An electrochemical sensor for detecting H1N1 influenza virus and its preparation and detection method

Abstract

The present invention relates to the field of electrochemical analysis and detection technology, and in particular to an electrochemical sensor for detecting H1N1 influenza virus and its preparation and detection method. An electrochemical sensor for detecting H1N1 influenza virus comprises an electrode, a CoFe2O4-Au@Pt nanocomposite, a biorecognition element, and a signal converter. The electrode is used to contact the HA protein in the H1N1 influenza virus and generate an electrochemical reaction. The CoFe2O4-Au@Pt nanocomposite is used as an electrode modification material to modify the electrode. The biorecognition element includes a DNA aptamer, which is used to specifically recognize the HA protein. The signal converter is used to convert the interaction between the biorecognition element and the HA protein into an electrical signal output. On the one hand, the CoFe2O4-Au@Pt nanocomposite in the present invention is covalently bonded to the DNA aptamer modified with the HS bond to form a Pt-S bond; on the other hand, through the bimetallic synergistic effect of Au NPs and Pt NPs, the electrocatalytic activity of the material is improved, the electron transfer rate is promoted, and the sensitivity of the sensor is thereby improved.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical analysis and detection, and in particular to an electrochemical sensor for detecting H1N1 influenza virus and a preparation and detection method thereof. Background Art

[0002] In recent decades, the threat posed by influenza pandemics to human health has increased significantly, and most countries worldwide have implemented relevant epidemic prevention plans. These influenza viruses, belonging to the Orthomyxoviridae family of single-stranded RNA viruses, are seasonally transmitted in birds, humans, and some mammals. Influenza viruses have two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are used for the classification of influenza viruses (influenza A, B, and C) and their pathogenesis. Influenza A viruses are divided into subtypes based on these two proteins, with a total of 18 different HA subtypes and 11 different NA subtypes. In particular, the H1N1 and H3N3 subtypes caused by influenza A are the most virulent human pathogens of the three influenza types and can cause severe epidemics. Therefore, rapid and accurate detection of influenza viruses is an effective means of providing timely medical treatment and controlling their large-scale spread.

[0003] Currently, influenza virus detection primarily relies on four methods: viral culture, serological testing, rapid influenza diagnostic tests (RIDTs), and reverse transcription polymerase chain reaction (RT-PCR). The former is the most commonly used in hospitals. However, in addition to expensive, large equipment, it also requires several days. Serological assays rely on antibody-viral antigen interactions, and antibody preparation is relatively time-consuming, labor-intensive, and expensive. Furthermore, multiple studies have reported that seasonal human influenza HA antibodies fail to detect certain influenza virus variants, potentially leading to false-negative diagnoses. RIDTs are enzymatic immunochromatographic assays that, requiring only 10–15 minutes, have become widely used in the clinic for treatment decisions. Although simple and rapid to perform, they achieve reasonable performance only when patient samples have a high viral load (10⁴–10⁶). This limited sensitivity (~80%) can lead to frequent false-negative diagnoses, necessitating further confirmation with other molecular tests (e.g., RT-PCR) recommended by the Centers for Disease Control and Prevention (CDC). Currently, PCR remains the most popular nucleic acid amplification method and is widely used for influenza virus diagnosis in hospitals. This detection method has extremely high sensitivity and specificity, and can exponentially amplify the target nucleic acid sequence (gene) until it reaches a detectable level. However, PCR requires a precise thermal cycler and well-trained personnel. In addition, the reaction may take more than 2-3 hours, which is too slow for clinical influenza A virus treatment.

[0004] Electrochemical sensors are a common detection method used in a wide range of fields, including food, medicine, chemicals, fermentation, and environmental monitoring. With the development of electrochemical sensors, the number of commercially available electrochemical sensors has gradually increased. However, existing electrochemical sensors suffer from low sensitivity, large detection errors, and poor stability, which can easily lead to errors when detecting the H1N1 virus, resulting in inaccurate test results. Summary of the Invention

[0005] In order to solve the technical problems of low sensitivity and large detection error of electrochemical sensors in the prior art, the present invention provides an electrochemical sensor for detecting H1N1 influenza virus.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: an electrochemical sensor for detecting H1N1 influenza virus, comprising an electrode, a CoFe2O4-Au@Pt nanocomposite, a biorecognition element, and a signal converter; the electrode is used to contact the HA protein in the H1N1 influenza virus to induce an electrochemical reaction; the CoFe2O4-Au@Pt nanocomposite is used as an electrode modification material to modify the electrode; the CoFe2O4-Au@Pt nanocomposite is added to the CoFe2O4-APTES solution to form a biorecognition element; and the biorecognition element is used to convert the HA protein in the H1N1 influenza virus into an electrochemical reaction. NPs, and then mechanically stirred for a period of time and washed and dried to obtain a biological recognition element; the biorecognition element includes a DNA aptamer, and the DNA aptamer is used to specifically recognize the HA protein in the H1N1 influenza virus, and the nucleotide sequence of the DNA aptamer is HS-C6-5-GGCCTACCGTAGTGTGCGTGGGCACATGTTCGCGCCACCGTGCTACAAC-3; the DNA aptamer is mixed with the CoFe2O4-Au@Pt nanocomposite material to obtain CoFe2O4-Au@Pt-aptamer; the CoFe2O4-Au@Pt nanocomposite material is connected to the DNA aptamer to form a Pt-S bond, which is used to fix the DNA aptamer on the surface of the electrode. When the HA protein of the H1N1 influenza virus to be detected is added to the electrode, the DNA aptamer in the CoFe2O4-Au@Pt-aptamer can specifically bind to the HA protein, thereby fixing the HA protein on the electrode; the signal converter is used to convert the interaction between the biorecognition element and the HA protein in the H1N1 influenza virus into an electrical signal output.

[0007] As a further improvement of the above scheme, the preparation process of the Au@Pt NPs is as follows: 0.5 mL of 1% wt HAuCl4 solution is added to 50 mL of an aqueous solution, and the aqueous solution is heated and stirred until boiling; then 0.8 mL of 1% wt sodium citrate is quickly added, and the aqueous solution is continued to be heated until the solution turns wine red, and then 0.1 M ascorbic acid and 1.25 mL of 1% wt H2PtCl6 are added. After heating for another 25 minutes, the solution finally turns dark gray, and the Au@Pt NPs are obtained.

[0008] As a further improvement of the above scheme, the maximum incorporation amount of the Au@Pt NPs is 90 mL.

[0009] As a further improvement of the above solution, the electrode is a magnetic glassy carbon electrode.

[0010] As a further improvement of the above solution, the preparation method of the CoFe2O4-APTES solution is as follows: CoFe2O4 nanoparticles are dissolved in ethanol, and then 3-aminopropyltriethoxysilane is added, and the mixture is mechanically stirred for a period of time to obtain a CoFe2O4-APTES solution.

[0011] As a further improvement of the above solution, the concentration of the 3-aminopropyltriethoxysilane is 0.5% to 4%.

[0012] As a further improvement of the above scheme, the preparation method of the CoFe2O4 nanoparticles is as follows: FeCl3·H2O, CoCl2·6H2O, urea and sodium citrate are added to ethylene glycol in sequence, the mixture is ultrasonically treated and fully dissolved to obtain a reaction solution 1; the reaction solution 1 is placed in a polytetrafluoroethylene liner and sealed in a reactor to react at 200°C for 10 hours; after the reactor is cooled to room temperature, the solid of the reaction solution 1 is recovered with a magnet, and the solid is washed with anhydrous ethanol and deionized water in sequence, and then the solid is dried after washing to obtain the CoFe2O4 nanoparticles with a spherical structure.

[0013] As a further improvement of the above solution, the detection concentration range of the electrochemical sensor for HA protein is 1 pg / mL to 1 μg / mL.

[0014] A preparation method of an electrochemical sensor for detecting H1N1 influenza virus comprises the following steps: washing the CoFe2O4-Au@Pt nanocomposite material in PBS buffer for multiple times, and resuspending the material in 250 μL of PBS buffer after washing; adding 20 μL of the DNA aptamer to the 250 μL PBS buffer to obtain a second mixed solution, and incubating the second mixed solution at room temperature for a period of time; removing the supernatant in the second mixed solution, collecting the solid in the second mixed solution with a magnet, and washing the collected solid in the second mixed solution with PBS buffer, and resuspending the washed solid in 250 μL of PBS buffer to obtain the CoFe2O4-Au@Pt-aptamer; and adding the CoFe2O4-Au@Pt nanocomposite material to the CoFe2O4-Au@Pt nanocomposite material. 1% BSA in PBS buffer was added to CoFe2O4-Au@Pt-aptamer and incubated for 30 minutes; the incubated solid was then washed with PBS buffer containing 1% Tween20, enriched with a magnet after washing, and the solid collected by the magnet was resuspended in 250 μL PBS buffer to obtain the CoFe2O4-Au@Pt-aptamer-BSA; 6 μL of the prepared CoFe2O4-Au@Pt-aptamer-BSA was dropped onto a clean electrode, and then different concentrations of HA protein were added. The concentration of HA protein was detected through the specific binding of the CoFe2O4-Au@Pt-aptamer-BSA to the HA protein.

[0015] A detection method for detecting H1N1 influenza virus adopts the above-mentioned electrochemical sensor for detecting H1N1 influenza virus for detection.

[0016] A detection method for detecting H1N1 influenza virus comprises the following steps: incubating the prepared electrochemical sensor with a series of HA proteins of known concentrations, and then measuring the current values ​​of the HA proteins at the series of concentrations by differential pulse voltammetry; plotting a standard working curve using the current values ​​corresponding to different HA protein concentrations, to obtain a standard curve of Y=-1.486X+29.149, where X is the logarithm of the concentration of the HA protein of known concentration, and Y is the current values ​​of the HA protein of different concentrations; incubating an HA protein of unknown concentration with the electrochemical sensor, and then measuring the current value of the HA protein at the concentration by electrochemical detection, and then calculating the concentration of the HA protein of unknown concentration using the standard working curve.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention constructs an electrochemical sensor based on CoFe2O4-Au@Pt nanocomposite material for measuring different concentrations of H1N1-HA protein. The excellent magnetic properties of the CoFe2O4-Au@Pt nanocomposite material can achieve rapid separation and enrichment of biomolecules under an external magnet, and can be firmly modified on the electrode, thereby amplifying the electronic signal on the electrode surface. In addition, the bimetallic synergistic effect of Au NPs and Pt NPs in the CoFe2O4-Au@Pt nanocomposite material can increase the effective electrochemical active area, improve the electrocatalytic activity of the material, promote the electron transfer rate, and ultimately improve the sensitivity of the sensor. Moreover, the CoFe2O4-Au@Pt nanocomposite material is doped with Au@Pt NPs, which can provide more binding sites and connect with the -HS modified DNA aptamer to form a Pt-S bond, thereby achieving effective fixation of the DNA aptamer.

[0019] (2) The use of a magnetic glassy carbon electrode facilitates the magnetic concentration of CoFe2O4-Au@Pt-aptamer-BSA on the electrode surface, thereby amplifying the electrochemical signal. Finally, label-free electrochemical voltammetry is used to detect HA protein. By forming a complex with HA protein and causing changes in the electronic signal on the electrode surface, HA protein can be rapidly detected. Due to the excellent electron transfer ability of the electrochemical sensor of the present invention, it has a wider detection range and can sensitively detect HA concentrations of 1 pg / mL-1 μg.

[0020] (3) This label-free electrochemical sensor based on CoFe2O4-Au@Pt nanocomposite was constructed, which can sensitively detect H1N1 protein using differential pulse voltammetry with a minimum detection limit of 0.945 pg / mL. The entire detection process takes only a few minutes. In addition, this electrochemical sensor construction method can be mass-produced and maintains detection activity and high specificity for three weeks.

[0021] (4) The electrochemical sensor prepared by the present invention has good anti-interference, stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the potential diagram after different concentrations of ATPES are added to CoFe2O4 in the present invention.

[0023] Figure 2 This is a high-resolution transmission image of Au@Pt NPs in the present invention under high magnification in a transmission microscope.

[0024] Figure 3 This is a high-resolution transmission image of the CoFe2O4-Au@Pt nanocomposite material of the present invention under high magnification in a transmission microscope.

[0025] Figure 4 This is a high-resolution transmission image of the CoFe2O4-Au@Pt nanocomposite material of the present invention under a transmission microscope at low magnification.

[0026] Figure 5 This is the element distribution diagram of the CoFe2O4-Au@Pt nanocomposite material in the present invention and the doping content ratio of Au and Pt elements.

[0027] Figure 6 Schematic diagram of the preparation of the electrochemical sensor of the present invention.

[0028] Figure 7 This is a DPV curve diagram of the electrochemical sensor prepared by the present invention detecting a series of HA proteins with known concentrations.

[0029] Figure 8 The figure is a standard curve diagram of the current and concentration of a series of HA proteins of known concentrations detected by the electrochemical sensor prepared by the present invention.

[0030] Figure 9 The figure shows an electrical signal diagram of the electrochemical sensor prepared by the present invention and detecting the electrical signals of some intermediate products by cyclic voltammetry.

[0031] Figure 10 The figure shows an electrical signal obtained by detecting the electrical signals of some intermediate products using the electrochemical sensor prepared by the present invention and the electrochemical impedance spectroscopy method.

[0032] Figure 11 This is the electrical signal diagram of the electrochemical sensor prepared by the present invention in electrolytes with different pH values.

[0033] Figure 12 The figure shows the results of stability test on the electrochemical sensor prepared by the present invention.

[0034] Figure 13 The figure shows the results of the reproducibility test of the electrochemical sensor prepared by the present invention. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0036] In the description of the present invention, it should be noted that for directional words, such as the terms "center", "transverse", "longitudinal", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like, indicating directions and positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and should not be understood as limiting the specific scope of protection of the present invention. The terms "first", "second", etc. in the description and claims of the present invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. The terms "including" and "having" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units that are not explicitly listed or inherent to these processes, methods, products or devices.

[0037] An embodiment of the present invention provides an electrochemical sensor for detecting H1N1 influenza virus, comprising an electrode, a CoFe2O4-Au@Pt nanocomposite material, a biorecognition element, and a signal converter.

[0038] The electrodes are used to contact the HA protein in the H1N1 influenza virus and produce an electrochemical reaction.

[0039] CoFe2O4-Au@Pt nanocomposite was used as an electrode modification material to modify the electrode. The CoFe2O4-Au@Pt nanocomposite was prepared by adding Au@Pt NPs to a CoFe2O4-APTES solution, mechanically stirring for 4 hours, then washing, and drying in a vacuum at 60°C.

[0040] The biorecognition element includes a DNA aptamer, which specifically recognizes the HA protein from the H1N1 influenza virus. The nucleotide sequence of the DNA aptamer is HS-C6-5-GGCCTACCGTAGTGTGCGTGGGCACATGTTCGCGCCACCGTGCTACAAC-3. The DNA aptamer is mixed with a CoFe2O4-Au@Pt nanocomposite to create a CoFe2O4-Au@Pt-aptamer. The Au@Pt NPs in the CoFe2O4-Au@Pt nanocomposite provide more binding sites and connect with the -HS-modified DNA aptamer to form Pt-S bonds, which are used to immobilize the DNA aptamer on the electrode surface. The DNA aptamer in this example can specifically recognize and bind to the HA protein in the H1N1 influenza virus. The DNA aptamer in this example is obtained by modifying the aptamer sequence with an alcohol-sulfhydryl group. It can be covalently immobilized on the CoFe2O4-Au@Pt nanocomposite material via Pt-S, ultimately modified onto the CoFe2O4-Au@Pt-aptamer and then modified onto the electrode via the CoFe2O4-Au@Pt-aptamer, thereby enabling detection of the HA protein. When the HA protein of the H1N1 influenza virus to be detected is added dropwise to the electrode, the DNA aptamer in the CoFe2O4-Au@Pt-aptamer can specifically bind to the HA protein, thereby immobilizing the HA protein on the electrode and effectively detecting the HA protein.

[0041] The CoFe2O4-Au@Pt nanocomposite material has excellent magnetic properties and can achieve rapid separation and enrichment of biomolecules under the action of an external magnet. It can also be firmly modified on a magnetic glassy carbon electrode to amplify the electronic signal on the electrode surface, thereby improving the sensitivity of the prepared electrochemical sensor.

[0042] The signal converter is used to convert the interaction between the biological recognition element and the HA protein in the H1N1 influenza virus into an electrical signal output.

[0043] In this embodiment, the electrode may be a magnetic glassy carbon electrode. Using a magnetic glassy carbon electrode facilitates magnetic concentration of the CoFe2O4-Au@Pt-aptamer on the electrode surface, thereby amplifying the electrochemical signal and improving the sensitivity and accuracy of the electrochemical sensor during detection.

[0044] In this embodiment, the specific preparation process of the CoFe2O4-Au@Pt nanocomposite material is as follows: take 0.1g CoFe2O4-ATPES, add 90mL of Au@Pt NPs, mechanically stir for 4h and wash, and then vacuum dry at 60°C to obtain the CoFe2O4-Au@Pt nanocomposite material.

[0045] In this example, the preparation process of Au@Pt NPs is as follows: 0.5 mL of a 1% wt HAuCl₄ solution is added to a 50 mL aqueous solution, and the solution is heated and stirred until boiling. Then, 0.8 mL of a 1% wt sodium citrate solution is quickly added. After further heating for several minutes, the solution changes color from light yellow to gray to black to purple to wine red, indicating the formation of AuNPs. 0.1 M ascorbic acid and 1.25 mL of a 1% wt H₂PtCl₆ solution are then added to the aqueous solution. After heating for another 25 minutes, the solution finally turns dark gray, resulting in the formation of Au@Pt NPs. The prepared Au@PtNPs provide a sufficient number of active sites that can covalently bind to specific -HS (alcoholthio)-modified DNA aptamers to form Pt-S bonds. Furthermore, the bimetallic synergy between the Au NPs and Pt NPs enhances the material's electrocatalytic activity, promotes electron transfer rates, and ultimately improves the sensitivity of the electrochemical sensor.

[0046] The maximum doping amount of Au@Pt NPs is 90 mL. During the actual preparation of the CoFe2O4-Au@Pt nanocomposite, the Au@Pt NPs form a dark gray solution. After adding the Au@Pt NPs to the CoFe2O4-ATPES and stirring to fully incorporate them, the CoFe2O4 doped with the Au@Pt NPs will separate from the solution under the application of a magnet. If the supernatant becomes clear, the Au@Pt NPs are fully incorporated into the CoFe2O4. At this point, Au@Pt NPs can be added further, stirred, and separated by magnet until the supernatant is no longer clear, indicating the maximum doping amount. When the supernatant is no longer clear after magnetic separation, the doping amount of Au@Pt NPs is 90 mL. The most highly doped Au@Pt NPs introduce more binding sites, thereby maximizing DNA aptamer immobilization.

[0047] In this embodiment, the preparation method of the CoFe2O4-APTES solution is as follows: CoFe2O4 nanoparticles are dissolved in ethanol, and then 3-aminopropyltriethoxysilane is added, and the mixture is mechanically stirred for 6 hours to obtain a CoFe2O4-APTES solution.

[0048] Among them, the chemical abbreviation of 3-aminopropyltriethoxysilane is APTES.

[0049] During the preparation of the CoFe2O4-APTES solution, the concentration of 3-aminopropyltriethoxysilane is 0.5% to 4%.

[0050] Therefore, it is necessary to conduct a series of experiments, add different concentrations of 3-aminopropyltriethoxysilane in each experiment, and then select a group of CoFe2O4-APTES solutions with the greatest amino group for subsequent operations.

[0051] The experimental procedure for screening for maximum amination is as follows:

[0052] (1) ATPES from 0.5% to 4% is divided into seven concentration gradients, specifically 0.5% ATPES, 1% ATPES, 1.5% ATPES, 2.5% ATPES, 3% ATPES, 3.5% ATPES, 4% ATPES, plus 0% APTES.

[0053] (2) Dissolve 1 g of CoFe2O4 in 100 mL of ethanol and prepare eight identical solutions;

[0054] (3) ATPES of different concentrations in step (1) were added to the solution in step (2) and mechanically stirred for 6 h. The Zeta potential of the eight solutions was then tested using a Zeta potential analyzer. The test results were as follows: Figure 1 As shown. By observing Figure 1 It can be seen that when the concentration of ATPES is 3%, the Zeta potential of the prepared CoFe2O4-APTES is the largest. The maximum Zeta potential indicates that the CoFe2O4-APTES has been maximized at this time.

[0055] In this example, ATPES was added to CoFe2O4 to achieve maximum amination of CoFe2O4. Maximum amination indicates that the ZeTa of CoFe2O4 is at its most positive potential. Au@Pt NPs, synthesized by reduction with sodium citrate and ascorbic acid, have a negative surface charge. Therefore, Au@Pt NPs can be incorporated into CoFe2O4-ATPES via electrostatic adsorption.

[0056] In this embodiment, the preparation method of CoFe2O4 nanoparticles is as follows: 1.51g FeCl3·H2O, 0.63g CoCl2·6H2O, 4.2g urea and 0.16g sodium citrate are added to 70mL ethylene glycol in sequence, and the mixture is sonicated to fully dissolve to obtain reaction solution 1, which is a brown solution;

[0057] The reaction solution 1 was placed in a polytetrafluoroethylene liner and sealed in a high-pressure reactor to react at 200°C for 10 hours. After the reactor was cooled to room temperature, the solid of the reaction solution 1 was recovered using a magnet and washed with anhydrous ethanol and deionized water in sequence. After washing, the solid was placed in a drying oven at 60°C to obtain spherical CoFe2O4 nanoparticles.

[0058] The prepared Au@Pt NPs and CoFe2O4-Au@Pt nanocomposites were placed under a transmission microscope for observation. Figure 2 This is a high-resolution transmission image of Au@Pt NPs at high magnification. Figure 3 This is a high-resolution transmission image of CoFe2O4-Au@Pt nanocomposite material under high magnification. Figure 4 This is a high-resolution transmission image of CoFe2O4-Au@Pt nanocomposite material at low magnification. Figure 5 This is the element distribution diagram of CoFe2O4-Au@Pt nanocomposite material and the ratio of Au and Pt element doping content.

[0059] from Figure 2 As can be seen in the figure, the prepared Au@Pt NPs are uniformly dispersed, with an average grain size of approximately 15nm and a sea urchin-like shape. The spherical material at the center of each particle is Au NPs, while the green petal-like material outside the sphere is Pt NPs, confirming that the prepared material is Au@Pt NPs.

[0060] from Figure 3 It can be seen that the CoFe2O4 nanoparticles in the prepared CoFe2O4-Au@Pt nanocomposite material are spherical and evenly dispersed, with an average grain size of about 150nm. The dark balls adsorbed on CoFe2O4 are Au@Pt.

[0061] Example 2

[0062] A method for preparing an electrochemical sensor for detecting H1N1 influenza virus comprises the following steps:

[0063] (1) Take 250 μL of the CoFe2O4-Au@Pt nanocomposite prepared in Example 1 with a concentration of 3 mg / mL, wash it three times in a PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.2, and resuspend it in 250 μL of PBS buffer solution after washing.

[0064] (2) Add 20 μL of DNA aptamer to 250 μL of PBS buffer to obtain mixed solution 2, and incubate mixed solution 2 at room temperature for 3 h. Remove the supernatant from mixed solution 2, collect the solid in mixed solution 2 with a magnet, and wash twice with PBS buffer to remove unbound biomolecules. The solid in mixed solution 2 is collected and resuspended in 250 μL of PBS buffer to obtain CoFe2O4-Au@Pt-aptamer.

[0065] (3) Add 1% BSA in PBS buffer to the CoFe2O4-Au@Pt-aptamer and incubate for 30 minutes. The purpose of this operation is to block nonspecific free sites. The incubated solid is then washed with PBS buffer containing 1% Tween20, enriched with a magnet after washing, and resuspended in 250 μL of PBS buffer and stored at 4°C to obtain CoFe2O4-Au@Pt-aptamer-BSA.

[0066] (4) Take 6 μL of the prepared CoFe2O4-Au@Pt-aptamer-BSA and drop it on a clean electrode. Then add different concentrations of HA protein to detect the concentration of HA protein through the specific binding of CoFe2O4-Au@Pt-aptamer-BSA and HA protein.

[0067] BSA, also known as insulator protein, is a purchased drug. It is commonly used as a blocking agent in biological detection tests. Its principle is to form a stable protein layer on the surface of the CoFe2O4 magnetic nanoparticles used in this patent through nonspecific adsorption. This layer can fill and cover the vacant positions on the CoFe2O4 magnetic nanoparticles where no DNA aptamers are adsorbed, preventing the HA protein to be detected from nonspecifically adsorbing to the CoFe2O4 magnetic nanoparticles, thereby affecting the test results.

[0068] In this embodiment, refer to Figure 6 To understand the preparation process of electrochemical sensors.

[0069] In this process, we need to verify some of the performance of the prepared electrochemical sensor, so we need to verify the prepared electrochemical sensor. The verification operation is as follows:

[0070] CoFe2O4-Au@Pt-aptamer was prepared according to steps (1) to (4) of the electrochemical sensor preparation method. The prepared CoFe2O4-Au@Pt-aptamer was then dropped onto a clean magnetic glassy carbon electrode to ensure a firm structure between the functionalized magnetic material and the electrode.

[0071] Prepare a series of HA proteins with known concentrations, ranging from 1pg / mL to 1μg / mL. Add these HA proteins with known concentrations to the electrodes respectively. Then measure the electrical signals of the HA proteins with known concentrations by DPV method to obtain the DPV curves of a series of HA protein concentrations. Please refer to Figure 7 By plotting the current and concentration of this series of HA proteins with known concentrations, a standard curve is obtained. Figure 8 shown.

[0072] pass Figure 7 It can be seen that the electrochemical sensor prepared in this example can sensitively detect the concentration of HA protein in the range of 1 pg / mL to 1 μg / mL, thereby verifying the sensitivity of the prepared electrochemical sensor.

[0073] pass Figure 8 It can be seen that the standard curve is Y = -1.486X + 29.149, where Y is the current value and X is the logarithm of the HA protein concentration. Figure 8 R in the standard curve 2 =0.988, where R 2 Indicates the degree of linear fitting, R 2 The closer it is to 1, the better the fitting degree of the standard curve is. 2 =0.988, indicating that the current values ​​of the electrochemical sensor prepared in this example responding to different concentrations of HA protein have a good linear relationship.

[0074] Electrochemical detection is generally a test from high concentration to low concentration, and the ultimate goal is to obtain the lowest detectable concentration of HA protein in H1N1. In this embodiment, the DPV method is used for detection. Figure 7 It can be seen that with the decrease of HA protein concentration, the DPV peak current gradually increased. Figure 8 It can be seen that the final current value has a linear relationship with the logarithm of the concentration, and the minimum detection limit is calculated accordingly. The electrochemical signals obtained by detecting different concentrations of HA protein are calculated according to the empirical formula of the minimum detection limit. The minimum detection limit LOD = 3σ / S sensitivity (S) is the slope of the linear fitting equation, and σ is the signal-to-noise ratio (standard deviation of the blank sample). Specifically, the signal before the addition of HA protein in the H1N1 influenza virus is the background signal. After adding different concentrations of HA protein, the signal changes accordingly. The detection limit is calculated by the difference in the change of different read-through signals. It can be calculated that the minimum concentration detection value of the electrochemical sensor prepared in this embodiment for HA protein is 0.945 pg / mL. The lower the detection limit, the higher the detection sensitivity of the prepared chemical sensor for HA protein. This proves that the sensitivity of the prepared electrochemical sensor is high.

[0075] Furthermore, before fabricating the electrochemical sensor of this example, we thoroughly investigated its assembly process, characterizing each modification step of the electrochemical sensor through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Specifically, during the fabrication of the electrochemical sensor, we conducted a series of experiments on the electrical signals of the intermediate products: CoFe2O4, CoFe2O4-Au@Pt nanocomposite, CoFe2O4-Au@Pt-aptamer, and CoFe2O4-Au@Pt-aptamer-BSA.

[0076] The electrical signals of the above products were detected by cyclic voltammetry and the following results were obtained: Figure 9 The electrical signal of the above product was detected by electrochemical impedance spectroscopy and the following results were obtained: Figure 10 results.

[0077] pass Figure 9 CV is used to describe the voltammetric curve of the electrode surface, and there is a clear redox peak at each functionalization step. Among them, the peak current of MGCE / CoFe2O4-Au@Pt is significantly higher than that of MGCE / CoFe2O4. This is because the Au@Pt nanoparticles have good electrochemical activity, which accelerates the transfer of electrons in the solution to the electrode surface. With the addition of DNA aptamers, the transfer of electrons is hindered, resulting in a significant decrease in the redox current of the electrode. In addition, due to the presence of the insulating protein molecule BSA, the electron transfer between the electrode and the electrolyte surface is further hindered, resulting in a continuous decrease in the redox current of the electrode. In general, these peak current reductions and potential shifts indicate the successful construction of the electrochemical sensor.

[0078] like Figure 10 The characterization of the electrochemical sensor was also verified by EIS. EIS was measured between 100kHz and 0.01Hz. The semicircle in the high-frequency part corresponds to the electron transfer resistance at the electrode interface. Compared with MGCE / CoFe2O4, MGCE / CoFe2O4-Au@Pt exhibits negligible electron transfer resistance. With the addition of DNA aptamers, [Fe(CN6)] 3- / 4- The redox barrier layer resulted in an increase in electron transfer resistance. Finally, the addition of BSA further hindered electron transfer. The CV and EIS results were largely consistent, demonstrating the successful fabrication of the electrochemical sensor and its feasibility for detecting the HA protein from the H1N1 influenza virus.

[0079] Before the electrochemical sensor is prepared and used, some parameters during its use need to be optimized.

[0080] 1. Optimize the performance of the prepared sensor in detecting HA protein in electrolytes with different pH values.

[0081] Before testing the concentration of HA protein, we need to prepare the electrolyte. During the test, the electrode with HA protein needs to be immersed in the electrolyte to conduct the experiment. The electrolyte commonly used in biological detection is 0.1M PBS as the solvent. Calculate 5mM [Fe(CN6)] 3- / 4- The required amount of solids and 0.1 M KCl were added to a volumetric flask containing 0.1 M PBS.

[0082] Prepare multiple electrochemical sensors with the same concentration of HA protein and configure electrolytes with different pH values. Place the electrodes of each sensor in electrolytes with different pH values, and then detect the current value corresponding to the HA protein at the concentration by DPV method. The results are as follows Figure 11 shown.

[0083] pass Figure 11 It can be seen that when the pH value of the electrolyte is 7.2, the current signal is the strongest, which shows that the prepared electrochemical sensor has the best detection signal when the pH value is 7.2.

[0084] 2. Optimization experiment on the recovery rate of HA protein in saliva samples

[0085] Saliva samples were spiked with a certain concentration of HA protein. The HA concentration in the saliva samples was then measured using the DPV method. The final current value was calculated based on the standard curve equation for HA protein. The results are shown in the table below.

[0086]

[0087]

[0088] As can be seen from the above table, the recovery rate of the electrochemical sensor prepared in this embodiment in the saliva sample is above 90%, and the detection performance of the electrochemical sensor is the best.

[0089] In addition, before the electrochemical sensor is used, we also conduct tests on the stability and reproducibility of the electrochemical sensor.

[0090] The stability of the prepared electrochemical sensor was tested as follows: the prepared sensor was stored in a refrigerator at 4°C for 30 days and the stability of the sensor was evaluated by testing HA (1 ng / mL) protein on days 1, 3, 5, 7, 14, 21, and 30. The test results are shown in Figure 2. Figure 12Test results showed that the sensor retained 94.15% of its initial value on day 14 and 78.08% on day 30. Although its current response decreased, the sensor's stability remained within an acceptable range. This demonstrates the excellent stability of the prepared electrochemical sensor.

[0091] The reproducibility of the prepared electrochemical sensor was tested as follows: six electrodes from the same batch were modified with MGCE / CoFe2O4-Au@Pt / ssDNA2 / BSA to detect HA protein at a concentration of 1 ng / mL. The test results obtained with different electrodes are shown in Figure 2. Figure 13 As shown. Figure 13 It can be seen that the structures measured by 6 different electrodes are basically consistent, and the relative standard deviation (RSD) is only 1.68%, which shows that the prepared sensor has high reproducibility.

[0092] Example 3

[0093] A detection method for detecting H1N1 influenza virus, which uses the electrochemical sensor for detecting H1N1 influenza as described in Examples 1 and 2 for detection.

[0094] A method for detecting H1N1 influenza virus, comprising the following steps:

[0095] A series of HA proteins with known concentrations were prepared and incubated separately, and then the current values ​​of the HA proteins at these concentrations were measured by differential pulse voltammetry;

[0096] The standard working curve was drawn by the current values ​​corresponding to different concentrations of HA protein, and the obtained standard curve was Y = -1.486X + 29.149, where X is the logarithm of the concentration of the known HA protein, and Y is the current value of different concentrations of HA protein;

[0097] An unknown concentration of HA protein is incubated with the electrochemical sensor, and then the current value of the HA protein at that concentration is measured by electrochemical detection method, and then the concentration of the unknown concentration of HA protein is calculated by the standard working curve.

[0098] Electrochemical detection methods include cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The scan rate of CV was 100 mV s -1 The EIS test applies an input voltage of less than 10mV within a frequency range of 0.1Hz to 100kHz. With a potential increase of 5mV, a pulse amplitude of 50mV, and a pulse width of 0.05s, the DPV scanning potential range is -0.2 to 0.6V.

[0099] Example 4

[0100] A kit comprising the electrochemical sensor of embodiment 1.

[0101] The above describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and description merely illustrate the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. An electrochemical sensor for detecting H1N1 influenza virus, characterized in that: It includes: an electrode, which is used to contact the HA protein in the H1N1 influenza virus to induce an electrochemical reaction; A CoFe2O4-Au@Pt nanocomposite material is used as an electrode modification material to modify the electrode; the CoFe2O4-Au@Pt nanocomposite material is prepared by adding Au@Pt NPs to the CoFe2O4-APTES solution, mechanically stirring for a period of time, and washing and drying; A biorecognition element, comprising a DNA aptamer, the DNA aptamer being used to specifically recognize the HA protein in the H1N1 influenza virus, the nucleotide sequence of the DNA aptamer being HS-C6-5-GGCCTACCGTAGTGTGCGTGGGCACATGTTCGCGCCACCGTGCTACAAC-3; the DNA aptamer being mixed with the CoFe2O4-Au@Pt nanocomposite to prepare a CoFe2O4-Au@Pt-aptamer; the CoFe2O4-Au@Pt nanocomposite being linked to the DNA aptamer to form a Pt-S bond for fixing the DNA aptamer to the surface of the electrode; and when the HA protein of the H1N1 influenza virus to be detected is added dropwise to the electrode, the DNA aptamer in the CoFe2O4-Au@Pt-aptamer can specifically bind to the HA protein, thereby fixing the HA protein on the electrode; A signal converter is used to convert the interaction between the biological recognition element and the HA protein in the H1N1 influenza virus into an electrical signal output.

2. The electrochemical sensor for detecting H1N1 influenza virus according to claim 1, wherein: The preparation process of the Au@Pt NPs is as follows: 0.5 mL of 1% wt HAuCl4 solution is added to 50 mL of an aqueous solution, and the aqueous solution is heated and stirred until boiling; then 0.8 mL of 1% wt sodium citrate is quickly added, and the aqueous solution is heated until the solution turns wine red. Then, 0.1 M ascorbic acid and 1.25 mL of 1% wt H2PtCl6 are added. After heating for another 25 minutes, the solution finally turns dark gray, and the Au@Pt NPs are obtained.

3. The electrochemical sensor for detecting H1N1 influenza virus according to claim 1, wherein: The maximum incorporation amount of the Au@Pt NPs was 90 mL; And / or, the electrode is a magnetic glassy carbon electrode.

4. The electrochemical sensor for detecting H1N1 influenza virus according to claim 1, wherein The preparation method of the CoFe2O4-APTES solution is as follows: CoFe2O4 nanoparticles are dissolved in ethanol, and then 3-aminopropyltriethoxysilane is added, and the mixture is mechanically stirred for a period of time to obtain the CoFe2O4-APTES solution.

5. The electrochemical sensor for detecting H1N1 influenza virus according to claim 4, wherein: The concentration of the 3-aminopropyltriethoxysilane is 0.5% to 4%.

6. The electrochemical sensor for detecting H1N1 influenza virus according to claim 4, wherein: The preparation method of the CoFe2O4 nanoparticles is as follows: FeCl3•H2O, CoCl2•6H2O, urea and sodium citrate are sequentially added to ethylene glycol, and the mixture is ultrasonicated to fully dissolve the mixture to obtain a reaction solution 1; The reaction solution 1 was placed in a polytetrafluoroethylene liner and sealed in a reactor to react at 200°C for 10 hours. After the reactor was cooled to room temperature, the solid of the reaction solution 1 was recovered using a magnet and washed with anhydrous ethanol and deionized water in sequence. After washing, the solid was dried to obtain the spherical CoFe2O4 nanoparticles.

7. The electrochemical sensor for detecting H1N1 influenza virus according to claim 4, wherein: The electrochemical sensor can detect HA protein in a concentration range of 1 pg / mL to 1 μg / mL.

8. A method for preparing an electrochemical sensor for detecting H1N1 influenza virus, characterized in that: It includes the following steps: The CoFe2O4-Au@Pt nanocomposite material in the electrochemical sensor for detecting H1N1 influenza virus according to claim 1 was washed multiple times in PBS buffer, and after washing, it was resuspended in 250 μL of PBS buffer; Add 20 μL of the DNA aptamer to 250 μL of PBS buffer to obtain a second mixed solution, and incubate the second mixed solution at room temperature for a period of time; The supernatant in the second mixed solution was removed, and the solid in the second mixed solution was collected using a magnet. The collected solid in the second mixed solution was washed with PBS buffer, and the washed solid was resuspended in 250 μL of PBS buffer to obtain the CoFe2O4-Au@Pt-aptamer. 1% BSA in PBS buffer was added to the CoFe2O4-Au@Pt-aptamer and incubated for 30 minutes; the incubated solid was then washed with PBS buffer containing 1% Tween20, and after washing, it was enriched with a magnet, and the solid collected by the magnet was resuspended in 250 μL of PBS buffer to obtain the CoFe2O4-Au@Pt-aptamer-BSA; 6 μL of the prepared CoFe2O4-Au@Pt-aptamer-BSA was dropped onto a clean electrode, and then different concentrations of HA protein were added. The concentration of HA protein was detected through the specific binding of the CoFe2O4-Au@Pt-aptamer-BSA to the HA protein.

9. A method for detecting H1N1 influenza virus, characterized in that: The detection is performed using the electrochemical sensor for detecting H1N1 influenza virus as claimed in claim 1.

10. The method for detecting H1N1 influenza virus according to claim 9, wherein: It includes the following steps: The prepared electrochemical sensor is incubated with a series of HA proteins of known concentrations, and then the current values ​​of the HA proteins under the series of concentrations are measured by differential pulse voltammetry; The standard working curve was drawn by the current values ​​corresponding to different concentrations of HA protein, and the obtained standard curve was Y=-1.486X+29.149, where X is the logarithm of the concentration of the known HA protein, and Y is the current value of different concentrations of HA protein; An HA protein of unknown concentration is incubated with the electrochemical sensor, and then the current value of the HA protein at the concentration is measured by electrochemical detection method, and then the concentration of the unknown HA protein is calculated by the standard working curve.

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