Virus detection platform based on combination of Fe3O4-coated UIO-66-NH2 core-shell structure MOF material and graphene field effect transistor and application thereof

Abstract

The invention discloses a virus detection platform based on combination of a Fe3O4-coated UIO-66-NH2 core-shell structure MOF material and a graphene field effect transistor and application of the virus detection platform. According to the virus detection platform, the Fe3O4-coated UIO-66-NH2 core-shell structure MOF material with magnetism is combined with a graphene field effect transistor biosensor; the porous structure, the high surface area and the excellent adsorption performance of the Fe3O4-coated UIO-66-NH2 core-shell structure MOF material are utilized, a sample with a complex environment can be efficiently captured through a capture antibody corresponding to a modified and fixed virus antigen under the condition that a sample solution does not need to be subjected to complex pretreatment, and through experimental verification, the virus can be efficiently captured through the capture antibody corresponding to the modified and fixed virus antigen. And the capture rate of the SARS-CoV-2 coronavirus is up to 78% or above. And the graphene field effect transistor modified with the labeled antibody is combined to specifically recognize the virus captured by the captured antibody, so that high-sensitivity detection of the virus is realized, a new idea is provided for detection of infectious disease viruses, especially on-site detection, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and specifically relates to a virus detection platform based on a Fe3O4@UIO-66-NH2 core-shell structure MOF material combined with a graphene field-effect transistor and its application. Background Art

[0002] The rapid spread and high pathogenicity of highly contagious viral diseases pose a significant threat to human health. Infectious diseases, caused by pathogenic microorganisms that invade the environment, are globally distributed. Compared to other diseases, highly contagious diseases can be transmitted and infected through a variety of vectors. Due to their high transmissibility, they can easily spread widely, posing a challenge to public health and safety. Timely, highly accurate, and on-site detection and diagnosis of viruses are effective means of preventing their further spread.

[0003] Current diagnostic methods for infectious disease viruses include polymerase chain reaction (PCR), flow cytometry, immunoassay, mass spectrometry, and enzyme-linked immunosorbent assays (ELISAs). However, these methods are complex, requiring expensive instruments and experienced operators. Some of them also require a series of pretreatments for the test samples, such as dilution, centrifugation, and filtration. These pretreatment measures are not only equipment-dependent and prone to increasing the error in the test results, but also prolong the overall detection time of the samples, which often limits their practical application.

[0004] In contrast, biosensors, especially graphene field-effect transistors (GFETs), have the advantages of low detection limits, simple operation, and rapid response. However, in practical applications, infectious viruses are often present in complex matrices (such as air, water, soil, food, blood, and animal bodies). These environments often contain a large amount of non-target biological substances (such as proteins, DNA fragments, and pollutants), which can seriously interfere with the specificity and sensitivity of sensor detection. Complex environments can seriously affect the specificity and sensitivity of sensor detection and pose great challenges to the separation and detection of viruses. Summary of the Invention

[0005] Based on the above background, the present invention provides a virus detection platform based on the combination of Fe3O4@UIO-66-NH2 core-shell structure MOF material and graphene field-effect transistor and its application. The present invention is based on the modification and fixation of capture antibodies corresponding to virus antigens on the Fe3O4@UIO-66-NH2 core-shell structure MOF material, so as to efficiently capture the virus and separate and enrich it; then, a graphene field-effect transistor modified with a labeled antibody is used to specifically bind to the virus captured by the capture antibody, and the virus is detected according to the change of the current-voltage curve.

[0006] The present invention improves the existing technology.

[0007] An infectious disease virus detection platform based on the Fe3O4@UIO-66-NH2 core-shell structure MOF material and graphene field-effect transistor, which includes the Fe3O4@UIO-66-NH2 core-shell structure MOF material, the graphene field-effect transistor, and paired antibodies corresponding to the antigen of the virus to be detected, wherein the paired antibodies include the capture antibody AntiE2 and the labeling antibody AntiE1;

[0008] The capture antibody AntiE2 is modified and fixed on the Fe3O4@UIO-66-NH2 core-shell structure MOF material;

[0009] The labeled antibody AntiE1 is modified on the channel surface of the graphene field effect transistor.

[0010] Furthermore, the preparation and modification of the Fe3O4@UIO-66-NH2 core-shell structure MOF material includes the following steps:

[0011] 1) Fe3O4, ZrCl4, and 2-aminoterephthalic acid are respectively added to an organic solvent to obtain a mixed solution;

[0012] 2) After the mixed solution is heated in two stages for reaction and cooled, the reaction product is subjected to magnetic separation to obtain a solid product which is washed to obtain the Fe3O4@UIO-66-NH2 core-shell structure MOF material.

[0013] 3) Adding the Fe3O4@UIO-66-NH2 core-shell structure MOF material and the capture antibody AntiE2 solution to the acetaldehyde solution and reacting them, after the reaction is completed, washing and drying can obtain the Fe3O4@UIO-66-NH2 core-shell structure MOF material modified with the capture antibody AntiE2.

[0014] Furthermore, the operation of the two-stage heating in step 2) is as follows:

[0015] First react at 70-95°C for 10-15h, then react at 100-110°C for 20-30h.

[0016] Furthermore, the steps of constructing and modifying the graphene field effect transistor are as follows:

[0017] ① Using PMMA to cover graphene on copper foil;

[0018] ②Use ferric chloride solution to corrode the copper substrate;

[0019] ③ Transfer the graphene-PMMA film to the Si / SiO2 substrate and dry it;

[0020] ④ Soak the PMMA / graphene / substrate in acetone solution and anneal to remove PMMA;

[0021] ⑤ Modify the conductive silver paste at both ends of the graphene as the source and drain of the sensor device, and use silicone rubber as the insulating electrode;

[0022] ⑥ Use Ag / AgCl as the gate electrode, add 1-pyridinic acid succinimide ester solution dropwise, react, and then wash;

[0023] ⑦ Add the labeled antibody AntiE1 solution dropwise to the reaction channel of the graphene field-effect transistor and wash after the reaction;

[0024] ⑧ Cover the graphene field effect transistor with ethanolamine.

[0025] Furthermore, the virus to be tested includes coronavirus.

[0026] Based on the same inventive concept, the present invention also provides the application of the above-mentioned virus detection platform based on the Fe3O4@UIO-66-NH2 core-shell structure MOF material and the graphene field-effect transistor in the preparation of products for detecting infectious viruses.

[0027] Based on the same inventive concept, the present invention also provides a non-diagnostic infectious virus detection method based on the above infectious virus detection platform, which comprises the following steps:

[0028] 1) Adding the Fe3O4@UIO-66-NH2 core-shell structure MOF material modified with the capture antibody AntiE2 to the sample solution and stirring to capture the virus to obtain a complex, magnetically collecting the complex and dispersing it in a buffer solution to obtain a dispersion;

[0029] 2) Using Ag / AgCl as a reference electrode, the dispersion obtained in step 1) is added dropwise to the channel surface of a graphene field-effect transistor modified with a labeled antibody AntiE1. The labeled antibody AntiE1 specifically binds to the viral antigen captured by the capture antibody AntiE2, and the virus is detected based on the change in the current-voltage curve.

[0030] Furthermore, the sample solution is selected from one of serum, soil solution, aerosol solution or water.

[0031] Specifically, the soil solution can be prepared by dispersing the solution in a PBS buffer solution.

[0032] The aerosol solution can be prepared by dispersing the aerosol in a PBS buffer solution.

[0033] Based on the same inventive concept, the present invention also provides a SARS-CoV-2 coronavirus detection platform based on a Fe3O4@UIO-66-NH2 core-shell structure MOF material combined with a graphene field-effect transistor, which includes a Fe3O4@UIO-66-NH2 core-shell structure MOF material, a graphene field-effect transistor, and paired antibodies corresponding to the antigens of the SARS-CoV-2 coronavirus, wherein the paired antibodies include a capture antibody AntiE2 and a labeling antibody AntiE1;

[0034] The capture antibody AntiE2 is modified and fixed on the Fe3O4@UIO-66-NH2 core-shell structure MOF material;

[0035] The labeled antibody AntiE1 is modified on the channel surface of the graphene field effect transistor.

[0036] Based on the same inventive concept, the present invention also provides the application of the above-mentioned SARS-CoV-2 coronavirus detection platform based on the Fe3O4@UIO-66-NH2 core-shell structure MOF material and the graphene field-effect transistor in the preparation of products for detecting SARS-CoV-2 coronavirus.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention combines the magnetic Fe3O4@UIO-66-NH2 core-shell structure MOF material with a graphene field-effect transistor biosensor. By utilizing the porous structure, high surface area and excellent adsorption performance of the Fe3O4@UIO-66-NH2 core-shell structure MOF material, viruses in samples from complex environments can be efficiently captured by modifying the capture antibodies corresponding to the fixed virus antigens without the need for complex pretreatment of the sample solution. Experimental verification shows that its capture rate for the SARS-CoV-2 coronavirus is as high as over 78%.

[0039] The present invention uses a graphene field-effect transistor biosensor modified with a labeled antibody to specifically identify the virus captured by the capture antibody, and then detects the virus according to the change in the current-voltage curve. The detection range is large and has high accuracy and sensitivity, so as to achieve high-sensitivity detection of the virus, providing new ideas for the detection of infectious viruses, especially on-site detection, and has broad application prospects.

[0040] The detection operation of the present invention is simple, and there is no need for complex processing operations such as pre-separation and filtration of complex samples. It is highly efficient and has a significant time effect. The present invention can be applied to the detection of viruses in complex environments, such as serum.

[0041] It has been verified that the graphene field-effect transistor of the present invention has good reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0043] Attachment Figure 1 TEM images and zeta potential diagrams of the intermediate and final products prepared for Fe3O4@UIO-66-NH2 / AntiE2:

[0044] Figure A is a transmission electron microscope (TEM) image of Fe3O4;

[0045] Figure B is the TEM image of Fe3O4@UIO-66-NH2;

[0046] Figure C is the TEM image of Fe3O4@UIO-66-NH2 / AntiE2;

[0047] Figures D and E are TEM element distribution maps of Fe3O4@UIO-66-NH2 / AntiE2;

[0048] Figure F shows the zeta potential of different samples.

[0049] Attachment Figure 2 The XPS characterization diagram of the intermediate product and the final product prepared by Fe3O4@UIO-66-NH2 / AntiE2, where:

[0050] Figure A is the XPS spectrum;

[0051] Figure B is the C1s spectrum of Fe3O4 nanoparticles.

[0052] Attachment Figure 3 Panel A shows Fourier transform infrared spectroscopy (FT-IR);

[0053] Attachment Figure 3 Figure B is the X-ray diffraction (XRD) spectrum;

[0054] Attachment Figure 3 Figure C shows the vibrating sample magnetometer (VSM) test results;

[0055] Attachment Figure 3 Figure D shows the UV-visible spectrum of PBS solution before and after the Fe3O4@UIO-66-NH2 / AntiE2 capture process.

[0056] Attachment Figure 4 A is the transfer curve of GFET with different concentrations from 0 to 10 ng / mL (0, 1 ag / mL, 100 ag / mL, 10 fg / mL, 1 pg / mL, 100 pg / mL, 10 ng / mL) detecting SARS-CoV-2 in PBS buffer in the presence of AntiE1;

[0057] Attachment Figure 4 B is a linear plot of VDirac of GFET and Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2 concentration in PBS buffer;

[0058] Attachment Figure 4 C is the transfer curve of GFET with different concentrations from 0 to 10 ng / mL (0, 1ag / mL, 100ag / mL, 10fg / mL, 1pg / mL, 100pg / mL, 10ng / mL) detecting SARS-CoV-2 in human serum in the presence of AntiE1;

[0059] Attachment Figure 4 D is the linear plot of VDirac of GFET and Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2 concentration in human serum.

[0060] Attachment Figure 5 is the specific surface area of ​​Fe3O4, Fe3O4@UIO-66-NH2 and Fe3O4@UIO-66-NH2 / AntiE2.

[0061] Attachment Figure 6 These are the electronic energy spectra of Fe3O4, Fe3O4@UIO-66-NH2 and Fe3O4@UIO-66-NH2 / AntiE2.

[0062] Attachment Figure 7 This is the transfer characteristic curve of the AntiE1 modification process of graphene field-effect transistor.

[0063] Attachment Figure 8 This is the UV spectrum before and after capturing the SARS-CoV-2 virus in the sample.

[0064] Attachment Figure 9 (a) Transfer characteristic curves of the GFET biosensor at different complex concentrations;

[0065] Attachment Figure 9 (b) Relationship between the complex concentration and the ΔVDirac shift of the GFET biosensor. DETAILED DESCRIPTION

[0066] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0067] Specifically, the present invention can be applied to the detection of SARS-CoV-2 virus.

[0068] Example: In this example, a combined sensor is prepared to detect SARS-CoV-2 coronavirus.

[0069] The materials used in this embodiment are as follows:

[0070] Ferric chloride hexahydrate (FeCl3·6H2O), sodium acetate (CH3COONa), ethylene glycol, ethanol, isopropanol, acetone, N,N-dimethylformamide (DMF), phosphate buffer solution (PBS), zirconium tetrachloride (ZrCl4), 2-aminoterephthalic acid (NH2-BDC), and glutaraldehyde were obtained from Bokachem Chemical Reagent Co., Ltd. (Shanghai, China);

[0071] Silicon / silicon dioxide (Si / SiO2) was from Sunson Electronics Co., Ltd. (Jiangsu, China);

[0072] Graphene was provided by Suzhou Tanfeng Graphene Technology Co., Ltd.;

[0073] SARS-CoV-2 coronavirus nucleocapsid protein (N protein) and anti-SARS-CoV-2 coronavirus nucleocapsid protein monoclonal antibodies were provided by Cusabio Bioengineering Co., Ltd. (Wuhan, China);

[0074] Monoethanolamine (MEA), 1-pyridinic acid succinimidyl ester (PASE), and silver paste (Ag) were from Sigma-Aldrich Trading Co., Ltd.;

[0075] Human serum was provided by the Affiliated Hospital of Inner Mongolia University for Nationalities and was approved by the hospital committee.

[0076] Among them, the anti-SARS-CoV-2 coronavirus nucleocapsid protein monoclonal antibodies include:

[0077] The labeled antibody with the catalog number CSB-DA701BmN⑦ (Mouse anti-SARS-CoV-2 coronavirus nucleocapsid protein (N protein) monoclonal antibody) is denoted as AntiE1.

[0078] The capture antibody with the catalog number CSB-DA701BmN⑧ (Mouse anti-SARS-CoV-2 coronavirus nucleocapsid protein (N protein) monoclonal antibody), denoted as AntiE2;

[0079] Characterization equipment:

[0080] The morphological characteristics of the samples were investigated using scanning electron microscopy (SEM, Hitachi S-4800, 5 kV, 5 μA) and transmission electron microscopy (TEM, 2100F / F200X);

[0081] The surface chemical composition and elemental state were analyzed using X-ray photoelectron spectroscopy (XPS, ESCALAB 250, Al Kα-radiation);

[0082] The structural and vibrational information was analyzed using Fourier transform infrared spectroscopy (FTIR, Nicolet 6700IR, 500–4000 cm-1);

[0083] Crystallographic properties were analyzed by X-ray diffraction (XRD, SmartLab Rigaku, 10°–80°);

[0084] Optical properties were measured using UV-visible spectroscopy;

[0085] The electrical properties are measured using BPN400 and 4200ASCS instruments to ensure accurate evaluation of the electrical properties.

[0086] The specific experimental operations of this embodiment are as follows:

[0087] 1. Fe3O4@UIO-66-NH2 core-shell structure MOF material

[0088] (1) The preparation and modification of Fe3O4@UIO-66-NH2 core-shell structure MOF materials include the following steps:

[0089] 1) Fe3O4, ZrCl4, and 2-aminoterephthalic acid are respectively added to an organic solvent to obtain a mixed solution;

[0090] Specifically, 25 mg of Fe3O4 was added to 10 mL of DMF and ultrasonicated for 30 min to obtain a first dispersion;

[0091] 37.5 mg ZrCl4 and 29 mg NH2-BDC were dissolved in 8 mL DMF to obtain a second dispersion;

[0092] Adding the first dispersion liquid to the second dispersion liquid and mixing them evenly to obtain a mixed liquid;

[0093] The preparation of Fe3O4 is as follows:

[0094] Dissolve 0.12M FeCl3·6H2O and 1.10M NaAC in 40mL of ethylene glycol, stir at room temperature for 30 minutes, then transfer to a reactor and heat at 200°C for 8 hours. After cooling to room temperature, wash with deionized water and ethanol, and dry at 60°C.

[0095] 2) After the mixed solution is heated in two stages for reaction and cooled, the reaction product is subjected to magnetic separation to obtain a solid product which is washed to obtain the Fe3O4@UIO-66-NH2 core-shell structure MOF material.

[0096] Specifically, the mixture obtained in step 1) is placed in a preheated oven at 80°C for 12 hours, then maintained at 100°C for 24 hours, and then cooled to room temperature. The obtained solid is magnetically separated and washed several times with ethanol and deionized water.

[0097] The Fe3O4@UIO-66-NH2 core-shell structure MOF material prepared in this step can be dispersed in phosphate buffer solution (PBS) and stored at 4°C for subsequent use.

[0098] 3) Add the Fe3O4@UIO-66-NH2 core-shell structure MOF material and AntiE2 solution (diluted to 100 ug / mL with PBS buffer) to the glutaraldehyde solution for reaction. After the reaction is completed, wash and dry to obtain the Fe3O4@UIO-66-NH2 core-shell structure MOF material modified with the capture antibody AntiE2.

[0099] Specifically, 10 mg of Fe3O4@UIO-66-NH2 and 500 μL of anti-SARS-CoV-2 (AntiE2) (100 μg / mL, diluted to 100 ug / mL with PBS buffer) were mixed and added to 2 ml of 1.5% glutaraldehyde solution, mixed and reacted for 2 hours. After magnetic separation, the mixture was washed three times with PBS buffer to obtain the Fe3O4@UIO-66-NH2 core-shell structure MOF material modified with the capture antibody AntiE2, which was recorded as Fe3O4@UIO-66-NH2 / AntiE2.

[0100] (2) Characterization of the intermediate product and final product of Fe3O4@UIO-66-NH2 / AntiE2 prepared above. The morphology and structural characteristics of Fe3O4@UIO-66-NH2

[0101] Transmission electron microscopy (TEM) was used to characterize the synthesized products. Figure 1As shown in A, the prepared Fe3O4 nanoparticles have a relatively regular spherical shape with an average particle size of 300nm. Fe3O4@UIO-66-NH2( Figure 1 B) A clear outer layer can be observed, demonstrating the core-shell structure of the prepared Fe3O4@UIO-66-NH2. After AntiE2 functionalization, the size and morphology of the Fe3O4@UIO-66-NH2 particles remained unchanged. TEM mapping of the functionalized particles clearly shows a uniform distribution of nitrogen, demonstrating successful functionalization. Figure 1 E represents the zeta potential variation of Fe3O4, Fe3O4@UIO-66-NH2, and Fe3O4@UIO-66-NH2 / AntiE2. The figure shows that the potential changes significantly at each step in the preparation of Fe3O4@UIO-66-NH2 / AntiE2, but remains positive. The specific surface areas of the prepared materials were determined through nitrogen adsorption-desorption experiments. Figure 5 The Brunauer-Emmett-Teller (BET) surface areas of Fe3O4, Fe3O4@UIO-66-NH2 and Fe3O4@UIO-66-NH2 / AntiE2 were 35.2, 199.9, and 96.5 m 2 / g, and the change in specific surface area indicates the successful synthesis and functionalization of Fe3O4@UIO-66-NH2.

[0102] X-ray photoelectron spectroscopy (XPS) was used to characterize and observe the surface element composition and state. Figure 2 and Figure 6 As shown, the peaks at binding energies of 717.4 eV, 532.4 eV, and 278.5 eV for Fe3O4 nanoparticles are attributed to Fe2p, O1s, and C1s, respectively. Compared to Fe3O4 nanoparticles and Fe3O4@UIO-66-NH2, Fe3O4@UIO-66-NH2 / AntiE2 exhibits an additional N1s peak at a binding energy of 402.6 eV, indicating that AntiE2 was successfully modified onto Fe3O4@UIO-66-NH2. Specifically, AntiE2 was modified onto Fe3O4@UIO-66-NH2 by forming a Schiff base via a glutaraldehyde reaction.

[0103] In addition, the X-ray diffraction (XRD) patterns of Fe3O4@UIO-66-NH2 and Fe3O4@UIO-66-NH2 / AntiE2 are basically consistent with those of Fe3O4 nanoparticles, indicating that the synthesis of Fe3O4@UIO-66-NH2 and the functional modification of AntiE2 have no effect on the crystal structure of Fe3O4 ( Figure 3 B).

[0104] The magnetic curves of the prepared samples were studied by vibrating sample magnetometer (VSM). Figure 3 As shown in Figure C, each sample exhibits superparamagnetism. Compared with Fe3O4, the magnetization intensity of Fe3O4@UIO-66-NH2 and Fe3O4@UIO-66-NH2 / AntiE2 is slightly reduced, mainly due to the non-magnetic shell UIO-66-NH2 and the modification of AntiE2. However, Fe3O4@UIO-66-NH2 / AntiE2 still maintains a relatively high magnetization intensity (45.5emu / g), which is beneficial for magnetic enrichment and separation.

[0105] b. Evaluation of the capture efficiency of Fe3O4@UIO-66-NH2 / AntiE2 prepared above

[0106] 2mL of 100μg / mL SARS-CoV-2 nucleocapsid protein (PBS solution) was added to 4mL of phosphate buffered saline (PBS), and then 5mg of Fe3O4@UIO-66-NH2 / AntiE2 was added to the aforementioned suspension. After the suspension was actively captured for 15 minutes using a magnetic stirrer, the magnetic composite material Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2 complex was separated using the magnetic separation principle. The supernatant of the complex before and after capture was taken and detected by UV-visible spectroscopy. The capture rate was calculated according to the relevant formula and the capture performance was evaluated. The results are shown in the attached figure. Figure 3 D. As Figure 3 As shown in D, the absorbance (A) of the Fe3O4@UIO-66-NH2 / AntiE2 solution decreased significantly after the capture process, indicating that the SARS-CoV-2 pathogen was successfully captured. According to the relevant formula (y = 7.015x - 0.1699R 2 =0.9167) and the capture rate was calculated to be 78.9%.

[0107] 2. Graphene field-effect transistor

[0108] (1) The construction and modification of graphene field-effect transistors (GFETs) include the following steps:

[0109] ① Using PMMA to cover graphene on copper foil;

[0110] Specifically, graphene is grown on copper (Cu) foil using chemical vapor deposition (CVD), and a layer of polymethyl methacrylate (PMMA) is spin-coated on the copper foil with graphene to cover it;

[0111] ②Use ferric chloride solution to corrode the copper substrate;

[0112] Specifically, the copper foil on which the graphene was deposited in step ① was placed in a 0.1M ferric chloride solution, and allowed to float on the surface to corrode the copper substrate;

[0113] ③ Transfer the graphene-PMMA film to the Si / SiO2 substrate and dry it;

[0114] ④ Soak the PMMA / graphene / substrate in acetone solution and anneal to remove PMMA;

[0115] Specifically, the films were immersed in acetone for 6 h and annealed in a H2 / Ar2 atmosphere at 380 °C for 2 h to fully remove the PMMA layer;

[0116] ⑤ Modify the conductive silver paste at both ends of the graphene as the source and drain of the sensor device, and use silicone rubber as the insulating electrode;

[0117] ⑥ Use Ag / AgCl as the gate electrode, add 1-pyridinic acid succinimide ester solution dropwise, react, and then wash;

[0118] Specifically, 20 μL of 1 M PASE was added to the surface of the graphene field-effect transistor channel, reacted at room temperature for 2 h, and then washed three times with deionized water (DI) and DMF solution.

[0119] ⑦ Add the labeled antibody AntiE1 solution (diluted to 100 μg / mL with PBS buffer) dropwise to the reaction channel of the graphene field-effect transistor and wash after the reaction;

[0120] Specifically, a solution of anti-SARS-CoV-2 coronavirus nucleocapsid protein monoclonal antibody (AntiE1) was added to the reaction channel of the graphene field-effect transistor, reacted at room temperature for 2 hours, and then washed three times with deionized water.

[0121] ⑧ Cover the graphene field effect transistor with monoethanolamine.

[0122] Specifically, the graphene surface was covered with 20% MAE at room temperature for 2 h to exclude the possibility of nonspecific adsorption.

[0123] (2) Testing the graphene field effect transistor prepared above

[0124] The performance of the sensing platform was evaluated by the Dirac point shift (ΔVDirac), which represents the change in carrier concentration due to the reaction between AntiE1 and Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2 on the surface of the sensing channel. The IV curve was recorded by a semiconductor parameter analyzer equipped with a probe station. All experimental tests were performed in the same 0.01× PBS buffer to ensure that the experimental conditions were completely consistent. Ag / AgCl gate electrodes were used to apply gate voltage (Vgs) to the 0.01× PBS buffer solution, and the Vgs sweep range was set from -0.5V to 1V, and the drain-source current (Ids) was measured at a specified drain-source voltage (Vds).

[0125] a. Figure 7 As shown, the GFET was functionalized. Before functionalization, the Dirac point voltage (VDriac) of the GFET was 0.62V, showing p-type doping characteristics. After the surface of the sensing channel was modified by PASE, Vgs shifted to the left to 0.14V (0.62V→0.48V), indicating that PASE modification induced n-type doping of the graphene channel. Subsequently, when the AntiE1 antibody bound to the surface of the sensor channel modified by PASE, the GFET exhibited p-type doping characteristics, and Vgs shifted (0.14→0.58V). With the addition of MEA, Vgs continued to shift. The above results indicate that the Vgs shift observed after the modification process confirms the successful fabrication and functionalization of the GFET biosensor.

[0126] b. The sensing performance of GFET was tested by enriching MOF materials with different concentrations in PBS buffer. The blank experiment used 30μL of PBS buffer solution, and then prepared Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2-PBS solutions with concentration gradients of 1ag / mL, 100ag / mL, 10fg / mL, 1pg / mL, 100pg / mL and 10ng / mL respectively. The prepared solutions were dropped onto the surface of the GFET channel and the changes in the current-voltage curve of the GFET were observed. Figure 4 As shown in Figure A, in the concentration range of 1ag / mL to 10ng / mL, as the concentration of Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2 increases, the Vgs of the GFET gradually shifts to the left, indicating that SARS-CoV-2 interacts with the Anti-SARS-CoV-2 on the surface of the sensing channel of the GFET, causing the GFET to exhibit n-type doping characteristics. In addition, the logarithmic plot of the change in Vgs and the MOF concentration shows a good linear relationship ( Figure 4B). The linear regression equation is y = 0.2314 + 0.0114 log C (R 2 =0.9980), and the limit of detection (LOD) was calculated to be 1.98 ag / mL based on 3σ / s.

[0127] c. Detection of SARS-CoV-2 in human serum samples

[0128] Unlike buffer solutions, serum is a very complex mixture formed by removing fibrinogen from plasma, which contains various plasma proteins, peptides, fats, carbohydrates, growth factors, hormones, inorganic substances, etc. The presence of these substances may significantly interfere with the detection sensitivity and accuracy of GFET. Before detection, Fe3O4@UIO-66-NH2 / AntiE2 was mixed with human serum containing 100μg / mL SARS-CoV-2 to evaluate its capture efficiency. Figure 8 As shown in Figure 2, after being captured by Fe3O4@UIO-66-NH2 / AntiE2, the absorbance (A) of the solution decreased significantly, and the capture rate was 53.4%. Figure 4 As shown in A, in the concentration range of 1ag / mL to 10ng / mL, as the concentration of Fe3O4@UIO-66-NH2 / AntiE2-SARS-CoV-2 increases, the Vgs of GFET gradually shifts to the left, indicating that SARS-CoV-2 interacts with Anti-SARS-CoV-2 on the surface of the sensing channel of GFET, causing n-type doping of GFET. In addition, the logarithmic plot of the change in Vgs and the concentration of MOF material shows a good linear relationship ( Figure 4 C). The linear regression equation is y = 0.2353 + 0.0115logC (R 2 =0.9960), and the limit of detection (LOD) was calculated to be 17.9 ag / mL ( Figure 4 D).

[0129] (3) Reproducibility of GFETs

[0130] The reproducibility of the upper GFET was evaluated.

[0131] After the SARS-CoV-2 virus in serum was detected, the GFET was washed with 0.01×PBST to remove the bound substances, and then re-modified with AntiE1 to observe the performance of the GFET. Figure 9As shown in the figure, as the concentration of the sample (Fe3O4@UIO-66-NH2 / AntiE2 complex) gradually increases, the Vgs of the GFET gradually shifts to the left, and n-type doping also appears, indicating that the GFET has excellent reproducibility. There is a good linear relationship between the concentration of Fe3O4@UIO-66-NH2 / AntiE2 and the VDirac shift.

[0132] This paper constructs a detection platform based on the combination of magnetic metal-organic frameworks (MOFs) and graphene field-effect transistors (GFETs), which can efficiently capture and highly sensitively detect SARS-CoV-2. The functionalized magnetic Fe3O4@UIO-66-NH2 / AntiE2 material exhibits excellent capture performance for the SARS-CoV-2 pathogen. This platform combines the rapid enrichment properties of the magnetic Fe3O4@UIO-66-NH2 / AntiE2 with the highly sensitive detection capabilities of the GFET, enabling efficient and rapid on-site detection of infectious viruses. The GFET's ΔVDirac exhibits a good linear relationship with MOF concentration, exhibiting a good linear response over a concentration range of 1 ag / mL to 10 ng / mL, with a detection limit of -1 ag / mL. The present invention is also applicable to the capture and detection of SARS-CoV-2 in serum containing complex substances, demonstrating the great potential of this biosensor for viral detection in clinical samples.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An infectious disease virus detection platform based on Fe3O4@UIO-66-NH2 core-shell structure MOF material and graphene field-effect transistor, characterized by: It includes Fe3O4@UIO-66-NH2 core-shell structure MOF material, graphene field-effect transistor and paired antibodies corresponding to the antigen of the virus to be detected, wherein the paired antibodies include capture antibody AntiE2 and labeling antibody AntiE1; The capture antibody AntiE2 is modified and fixed on the Fe3O4@UIO-66-NH2 core-shell structure MOF material; The labeled antibody AntiE1 is modified on the channel surface of the graphene field effect transistor.

2. The infectious disease virus detection platform based on the Fe3O4@UIO-66-NH2 core-shell structure MOF material and graphene field-effect transistor according to claim 1 is characterized in that: The preparation and modification of the Fe3O4@UIO-66-NH2 core-shell structure MOF material includes the following steps: 1) Fe3O4, ZrCl4, and 2-aminoterephthalic acid are respectively added to an organic solvent to obtain a mixed solution; 2) The mixed solution is heated in two stages for reaction, cooled, and then the reaction product is magnetically separated to obtain a solid product which is washed to obtain the Fe3O4@UIO-66-NH2 core-shell structure MOF material. 3) The Fe3O4@UIO-66-NH2 core-shell structure MOF material and the capture antibody AntiE2 solution are added to the glutaraldehyde solution for reaction. After the reaction is completed, the Fe3O4@UIO-66-NH2 core-shell structure MOF material modified with the capture antibody AntiE2 is obtained after washing and drying.

3. The infectious disease virus detection platform based on the Fe3O4@UIO-66-NH2 core-shell structure MOF material and graphene field-effect transistor according to claim 2, characterized in that: The operation of the two-stage heating in step 2) is as follows: First react at 70-95°C for 10-15h, then react at 100-110°C for 20-30h.

4. The infectious disease virus detection platform based on the Fe3O4@UIO-66-NH2 core-shell structure MOF material and graphene field-effect transistor according to claim 1, characterized in that: The steps of constructing and modifying the graphene field effect transistor are as follows: ① Using PMMA to cover graphene on copper foil; ②Use ferric chloride solution to corrode the copper substrate; ③ Transfer the graphene-PMMA film to the Si / SiO2 substrate and dry it; ④ Soak the PMMA / graphene / substrate in acetone solution and anneal to remove PMMA; ⑤ Modify the conductive silver paste at both ends of the graphene as the source and drain of the sensor device, and use silicone rubber as the insulating electrode; ⑥ Use Ag / AgCl as the gate electrode, add 1-pyridinic acid succinimide ester solution dropwise, react, and then wash; ⑦ Add the labeled antibody AntiE1 solution dropwise to the reaction channel of the graphene field-effect transistor and wash after the reaction; ⑧ Cover the graphene field effect transistor with ethanolamine.

5. The infectious disease virus detection platform according to any one of claims 1 to 3, characterized in that: The viruses to be tested include coronaviruses.

6. Use of a virus detection platform based on a Fe3O4@UIO-66-NH2 core-shell structure MOF material combined with a graphene field-effect transistor as described in any one of claims 1 to 5 in the preparation of a product for detecting infectious viruses.

7. A method for detecting infectious diseases for non-diagnostic purposes based on the infectious disease virus detection platform according to any one of claims 1 to 5, characterized in that: It includes the following steps: 1) Adding the Fe3O4@UIO-66-NH2 core-shell structure MOF material modified with the capture antibody AntiE2 to the sample solution and stirring to capture the virus to obtain a complex, magnetically collecting the complex and dispersing it in a buffer solution to obtain a dispersion; 2) Using Ag / AgCl as a reference electrode, the dispersion obtained in step 1) is added dropwise to the channel surface of a graphene field-effect transistor modified with a labeled antibody AntiE1. The labeled antibody AntiE1 specifically binds to the viral antigen captured by the capture antibody AntiE2, and the virus is detected based on the change in the current-voltage curve.

8. A virus detection method for non-diagnostic purposes according to claim 7, characterized in that: The sample solution is selected from one of serum, soil solution, aerosol solution or water.

9. A SARS-CoV-2 coronavirus detection platform based on Fe3O4@UIO-66-NH2 core-shell structure MOF material combined with graphene field-effect transistor, characterized in that: It includes Fe3O4@UIO-66-NH2 core-shell structure MOF material, graphene field-effect transistor and paired antibodies corresponding to the antigen of SARS-CoV-2 coronavirus, wherein the paired antibodies include capture antibody AntiE2 and labeling antibody AntiE1; The capture antibody AntiE2 is modified and fixed on the Fe3O4@UIO-66-NH2 core-shell structure MOF material; The labeled antibody AntiE1 is modified on the channel surface of the graphene field effect transistor.

10. Use of a SARS-CoV-2 coronavirus detection platform based on a Fe3O4@UIO-66-NH2 core-shell structure MOF material combined with a graphene field-effect transistor as described in claim 9 in the preparation of a product for detecting SARS-CoV-2 coronavirus.

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