CN113155924A - Detection method of norovirus - Google Patents
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Abstract
The invention relates to a method for detecting norovirus, which comprises the utilization of AuNPs @ ZnFe2O4@COF、Apt@AuNPs@ZnFe2O4@COF、AuNPs@BP@Ti3C2MXene three substances. The method comprises the steps of 1) synthesizing ZnFe2O4@ COF; synthesis of AuNPs @ ZnFe2O4@ COF; synthesis of Apt @ AuNPs @ ZnFe2O4@ COF; 2) stripping black phosphorus BP; synthesis of BP @ Ti3C2MXene; synthesis of AuNPs @ BP @ Ti3C2MXene; immobilization of the polypeptide (NoroBP); 3) constructing an electrochemical sensor of norovirus. The invention constructs an electrochemical sensor capable of quickly and effectively detecting the concentration of norovirus in a sample.
Description
Technical Field
The invention belongs to the technical field of aptamer electrochemical sensing, and particularly belongs to the technical field of norovirus detection methods.
Background
Norovirus (NoV) is a linear, single-stranded, forward RNA virus belonging to the family caliciviridae, the major pathogen that can cause epidemics and outbreaks of acute gastroenteritis in humans following rotaviruses. NoV are divided into 7 genomes (GI to GVIII) according to the difference of VP protein coding sequences, wherein the GI, GII and GIV genomes mainly cause human infection, the GI genome comprises 9 different genotypes, the GII genome comprises 22 different genotypes, and the GIV genome comprises 2 different genotypes, wherein the acute gastroenteritis of human caused by the GII genome is most common. Worldwide, the gii.4 genotype has dominated the NoV outbreak of infection.
The traditional detection methods are ELISA, immunochromatography and Real-time RT-PCR. ELISA is enzyme-linked immunosorbent assay, norovirus detection is mainly through antigen-antibody reaction, the method saves time and cost, is simple and convenient to operate, but has poor specificity and sensitivity and high cost. The immunochromatography technique utilizes a test strip on which a monoclonal antibody or polyclonal antibody prepared from norovirus antigens is used for detection. The method has the advantages of time saving and convenient operation, thus being widely used for clinical detection, but has the defect of higher false negative rate due to lower sensitivity. The Real-time RT-PCR is to extract virus RNA, design and synthesize primers and probes, and perform fluorescence quantification by using a quantitative PCR instrument; the Real-time RT-PCR method has the characteristics of high detection sensitivity and accurate result, but is more complicated to detect the multi-virus subtype, high in detection cost and needs expensive instruments and equipment and professional personnel for operation.
Disclosure of Invention
The present invention provides a method for detecting norovirus, which solves the above-mentioned problems. The invention is realized by adopting the following technical scheme.
The material is AuNPs @ ZnFe2O4@COF；
The synthesis steps of the material comprise: (1) synthesis of AuNPs from HAuCl4Adding the solution into deionized water, heating and stirring, adding sodium citrate solution, refluxing until the solution turns into wine red, naturally cooling to room temperature, and storing at 4 deg.C for use;
(2)ZnFe2O4dispersing the @ COF powder in deionized water, ultrasonic treating for 1-4h, and removing larger particles by centrifugation to obtain ZnFe2O4Suspending liquid of @ COF, centrifuging, freeze drying, and storing at 4 deg.C for use;
(3) ZnFe to be obtained2O4@ COF ZnFe prepared by adding water2O4@ COF solution, taking ZnFe2O4Dropping the @ COF solution into the gold nanoparticle solution, stirring at 4 ℃, and centrifugally washing to obtain AuNPs @ ZnFe2O4@ COF nanocomposites.
The other material is prepared from Apt @ AuNPs @ ZnFe2O4@ COF; the preparation method of the material comprises the following steps: adding norovirus aptamer (Apt) 5-30 μ L10 μmol/L to AuNPs @ ZnFe 500 μ L0.1-5 mg/mL2O4Oscillating for 12h at room temperature in the @ COF nano composite material solution, and centrifugally washing to obtain Apt @ AuNPs @ ZnFe2O4@ COF composite probe material.
The other material is AuNPs @ BP @ Ti3C2 MXene；
The synthesis steps of the material comprise: BP @ Ti3C2Preparing BP @ Ti by adding water to MXene3C2MXene solution, taking BP @ Ti3C2Dropwise adding the MXene solution into the gold nanoparticle solution, stirring at 4 ℃, and centrifugally washing to obtain AuNPs @ BP @ Ti3C2MXene nanocomposites.
The three materials are the most important three substances in the detection method of the invention, and the three substances are not described in the current publication, so the three substances and the synthesis method are one of the core technologies of the technology of the invention. Furthermore, the subsequent research of using methods of the three substances becomes a new research of science and technology.
The method of using the three materials is to specifically recognize GII.4 norovirus in a sandwich manner by using polypeptide (NoroBP) and aptamer. Through research, the method related to the invention is one of the using methods of the core material of the invention, and is not only one.
The method for sandwich-type specific recognition of the GII.4 norovirus by using the polypeptide (NoroBP) and the aptamer detects the range of 0.01 to 105copies/mL。
The detection limit of the detection range of the invention is 0.01copy/mL at least. The minimum limit value which can be detected by the invention is lower than the minimum limit values disclosed by other methods at present, and the invention has higher innovation height.
The method of the invention comprises the following steps:
1) synthesis of ZnFe2O4@ COF; synthesis of AuNPs @ ZnFe2O4@ COF; synthesis of Apt @ AuNPs @ ZnFe2O4@COF；
2) Stripping black phosphorus BP; synthesis of BP @ Ti3C2MXene; synthesis of AuNPs @ BP @ Ti3C2MXene; immobilization of the polypeptide (NoroBP);
3) constructing the norovirus electrochemical immunosensor. The three steps are the core steps of the method steps of the invention.
Detailed description: the method of the invention comprises the following steps:
(1) al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, and sequentially ultrasonically cleaning the mirror surface in 50% nitric acid solution, 50% absolute ethyl alcohol and ultrapure water; blowing the surface of the electrode by using nitrogen, and dropwise adding 10 mu L of 1mg/mL AuNPs @ BP @ Ti3C2The MXene nano composite material is naturally dried to form a layer of basement membrane on the surface of the electrode;
(2) preparing 1mg/mL polypeptide (NoroBP) solution, dropwise adding 10-30 μ L solution on the treated electrode surface, and incubating at 4 deg.C for 1-4 h;
(3) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10-30 μ L1 mmol/L1-Hexanethiol (HT), and incubating at room temperature for 30 min;
(4) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, drying the electrode, and adding 10-30 μ L PBS with concentration of 10-2-105Dripping a series of NoV solutions with different concentrations of copies/mL on the surface of the glassy carbon electrode treated in the step (2), and incubating for 0.5-4h at 4 ℃;
(5) by usingWashing the electrode with 0.2mol/L pH 7.0PBS for 3 times, and after the electrode is dried, washing 10 mu L of Apt @ AuNPs @ ZnFe with the concentration of 1mg/mL2O4Dripping the @ COF nano composite material on the surface of the electrode, and incubating for 0.5-4h at room temperature;
(6) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10-30 μ L10mmol/L Toluidine Blue (TB) solution on the surface of the electrode, keeping the electrode wet, and incubating at room temperature for 10-120 min;
(7) and (3) washing the electrode with 0.2mol/L of PBS (pH 7.0) for 3 times, after the electrode is dried, detecting an analyte in 10mL of PBS (pH 7.0) by using pulse voltammetry (DPV), wherein the scanning voltage range is-0.6-0V, the pulse amplitude is 0.05V, the pulse width is 0.05s, and drawing a working curve.
The NoV solution is prepared by collecting feces suspected to contain norovirus and adding water for dilution. In fact, any excrement, saliva, etc. which is separated from the human body itself can be used as the NoV solution.
In addition to the above technical solutions, the molecule that can be used to identify NoV according to the present invention can be other biomolecules besides aptamers, such as NoV antibodies (mono-or poly-antibodies) and other polypeptides that have been modified to have the same sequence as the main sequence of the polypeptide (NoroBP). COFs in the present invention may also be replaced with other covalent organic backbone materials.
Some principles/methodologies of the present invention are briefly described as follows: in the invention, the magnetic composite covalent organic framework nano material (AuNPs @ ZnFe)2O4@ COF) as a new generation of magnetic composite nano material, has good adsorbability, can be dissolved in water, and has the characteristics of binding with biomolecules with recognition effects such as antibodies, aptamers, polypeptides and the like.
And AuNPs @ ZnFe2O4The @ COF composite material has the characteristics of good conductivity and magnetic separation, and has an excellent enrichment effect on signal molecule Toluidine Blue (TB).
Meanwhile, in the present invention, 2D black phosphorus BP and Ti are stacked by using van der waals force3C2To prepare BP/MXene (Ti)3C2) Heterostructure for use as electrode substrate material to increase conductivity and sensitivity of sensorHowever, there is no report of using this heterostructure in electrochemical sensors.
NoroBP is a polypeptide that specifically recognizes norovirus and has been used in recent years for the establishment of norovirus detection methods. The characteristic that the sensor can be combined with gold nanoparticles on a substrate material and can specifically recognize the GII.4 subtype norovirus and the magnetic composite covalent organic framework nano material are utilized to jointly act to construct a norovirus electrochemical sensor with the advantages of high sensitivity, wide detection range, quick detection, low detection limit, convenience in operation and the like.
The object of the present invention is to assemble a polypeptide (NoroBP) into BP @ Ti3C2MXene @ Au coated electrode, which forms an electrode surface capable of capturing NoV GII.4, and capturing and fixing virus particles on the electrode surface. Then, the synthesized magnetic composite covalent organic framework nano material which is loaded with the NoV GII.4 aptamer and has excellent electrochemical performance is used for identifying virus particles on the surface of the electrode, meanwhile, toluidine blue solution is dripped on the surface of the electrode for incubation by utilizing the excellent adsorption capacity of the composite material to toluidine blue, and finally, the test is carried out in 10mL PBS buffer solution with the concentration of 0.2mol/L and the pH value of 7.0, thus forming the electrochemical sensor which can quickly and effectively detect the concentration of the norovirus in the sample.
The invention has the beneficial effects that 1) AuNPs @ ZnFe2O4The @ COF nanocomposite material was successfully prepared and was demonstrated by infrared spectroscopy, scanning electron microscopy images, transmission electron microscopy images, EDS elemental analysis, Zeta potential, XRD powder X-ray diffraction pattern and XPS photoelectron spectroscopy.
2) COFs, while having potential as probe molecules, are poorly conductive; ZnFe2O4@ COF has more catalytically active sites than COF alone, is more electrically conductive, but lacks sites to bind Apt; AuNPs @ ZnFe2O4The @ COF not only has Au sites with the function of combining with Apt, but also has more excellent conductivity, AuNPs @ ZnFe2O4The synthesis of the @ COF nano composite material effectively combines the excellent performances of the three materials together and fully exerts the functionsIs the excellent performance of the electrochemical sensor probe.
3) As the number of the electrode surface assembly layers increases, the concentration is 1mmol/L K at 0.1mol/L KCl4[Fe(CN)6]/K3[Fe(CN)6]And performing Electrochemical Impedance Spectroscopy (EIS) analysis in the solution, and simultaneously performing corresponding Cyclic Voltammetry (CV) verification, wherein the verification result is that gold is successfully enriched, polypeptides are successfully modified, the HT blocking effect is good, NoV in the solution can be successfully captured, and the probe combination is stable.
4) As the virus concentration increases, the electrical signal detected by pulsed voltammetry (DPV) increases and is at 10-2-105The norovirus concentration range of copies/mL has a good linear relationship.
5) The substitution of NoV with other substances, such as saline solutions, biological protein molecules, and viruses that also cause intestinal disease (rotavirus and enteroadenovirus), did not detect the corresponding electrochemical signals. The electrochemical sensor constructed by the method has higher specificity.
6) The invention successfully prepares AuNPs @ ZnFe2O4@ COF nanocomposites. The results of Electrochemical Impedance Spectroscopy (EIS) and corresponding Cyclic Voltammetry (CV) verification show that the polypeptide (NoroBP) can be assembled through AuNPs @ BP @ Ti3C2The MXene nano composite material is modified on an electrode, and can effectively capture NoV, AuNPs @ ZnFe2O4The @ COF nano composite material can specifically recognize norovirus through the connection effect of Apt and is assembled on an electrode, and AuNPs @ ZnFe has TB adsorption capacity and catalytic performance2O4The @ COF nanocomposite can be used as an excellent probe for detecting NoV. Meanwhile, the screening effect of the polypeptide (NoroBP) and the norovirus aptamer (Apt) on NoV enables the constructed electrochemical sensor to have the specific recognition capability on NoV, and the linear detection range of the prepared sensor is 0.01-105copies/mL, with a minimum detection Limit (LOD) of 0.01copy/mL, which is the lowest value reported so far, and is expected to be applied in low virus concentration (load) sample detection.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is ZnFe2O4TEM images of the nanoparticles.
FIG. 2 is ZnFe2O4SEM images of nanoparticles.
FIG. 3 is ZnFe2O4TEM image of @ COF.
FIG. 4 is ZnFe2O4SEM picture of @ COF.
FIG. 5 is AuNPs @ ZnFe2O4TEM image of @ COF.
FIG. 6 is AuNPs @ ZnFe2O4EDS plot of @ COF.
FIG. 7 is ZnFe2O4、COF、ZnFe2O4@ COF and ZnFe2O4Zeta potential diagram of @ COF @ Au.
Fig. 8 is a TEM image of black phosphorus BP.
Fig. 9 is an SEM image of black phosphorus BP.
FIG. 10 is an EDS map of AuNPs @ BP @ Ti3C2 MXene.
FIG. 11 is a current diagram for material optimization, ZnFe2O4ZnFe in @ COF2O4The relationship with the COF reaction raw material input proportion.
FIG. 12 is a graph of the current for material optimization, BP: Ti3C2BP and Ti in MXene3C2MXene reaction raw material input proportion relation diagram.
FIG. 13 is a graph of the linear detection range of the sensor of the present invention.
FIG. 14 is a schematic diagram of the sensor detection of the present invention.
FIG. 15 is a block diagram of a process flow of the present invention.
FIG. 16 is a prior art synthesis of ZnFe2O4@ COF.
Detailed Description
For further disclosure, but not limitation, the present invention is described in further detail below with reference to examples.
The chemical reagents and solvents used in the examples were all analytical grade; the used polypeptide and aptamer are obtained by a commercial method; the instrument used for electrochemical detection was electrochemical workstation CHI 660D.
1. ZnFe synthesis according to previous literature reports2O4@ COF, and characterized.
(1) Synthesis of ZnFe2O4Ferric chloride hexahydrate (1.0-5.0g) and anhydrous zinc chloride (0.1-0.5g) were dissolved in ethylene glycol (40mL) to form a clear solution, followed by addition of sodium acetate (3.6g) and polyethylene glycol 20000(0.1-2.0 g). After the mixed solution is vigorously stirred for 0.5h, the mixed solution is transferred into a stainless steel autoclave (the volume is 50mL) to react for 8-24h at the temperature of 200 ℃, and after the reaction is finished, the mixed solution is naturally cooled to the room temperature. The product was washed several times with ethanol and water and the black product was dried under vacuum at 60 ℃ for 6 h.
(2) Further synthesizing ZnFe2O4@ COF, 1,3, 5-tris (4-aminophenyl) benzene (TAPB,0.1-0.8g), terephthalaldehyde (TPA,0.01-0.2g) and ZnFe2O4(0.1-1.0g) was dissolved in 50mL of dimethyl sulfoxide. Dispersing for 5min by ultrasonic wave, adding 1.0-5.0mL of anhydrous acetic acid under ultrasonic wave, and reacting for 15min at room temperature. Magnet collection ZnFe2O4@ COF, washed three times with tetrahydrofuran and methanol, respectively, and dried under vacuum at 60 ℃.
The characterization method comprises the following steps: infrared spectroscopy, scanning electron microscopy images, transmission electron microscopy images, EDS elemental analysis, Zeta potential, XRD powder X-ray diffraction patterns, XPS photoelectron spectroscopy.
2.AuNPs@ZnFe2O4Synthesis and characterization of @ COF nanocomposites.
(1) Synthesizing AuNPs, adding 1.0mL HAuCl with concentration of 5.0-30.0mmol/L4Adding the solution into 100mL deionized water, heating and stirring at 110 deg.C for 10min, adding 10mL sodium citrate solution with concentration of 2.0-20.0mmol/L, refluxing for 30min until the solution turns wine red, naturally cooling to room temperature, and storing at 4 deg.C for use.
(2)ZnFe2O4The @ COF powder was dispersed in deionized water and sonicated for 1-4 h. Then, ZnFe was obtained by centrifugation2O4Suspension of @ COF for removing larger particles, freeze-drying for 12h after centrifugation, and storing at 4 deg.CAnd (5) standby.
(3) ZnFe to be obtained2O4@ COF ZnFe with concentration of 0.1-5mg/mL is prepared by adding water2O4@ COF solution, 1mL ZnFe2O4Dropping the @ COF solution into 10mL gold nanoparticle solution with the concentration of 0.2-1.0mmol/L, stirring for 12h at 4 ℃, and centrifugally washing to obtain AuNPs @ ZnFe2O4@ COF nanocomposites.
The characterization method comprises the following steps: transmission electron microscope images, EDS elemental analysis, Zeta potential, XRD powder X-ray diffraction spectrum, XPS photoelectron spectrum.
3.Apt@AuNPs@ZnFe2O4Synthesis of @ COF nanocomposites.
Adding norovirus aptamer (Apt) 5-30 μ L10 μmol/L to AuNPs @ ZnFe 500 μ L0.1-5 mg/mL2O4And (3) shaking the solution of the @ COF nano composite material at room temperature for 12 hours, and centrifuging and washing for later use.
4. BP/MXene (Ti) synthesis according to previous literature reports3C2) And characterizing.
(1) Preparing BP nano-sheets, dispersing 5-50mg of blocky black phosphorus in 100mL of water under the action of argon, and carrying out ultrasonic treatment on the suspension for 4-24 h. And centrifuging the stable suspension at 3000rpm for 3min to finally obtain the BP nanosheet dispersion.
(2) Taking a proper amount of Ti3C2Adding the mixture into BP nano-sheet dispersion liquid, stirring the mixture for 0.5-4h, transferring the suspension into a stainless steel autoclave, reacting for 1-3h at the temperature of 120-.
The characterization method comprises the following steps: scanning electron microscope image, transmission electron microscope image, Zeta potential.
5.AuNPs@BP@Ti3C2And synthesizing and characterizing the MXene nano composite material.
The obtained BP @ Ti3C2MXene is prepared into BP @ Ti with the concentration of 0.5-5mg/mL by adding water3C2Taking 1mL of BP @ Ti as MXene solution3C2Dropping MXene solution into 10mL gold nanoparticle solution with concentration of 0.1-1.0mmol/L, stirring at 4 deg.C for 12h, centrifuging and washing to obtain AuNPs @ BP @ Ti3C2MXene nano-scaleA composite material.
The characterization method comprises the following steps: scanning electron microscope image, transmission electron microscope image, Zeta potential, Mapping.
6. A method for preparing a working electrode.
Al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, and sequentially ultrasonically cleaning the mirror surface in 50% nitric acid solution, 50% absolute ethyl alcohol and ultrapure water; blowing the surface of the electrode by nitrogen, and dripping 10-20 mu L of AuNPs @ BP @ Ti with the concentration of 0.5-5.0mg/mL3C2And (4) naturally airing the MXene nano composite material.
7. And screening the optimal reaction conditions of the working electrode.
(1) Preparing 0.1-1mg/mL polypeptide (NoroBP) solution, adding 10 μ L dropwise onto the treated electrode surface, incubating at 4 deg.C for 0-3h, washing the electrode with 0.2mol/L pH 7.0PBS for 3 times, drying the electrode, and washing with 0.1mol/L KCl, 1mmol/L K4[Fe(CN)6]/K3[Fe(CN)6]Electrochemical Impedance Spectroscopy (EIS) was performed in solution with corresponding Cyclic Voltammetry (CV) validation.
(2) Preparing 0.1-4mg/mL polypeptide (NoroBP) solution, dripping 10 μ L solution on the treated electrode surface, incubating at 4 deg.C for 1-8h, dripping 10-50 μ L1 mmol/L1-Hexanethiol (HT), incubating at room temperature for 30min, washing the electrode with 0.2mol/L pH 7.0PBS for 3 times, drying the electrode, and sucking 10 μ L solution-2-105Dripping NoV solution of copies/mL onto the treated electrode, incubating at 4 deg.C for 0-4 hr, washing the electrode with 0.2mol/L pH 7.0PBS for 3 times, drying, and adding KCl at 0.1mol/L and 1mmol/L K4[Fe(CN)6]/K3[Fe(CN)6]Electrochemical Impedance Spectroscopy (EIS) was performed in solution with corresponding Cyclic Voltammetry (CV) validation.
(3) 10 mu L of Apt @ AuNPs @ ZnFe with the concentration of 1mg/mL2O4Dropping @ COF nanocomposite on the surface of the electrode treated by polypeptide/HT/NoV, raising the temperature at room temperature for 0-3h, washing the electrode with 0.2mol/L pH 7.0PBS for 3 times, drying the electrode, and adding the electrode in 0.1mol/L KCl, 1mmol/L K4[Fe(CN)6]/K3[Fe(CN)6]Electrochemical impedance spectroscopy in solution(EIS) while performing corresponding Cyclic Voltammetry (CV) verification.
8. And (3) detecting norovirus.
(1) Al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, and sequentially ultrasonically cleaning the mirror surface in 50% nitric acid solution, 50% absolute ethyl alcohol and ultrapure water; blowing the surface of the electrode by using nitrogen, and dripping 10 mu L of AuNPs @ BP @ Ti of 1mg/mL3C2The MXene nano composite material is naturally dried to form a layer of basement membrane on the surface of the electrode;
(2) preparing 1mg/mL polypeptide (NoroBP) solution, dropwise adding 10-30 μ L solution on the treated electrode surface, and incubating at 4 deg.C for 1-4 h;
(3) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10-30 μ L1 mmol/L1-Hexanethiol (HT), and incubating at room temperature for 30 min;
(4) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, drying the electrode, and adding 10-30 μ L PBS with concentration of 10-2-105Dripping a series of NoV solutions with different concentrations of copies/mL on the surface of the glassy carbon electrode treated in the step (2), and raising the temperature for 0.5-4h at 4 ℃;
(5) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, and after the electrode is dried, washing 10 microliter Apt @ AuNPs @ ZnFe with the concentration of 1mg/mL2O4Dripping the @ COF nano composite material on the surface of the electrode, and raising the temperature for 0.5-4h at room temperature;
(6) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10-30 μ L10mmol/L Toluidine Blue (TB) solution on the surface of the electrode, keeping the electrode wet, and incubating at room temperature for 10-120 min;
(7) and (3) washing the electrode with 0.2mol/L of PBS (pH 7.0) for 3 times, after the electrode is dried, detecting an analyte in 10mL of PBS (pH 7.0) by using pulse voltammetry (DPV), wherein the scanning voltage range is-0.6-0V, the pulse amplitude is 0.05V, the pulse width is 0.05s, and drawing a working curve.
Detailed experimental examples:
example 1: ZnFe2O4The preparation method of @ COF is as follows:
(1)ZnFe2O4preparation of nanoparticles: ferric chloride hexahydrate (1.0g) was weighedAnd anhydrous zinc chloride (0.5g) dissolved in ethylene glycol (60mL) to form a clear solution, followed by addition of sodium acetate (5.0g) and polyethylene glycol 20000(0.1 g). After the mixture was vigorously stirred for 2 hours, the mixture was transferred to a stainless autoclave (capacity 100mL) and reacted at 200 ℃ for 12 hours, and after the reaction was terminated, the mixture was naturally cooled to room temperature to obtain a suspension containing black precipitates. The precipitate was washed several times with ethanol and water and the black product was dried under vacuum at 60 ℃ for 6 h. Obtaining powdered ZnFe2O4And (3) nanoparticles.
(2) Further synthesizing ZnFe2O4@ COF: taking 1,3, 5-tri (4-aminophenyl) benzene (TAPB,0.1g), terephthalaldehyde (TPA,0.06g) and ZnFe2O4(2.0g) was dissolved in 50mL of dimethyl sulfoxide. Ultrasonically dispersing for 60min, adding 10mL of anhydrous acetic acid under ultrasonic, and reacting for 60min at room temperature. Magnet collection ZnFe2O4@ COF washed with tetrahydrofuran and methanol, respectively, three times to give a brown solid, and vacuum dried at 60 deg.C to give ZnFe2O4@ COF brown powder.
Example 2: preparation and characterization of ZnFe2O4@ COF is as follows:
(1)ZnFe2O4preparation of nanoparticles: ferric chloride hexahydrate (3.0g) and anhydrous zinc chloride (1.0g) were weighed, dissolved in ethylene glycol (80mL) to form a clear solution, followed by the addition of sodium acetate (2.0g) and polyethylene glycol 20000(0.1 g). After the mixture was vigorously stirred for 0.5 hour, the mixture was transferred to a stainless autoclave (capacity 100mL) and reacted at 200 ℃ for 24 hours, and after the reaction was terminated, the mixture was naturally cooled to room temperature to obtain a suspension containing black precipitates. The precipitate was washed several times with ethanol and water and the black product was dried under vacuum at 60 ℃ for 6 h. Obtaining powdered ZnFe2O4And (3) nanoparticles. ZnFe2O4Fig. 1TEM and fig. 2SEM representation of nanoparticles.
(2) Further synthesizing ZnFe2O4@ COF: taking 1,3, 5-tri (4-aminophenyl) benzene (TAPB,0.3g), terephthalaldehyde (TPA,0.03g) and ZnFe2O4(1.0g) was dissolved in 50mL of dimethyl sulfoxide. Ultrasonically dispersing for 30min, adding 1.5mL of anhydrous acetic acid under ultrasonic, and reacting for 15min at room temperature. Magnet collection ZnFe2O4@COF，Washing with tetrahydrofuran and methanol for three times to obtain brown solid, and vacuum drying at 60 deg.C to obtain ZnFe2O4@ COF brown powder. ZnFe2O4FIG. 3TEM and FIG. 4SEM representation of @ COF.
Example 3: AuNPs @ ZnFe2O4Preparation of @ COF nanocomposites:
(1) preparation of AuNPs: 50mL of HAuCl with a concentration of 20mmol/L were added with stirring4Adding the solution into 200mL boiling deionized water, heating and stirring at 110 deg.C for 30min, slowly adding 50mL sodium citrate solution with concentration of 20mmol/L dropwise, condensing and refluxing for 60min until the solution turns into wine red, stirring at room temperature for 60min, and storing at 4 deg.C.
(2) ZnFe is obtained in the last step2O4Preparation of @ COF ZnFe with concentration of 5mg/mL by adding water2O4@ COF solution, 2mL ZnFe2O4Dropping the @ COF solution into a gold nanoparticle solution with the concentration of 0.2 mmol/L50 mL, stirring for 24h at 4 ℃, collecting by a magnet, removing supernatant, and continuously centrifugally washing for 3 times by deionized water to obtain AuNPs @ ZnFe2O4@ COF nanocomposites.
Example 4: AuNPs @ ZnFe2O4Preparation and characterization of @ COF nanocomposites:
(1) preparation of AuNPs: 20mL of HAuCl with a concentration of 20mmol/L were added under stirring4Adding the solution into 150mL of boiling deionized water, heating and stirring at 110 ℃ for 60min, slowly dropwise adding 30mL of 50mmol/L sodium citrate solution, condensing and refluxing for 60min until the solution turns into wine red, stirring the solution at room temperature for 60min, and storing at 4 ℃.
(2) ZnFe is obtained in the last step2O4Preparation of water of @ COF ZnFe with concentration of 2mg/mL2O4@ COF solution, 5mL ZnFe2O4Dropping the @ COF solution into 10mL gold nanoparticle solution with the concentration of 0.1mmol/L, stirring for 8h at 4 ℃, collecting by a magnet, removing supernatant, and continuously centrifugally washing for 3 times by deionized water to obtain AuNPs @ ZnFe2O4@ COF nanocomposites.
Adopting transmission electron microscope image and EDS element analysis to AuNPs @ Fe3O4The @ COF nanocomposite was characterized: from the transmission electron micrograph, ZnFe2O4A layer of flocculent COF structure is wrapped outside the nano particles, small granular AuNPs are uniformly distributed in the COF structure, and meanwhile, EDS (electronic data System) element analysis shows that Au elements are added in elements of the nano compound after the AuNPs is added, so that successful synthesis of the nano compound can be shown. Example 5: in AuNPs @ ZnFe2O4@ COF modified Apt:
apt synthesized by the company was diluted to 10. mu. mol/L with deionized water according to the dilution method described in the specification, and then 30. mu.L of 10. mu. mol/L norovirus aptamer (Apt) was added to 500. mu.L of 1mg/mL AuNPs @ ZnFe2O4And (3) oscillating the solution of the @ COF nano composite material at room temperature for 12 hours, and removing supernatant after magnet enrichment and washing the supernatant with deionized water for later use.
Example 6: the electrochemical performance research of probe materials with different synthesis ratios comprises the following specific steps:
exploration of synthesis of ZnFe2O4ZnFe at @ COF2O4Whether the ratio of the COF reaction raw material to the COF reaction raw material has obvious influence on the performance of the probe material. Firstly, ZnFe is weighed respectively2O410mg \ TAPB20mg \ TPA50mg to synthesize ZnFe2O4Respectively adding 5mL of dimethyl sulfoxide to the raw material of @ COF for dissolving, performing ultrasonic treatment for 5min, then respectively adding 150 mu L of anhydrous acetic acid to obtain a solution with dark-to-light gray-brown color, reacting for 30min under the environmental condition, collecting a product by a magnet, cleaning with methanol for three times, and performing vacuum drying at 60 ℃ to obtain ZnFe2O4@ COF brown powder.
Al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, sequentially ultrasonically cleaning the mirror surface in 50% nitric acid solution, 50% absolute ethyl alcohol and ultrapure water, and drying the surface of an electrode to obtain 10 mu L of four ZnFe materials2O4ZnFe of 2mg/mL in a different ratio to COF2O4Dropping @ COF on the surface of electrode, drying, dropping 20 μ L10mmol/L Toluidine Blue (TB) solution on the surface of electrode, keeping it wet, incubating at room temperature for 30minThe analyte is detected by pulse voltammetry (DPV) as ZnFe on the electrode surface2O4ZnFe synthesized in a ratio of TAPB + TPA of 10:12O4The adsorption capacity of @ COF for TB is the most excellent.
Example 7: the preparation method of the norovirus electrochemical immunosensor working electrode comprises the following specific steps:
(1) al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, and sequentially ultrasonically cleaning the mirror surface in 50% nitric acid solution, 50% absolute ethyl alcohol and ultrapure water; blowing the surface of the electrode by using nitrogen, and dripping 20 mu L of AuNPs @ BP @ Ti of 2mg/mL3C2Airing the MXene nano composite material to form a base film on the surface of the electrode;
(2) preparing 0.1mg/mL polypeptide (NoroBP) solution, dropwise adding 15 μ L solution on the treated electrode surface, and incubating at 4 deg.C for 30 min;
(3) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10 mu L of 1-Hexanethiol (HT) of 0.1mmol/L, and incubating for 30min at room temperature;
(4) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, drying the electrode, and adding 20 μ L PBS with a concentration of 10-2-105Dripping a series of NoV solutions with different concentrations of copies/mL on the surface of the glassy carbon electrode treated in the step (2), and incubating for 30min at 4 ℃;
(5) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, and after the electrode is dried, adding 10 mu L Apt @ AuNPs @ ZnFe with the concentration of 0.5mg/mL2O4Dripping the @ COF nano composite material on the surface of the electrode, and incubating for 4h at room temperature;
(6) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 20 mu L10mmol/L Toluidine Blue (TB) solution on the surface of the electrode, keeping the electrode wet, and incubating for 30min at room temperature;
(7) and (3) washing the electrode with 0.2mol/L of PBS (pH 7.0) for 3 times, after the electrode is dried, detecting an analyte in 20mL of PBS (pH 7.0) by using pulse voltammetry (DPV), wherein the scanning voltage range is-0.6-0V, the pulse amplitude is 0.05V, the pulse width is 0.05s, and drawing a working curve. The sensor detection principle is shown in the figure.
The above description is only a part of specific embodiments of the present invention (since the formula of the present invention belongs to the numerical range, the embodiments are not exhaustive, and the protection scope of the present invention is subject to the numerical range and other technical point ranges), and the detailed contents or common knowledge known in the schemes are not described too much. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (9)
1. The material is characterized in that the material is AuNPs @ ZnFe2O4@COF；
The synthesis steps of the material comprise: (1) synthesis of AuNPs from HAuCl4Adding the solution into deionized water, heating and stirring, adding sodium citrate solution, refluxing until the solution turns into wine red, naturally cooling to room temperature, and storing at 4 deg.C for use;
(2)ZnFe2O4dispersing the @ COF powder in deionized water, ultrasonic treating for 1-4h, and removing larger particles by centrifugation to obtain ZnFe2O4Suspending liquid of @ COF, centrifuging, freeze drying, and storing at 4 deg.C for use;
(3) ZnFe to be obtained2O4@ COF ZnFe prepared by adding water2O4@ COF solution, taking ZnFe2O4Dropping the @ COF solution into the gold nanoparticle solution, stirring, and centrifugally washing to obtain AuNPs @ ZnFe2O4@ COF nanocomposites.
2. A material prepared using the material of claim 1, wherein the material is Apt@AuNPs@ZnFe2O4@ COF; the preparation method of the material comprises the following steps: 5-30 μ L of 10 μmol/L norovirus aptamer Apt was added to 500 μ L of 0.1-5mg/mL AuNPs @ ZnFe2O4Oscillating for 12h at room temperature in the @ COF nano composite material solution, and centrifugally washing to obtain Apt @ AuNPs @ ZnFe2O4@ COF composite probe material.
3. The material is characterized in that the material is AuNPs @ BP @ Ti3C2 MXene；
The synthesis steps of the material comprise: BP @ Ti3C2Preparing BP @ Ti by adding water to MXene3C2MXene solution, taking BP @ Ti3C2Dropping MXene solution into gold nanoparticle solution, stirring at 4 ℃, and centrifugally washing to obtain AuNPs @ BP @ Ti3C2MXene nanocomposites.
4. A method of using the material of claim 1 and claims 2 and 3, wherein the method comprises sandwich specific recognition of gii.4 norovirus using the polypeptide NoroBP and the aptamer.
5. The method of claim 4, wherein the method for sandwich-type specific recognition of GII.4 norovirus using the polypeptide NoroBP and the aptamer detected in the range of 0.01 to 105copies/mL。
6. The method of claim 5, wherein the detection limit of the detection range is at least 0.01 copy/mL.
7. Method according to claim 4, characterized in that it comprises the following steps:
1) synthesis of ZnFe2O4@ COF; synthesis of AuNPs @ ZnFe2O4@ COF; synthesis of Apt @ AuNPs @ ZnFe2O4@COF；
2) Stripping black phosphorus BP; synthesis of BP @ Ti3C2MXene; synthesis ofAuNPs@BP@Ti3C2MXene; immobilization of the polypeptide NoroBP;
3) constructing an electrochemical sensor of norovirus.
8. Method according to claim 7, characterized in that it comprises the following steps:
(1) al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, and sequentially ultrasonically cleaning the mirror surface in 50% nitric acid solution, 50% absolute ethyl alcohol and ultrapure water; blowing the surface of the electrode by using nitrogen, and dropwise adding 10 mu L of 1mg/mL AuNPs @ BP @ Ti3C2The MXene nano composite material is naturally dried to form a layer of basement membrane on the surface of the electrode;
(2) preparing 1mg/mL polypeptide NoroBP solution, dropwise adding 10-30 mu L of the solution on the surface of the treated electrode, and incubating for 1-4h at 4 ℃;
(3) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10-30 mu L1 mmol/L1-hexanethiol HT, and incubating for 30min at room temperature;
(4) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, drying the electrode, and adding 10-30 μ L PBS with concentration of 10-2-105Dripping a series of NoV solutions with different concentrations of copies/mL on the surface of the glassy carbon electrode treated in the step (2), and incubating for 0.5-4h at 4 ℃;
(5) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, and after the electrode is dried, washing 10 microliter Apt @ AuNPs @ ZnFe with the concentration of 1mg/mL2O4Dripping the @ COF nano composite material on the surface of the electrode, and incubating for 0.5-4h at room temperature;
(6) washing the electrode with 0.2mol/L PBS (pH 7.0) for 3 times, after the electrode is dried, dripping 10-30 μ L10mmol/L toluidine blue TB solution on the surface of the electrode, keeping the electrode wet, and incubating for 10-120min at room temperature;
(7) and (3) washing the electrode for 3 times by using 0.2mol/L PBS (pH 7.0), after the electrode is dried, detecting the analyte by using pulse voltammetry DPV in 10mL PBS (pH 7.0), wherein the scanning voltage range is-0.6-0V, the pulse amplitude is 0.05V, and the pulse width is 0.05s, and drawing a working curve.
9. The method of claim 8, wherein the NoV solution is prepared by diluting collected feces suspected of containing norovirus with water.
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