely some specific embodiments of the present invention. Commonly known details or common knowledge in the solutions are not described in detail here (including but not limited to abbreviations, acronyms, and units conventionally used in the art). It should be noted that the above embodiments do not limit the present invention in any way. For those skilled in the art, any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting hepatitis B virus using a biosensor constructed based on a magnetic covalent organic framework material, characterized in that... It lies in, including: Step 1) Synthesize ZnFe2O4@COF and characterize it; Step 2) Synthesize and characterize Au@ZnFe2O4@COF nanocomposites; Step 3) Synthesize CP@Au@ZnFe2O4@COF nanocomposite material; specifically, step 3) involves adding the hepatitis B virus capture probe CP to... Add the Au@ZnFe2O4@COF nanocomposite solution, shake at 4℃ for 12h, add hexamethylenetetramine (HT) for sealing, and wash with magnetic adsorption for later use; Step 4) Synthesize CuO nanomaterials; Step 5) Synthesize LP@CuO, specifically by adding the hepatitis B virus signal probe LP to a CuO nanomaterial solution and shaking at room temperature for 8 hours. Add hexamethylenetetramine to seal, centrifuge and wash for later use.

2. The method for detecting hepatitis B virus based on a biosensor constructed using a magnetic covalent organic framework material according to claim 1, characterized in that, Step 1) specifically refers to: (1) To synthesize ZnFe2O4, ferric chloride hexahydrate and anhydrous zinc chloride were dissolved in ethylene glycol to form a transparent solution. Sodium acetate and polyethylene glycol 20000 were then added. After vigorous stirring, the mixture was transferred to a stainless steel autoclave and reacted at 200°C for 6-10 h. After the reaction was terminated, the mixture was allowed to cool to room temperature naturally. The product was washed several times with ethanol and water. The black product was dried in a vacuum environment at 60°C for 4-8 h. The samples were collected for subsequent experiments and characterization. (2) Further synthesis of ZnFe2O4@COF: 1,3,5-tris(4-aminophenyl)benzene (TAPB), terephthalaldehyde (TPA) and ZnFe2O4 were dissolved in dimethyl sulfoxide; ultrasonically dispersed, anhydrous acetic acid was added under ultrasonication, and the reaction was carried out at room temperature; ZnFe2O4@COF was collected with a magnet, washed with tetrahydrofuran and methanol respectively, dried under vacuum at 60℃, and the sample was collected for subsequent experiments and characterization.

3. The method for detecting hepatitis B virus based on a biosensor constructed from a magnetic covalent organic framework material according to claim 1, characterized in that, Step 2) specifically refers to: (1) To synthesize AuNPs, add HAuCl4 solution to deionized water, heat and stir at 80-120℃, add sodium citrate solution, reflux for 20-40 min until the solution turns wine red, cool naturally to room temperature, and store at 4℃. (2) The ZnFe2O4@COF powder sample was prepared into a ZnFe2O4@COF solution by adding water. The ZnFe2O4@COF solution was added dropwise to the gold nanoparticle AuNPs solution, stirred at 4℃ for 12h, centrifuged and washed to obtain Au@ZnFe2O4@COF nanocomposite material.

4. The method for detecting hepatitis B virus based on a biosensor constructed from a magnetic covalent organic framework material according to claim 1, characterized in that, Step 3) specifically involves adding the hepatitis B virus capture probe CP to the Au@ZnFe2O4@COF nanocomposite solution, shaking at 4°C for 12 hours, adding hexamethylenetetramine (HT) for blocking, and washing with magnetic adsorption for later use.

5. The method for detecting hepatitis B virus based on a biosensor constructed from a magnetic covalent organic framework material according to claim 1, characterized in that, The steps for detecting the hepatitis B virus are as follows: (1) Add CP@Au@ZnFe2O4@COF to a centrifuge tube, add HBV DNA target, incubate at room temperature, and wash with PBST solution using magnetic adsorption. (2) Add LP@CuO, incubate at room temperature, and clean with PBST magnetic adsorption. (3) Add MES buffer solution, and add 4-AP and 2,4-DP at the same time, and develop color at a certain temperature. (4) Take the supernatant and use a UV spectrophotometer to measure the absorption peak at 400-700 nm, and record the highest absorption peak at 510 nm. Record and plot the working curve.

6. The method for detecting hepatitis B virus based on a biosensor constructed from a magnetic covalent organic framework material according to claim 5, characterized in that, The pH of the MES buffer solution is 6.

8.

7. A biosensor for detecting hepatitis B based on a magnetic covalent organic framework material, as described in claim 5. The method of using the antiviral agent is characterized by, The specified temperature environment is 60℃.

8. The method for detecting hepatitis B virus based on a biosensor constructed from a magnetic covalent organic framework material according to claim 5, characterized in that, The color development time is 15 minutes.

9. The method for detecting hepatitis B virus based on a biosensor constructed from a magnetic covalent organic framework material according to claim 5, characterized in that, The room temperature incubation time is 15 minutes.

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