A detection method for detecting H1N1 influenza virus

Abstract

The application relates to the technical field of electrochemical analysis, in particular to a detection method for detecting H1N1 influenza virus. The detection method for detecting H1N1 influenza virus comprises the following steps: adding HA of a sample to be detected into MB-Au-Apt1 dispersion to obtain mixed solution one; suspending the solid in the mixed solution one in PBS buffer solution; adding AuPtNFs-Apt2 dispersion to obtain mixed solution two; resuspending the solid in the mixed solution two in 1 / 4 SSC buffer solution; adding AuPtNFs-Probe dispersion to obtain mixed solution three; resuspending the solid in the mixed solution three in PBS buffer solution to obtain sample treatment solution; and detecting the HA of the sample treatment solution by differential pulse voltammetry on an electrochemical platform to obtain the concentration of the HA of the sample to be detected. The catalytic property of AuPtNFs is used to amplify the detection signal, the property of nucleic acid and the base complementary ligand principle of the aptamer are used to amplify the number of AuPt nanoflowers and to amplify the detection signal again, and finally the HA protein in the H1N1 influenza virus is sensitively detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical analysis, in particular to a detection method for detecting H1N1 influenza virus. BACKGROUND

[0002] Influenza viruses are spreading worldwide and causing epidemics every year, which has a significant negative impact on public health and the global economy. Since 2010, the number of seasonal influenza cases has increased dramatically from 9 million to 45 million, with 65,000 deaths per year. The influenza A virus, known as H1N1, is transmitted through the air and is highly contagious in the human population. Hemagglutinin, or HA, is a rich and tiny membrane protein (Stokes radius: 8.5 nm) that confers the ability to infect H1N1. Therefore, in order to reduce the impact of influenza viruses, it is essential to obtain a rapid, sensitive and portable diagnostic method.

[0003] The identification of influenza viruses uses conventional viral diagnostic techniques, including virus culture, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Among them, virus culture requires well-trained laboratory personnel, sterile laboratory tables and longer culture time. Although polymerase chain reaction is a commonly used viral detection technique, it is labor-intensive and requires large instruments. For ELISA technology, it mainly relies on antibodies to capture viruses for detection, which is mainly limited by sensitivity and specificity.

[0004] The above detection methods can detect influenza viruses, but these methods require contact with live viruses, or are complex, time-consuming and labor-intensive, or have few detection sites and low sensitivity, making the above detection methods unable to meet the actual detection requirements. SUMMARY

[0005] In order to solve the problem of few detection sites and low sensitivity in detecting H1N1 influenza virus in the prior art, the present application provides a detection method for detecting H1N1 influenza virus, which can double-amplify the detection signal, increase the detection site, improve the detection speed and sensitivity.

[0006] To achieve the above purposes, the technical solution adopted by the present application is as follows: a detection method for detecting H1N1 influenza virus, characterized in that it comprises the following steps:

[0007] The HA to be detected sample is treated: the HA to be detected sample is added to the MB-Au-Apt1 dispersion liquid and mixed completely to obtain a mixed liquid one; then the solid in the mixed liquid one is separated by an external magnetic field, and the separated solid is resuspended in a PBS buffer; then the AuPtNFs-Apt2 dispersion liquid is added to the PBS buffer and mixed to obtain a mixed liquid two; the solid in the mixed liquid two is separated by an external magnetic field, and the MB-Au-Apt1@HA@AuPtNFs-Apt2 is separated and resuspended in 1 / 4 SSC buffer; then the AuPtNFs-Probe dispersion liquid is added to the 1 / 4 SSC buffer and mixed to obtain a mixed liquid three, and the solid in the mixed liquid three is separated by an external magnetic field, and the separated solid is resuspended in a PBS buffer to obtain a sample treatment liquid;

[0008] The prepared sample treatment liquid is added dropwise to the electrode of the electrochemical platform for detecting HA, and then the electrode is immersed in a PBS buffer containing HQ and H2O2 for a period of time.

[0009] Then, a DPV potential scan is performed from -0.1V to 0.6V at a pulse amplitude of 50mV, a pulse period of 500ms, and a quiet time of 2s.

[0010] The concentration of the HA to be detected sample can be obtained from the results of the potential scan of the electrochemical platform.

[0011] As a further improvement of the above scheme, during the treatment of the HA to be detected sample, the aptamer one Apt1 in the MB-Au-Apt1 dispersion liquid is used to capture HA and form a sandwich structure MB-Au-Apt1@HA@AuPtNFs-Apt2 with the aptamer two Apt2 in the AuPtNFs-Apt2 dispersion liquid.

[0012] As a further improvement of the above scheme, the AuPtNFs-Probe in the AuPtNFs-Probe dispersion liquid is connected to the AuPtNFs-Apt2 on the MB-Au-Apt1@HA@AuPtNFs-Apt2 by base complementary pairing to form the MB-Au-Apt1@HA@AuPtNFs-Apt2-AuPtNFs-Probe with multiple AuPtNFs-Probe.

[0013] As a further improvement of the above scheme, a nanocomposite material needs to be prepared before the treatment of the HA to be detected sample, and the nanocomposite material includes an MB-Au dispersion liquid and an AuPtNFs dispersion liquid.

[0014] As a further improvement of the above scheme, the preparation method of the MB-Au dispersion liquid comprises the following steps:

[0015] Preparation of Fe3O4 dispersion liquid;

[0016] The prepared Fe3O4 dispersion liquid is dispersed into a mixture of ethanol and water, and ultrasonic dispersion is performed, then 25% ammonia water is added, and tetraethyl orthosilicate is added dropwise, and stirring is performed at room temperature to obtain suspension one;

[0017] The suspension one is separated by a magnet, and is washed with ethanol and deionized water respectively for multiple times to obtain Fe3O4@SiO2;

[0018] The prepared Fe3O4@SiO2 is dispersed in a mixture of ethanol and deionized water, and ultrasonic is performed, then 3-aminopropyltriethoxysilane is added dropwise after ultrasonic, and stirring is performed at room temperature under nitrogen protection for a period of time to obtain suspension two;

[0019] The suspension two is separated by a magnet, and is washed with ethanol and deionized water respectively for multiple times to obtain Fe3O4@SiO2-NH2, and is dispersed in 50 mL of deionized water to obtain MB dispersion liquid;

[0020] A certain amount of the MB dispersion liquid is taken and ultrasonic treatment is performed, then HAuCl4 aqueous solution and sodium citrate aqueous solution are added respectively, and stirring is performed, then sodium borohydride is added after stirring for a period of time to perform reaction, and suspension three is obtained;

[0021] The suspension three is separated by a magnet, and is washed with ethanol and deionized water for multiple times to obtain MB-Au, and is dispersed in 50 mL of deionized water to obtain the MB-Au dispersion liquid.

[0022] As a further improvement of the above scheme, the preparation method of the AuPtNFs dispersion liquid comprises the following steps:

[0023] HAuCl4 solution is added to a container containing water to obtain mixture four, and mixture four is heated to boiling;

[0024] Sodium citrate solution is quickly added to the boiling mixture four, and stirring and boiling are performed,

[0025] After boiling for a period of time, ascorbic acid and H2PtCl6 are added respectively, and heating is continued for 20 min to obtain the AuPtNFs dispersion liquid.

[0026] As a further improvement of the above scheme, the preparation method of the MB-Au-Apt1 dispersion liquid comprises the following steps:

[0027] Take a certain amount of the prepared MB-Au dispersion liquid, and add aptamer Apt1 to the MB-Au dispersion liquid, and incubate at 25°C for 2 hours to obtain suspension four;

[0028] Remove the supernatant of the suspension four with a magnet to obtain precipitate one; wash the precipitate one with PBS buffer three times;

[0029] Resuspend the washed precipitate one in 200 μL of PBS buffer containing 2% BSA, and oscillate at 25°C for 0.5h to obtain suspension five;

[0030] Separate the suspension five by magnet to obtain MB-Au-Apt1, and resuspend it in 200 μL of PBS solution to obtain the MB-Au-Apt1 dispersion liquid.

[0031] As a further improvement of the above scheme, the preparation of the AuPtNFs-Apt2 also includes the following steps:

[0032] Take a certain amount of the prepared AuPtNFs dispersion liquid, and add aptamer Apt2 to the AuPtNFs dispersion liquid, and incubate at 25°C for 2h to obtain suspension six;

[0033] Remove the supernatant of the mixture six with a magnet to obtain precipitate two; wash the precipitate two with PBS buffer three times;

[0034] Resuspend the washed precipitate two in 200 μL of PBS buffer containing 2% BSA, and oscillate at 25°C for 0.5h to obtain suspension seven;

[0035] Separate the suspension seven by magnet to obtain AuPtNFs-Apt2, and resuspend it in 200 μL of PBS solution to obtain the AuPtNFs-Apt2 dispersion liquid.

[0036] As a further improvement of the above scheme, the preparation of the AuPtNFs-Probe dispersion liquid also includes the following steps:

[0037] Take a certain amount of the prepared AuPtNFs dispersion liquid, and add DNA probe Probe to the AuPtNFs dispersion liquid, and incubate at 25°C for 2h to obtain suspension eight;

[0038] Remove the supernatant of the suspension eight with a magnet to obtain precipitate three; wash the precipitate three with PBS buffer;

[0039] The precipitate after washing is triple-suspended in 200 μL PBS buffer containing 2% BSA and shaken at 25°C for 0.5 h to obtain a suspension nine;

[0040] The suspension nine is separated by a magnet to obtain AuPtNFs-Probe which is resuspended in 200 μL PBS solution, thereby obtaining the AuPtNFs-Probe dispersion.

[0041] As a further improvement of the above scheme, the DNA nucleotide sequence of the nucleic aptamer one Apt1 is 5'-HS-(CH2)6-TACTGCACACGACACCGACTGTCACCATCACCTCGGCGCA-3'.

[0042] As a further improvement of the above scheme, the DNA nucleotide sequence of the nucleic aptamer two Apt2 is 5'-HS-(CH2)6-GGCCTACCGTAGTGTGCGTGGGCACATGTTCGCGCCACCGTGCTACAAC-3'.

[0043] Further, the nucleotide sequence of the DNA probe Probe is 5'-HS-(CH2)6-GTTGTAGCACG-3'.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] (1) The nucleic aptamer one Apt1 in the MB-Au-Apt1 dispersion is used to capture HA and form a sandwich structure MB-Au-Apt1@HA@AuPtNFs-Apt2 with the nucleic aptamer two Apt2 in the AuPtNFs-Apt2 dispersion. The material in the sandwich structure is dropped on an electrode, and the detection signal of HA can be amplified in the HQ / H2O2 and PBS buffer system by differential pulse voltammetry. Meanwhile, the AuPtNFs-Probe in the AuPtNFs-Probe dispersion can also be connected to the AuPtNFs-Apt2 on the MB-Au-Apt1@HA@AuPtNFs-Apt2 by base complementary pairing to form MB-Au-Apt1@HA@AuPtNFs-Apt2-AuPtNFs-Probe with multiple AuPtNFs-Probe, thereby increasing the detection site in the HA detection process and realizing the re-amplification of the HA detection signal. Through this double signal amplification strategy, rapid and sensitive detection of HA can be realized, and the detection effect is improved.

[0046] (2) The application can realize the detection of low concentration HA and further improve the sensitivity in the detection of HA by the double signal amplification strategy for the HA detection signal.

[0047] (3) The application can effectively reduce the detection limit in the detection of HA by the double signal amplification strategy for the HA detection signal, and the lower the detection limit, the higher the detection sensitivity of the method. Through the analysis of the detection limit, it can be known that the detection limit of the twice amplified HA detection signal is about 8 times smaller than that of the once amplified HA detection signal, so that the twice amplified HA detection signal can realize further sensitive detection of HA. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a schematic diagram for processing the HA of the sample to be detected in the embodiment 1 of the application.

[0049] Figure 2 It is a preparation flow chart of the MB-Au-Apt1 dispersion liquid and the AuPtNFs-Apt2 dispersion liquid in the embodiment 1 of the application.

[0050] Figure 3 It is a DPV signal diagram of HA under different processing modes on the electrochemical platform.

[0051] Figure 4 It is a transmission electron microscope (TEM) image of different products of the MB-Au dispersion liquid in the preparation stage.

[0052] Figure 5 It is a transmission electron microscope (TEM) image of different products of the AuPtNFs dispersion liquid in the preparation stage.

[0053] Figure 6 It is a DPV signal diagram of a series of HA with known concentrations in the experiment 1 of the application.

[0054] Figure 7 It is a standard curve diagram between the concentration and the current of HA in the experiment 1 of the application.

[0055] Figure 8 It is a DPV signal diagram of a series of HA with known concentrations in the experiment 2 of the application.

[0056] Figure 9 It is a standard curve diagram between the concentration and the current of HA in the experiment 2 of the application.

[0057] Figure 10 It is two groups of DPV signal diagrams obtained under the conditions of the experiment 1 and the experiment 2 when the concentration of HA is 50 pg / mL in the application.

[0058] SEQUENCE LIST DESCRIPTION (SEQUENCE LIST CONTENT IS PROVIDED SEPARATELY)

[0059] SEQ ID NO. 1 is the DNA nucleotide sequence of the nucleic aptamer Apt1 described in the embodiments of the present application;

[0060] SEQ ID NO. 2 is the DNA nucleotide sequence of the nucleic aptamer Apt2 described in the embodiments of the present application;

[0061] SEQ ID NO. 3 is the nucleotide sequence of the DNA probe Probe described in the embodiments of the present application. DETAILED DESCRIPTION

[0062] Hereinafter, the present application will be further described in conjunction with the specific embodiments, and it should be noted that the following described embodiments or technical features can be combined with each other to form new embodiments without conflict.

[0063] In the description of the present application, it should be noted that for orientation words, such as the terms "center", "transverse", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. indicate the orientation and positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and cannot be understood as limiting the specific protection scope of the present application. The terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, and do not necessarily describe a specific order or sequence. The terms "include" and "have" and any variations thereof in the specification and claims of the present application are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device that includes a series of steps or units does not necessarily limit to those steps or units clearly listed, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0064] Embodiment 1

[0065] Referring to Figure 1 As shown in the drawings, one embodiment of the present application provides a detection method for detecting H1N1 influenza virus, which comprises the following steps:

[0066] (1) The HA sample to be detected is treated: the HA sample to be detected is added to the MB-Au-Apt1 dispersion liquid and mixed completely to obtain a mixed liquid one. Then the solid in the mixed liquid one is separated by an external magnetic field, and the separated solid is resuspended in a PBS buffer. The AuPtNFs-Apt2 dispersion liquid is added to the PBS buffer and mixed to obtain a mixed liquid two. The solid in the mixed liquid two is separated by an external magnetic field, and the MB-Au-Apt1@HA@AuPtNFs-Apt2 is separated and resuspended in a 1 / 4 SSC buffer. The AuPtNFs-Probe dispersion liquid is added to the 1 / 4 SSC buffer and mixed to obtain a mixed liquid three. The solid in the mixed liquid three is separated by an external magnetic field, and the separated solid is resuspended in a PBS buffer to obtain a sample treatment liquid.

[0067] (2) The prepared sample treatment liquid is added dropwise to the electrode of the electrochemical platform for detecting HA, and then the electrode is immersed in a PBS buffer containing HQ and H2O2 for a period of time.

[0068] (3) Then, DPV potential scanning is performed from -0.1 V to 0.6 V at a pulse amplitude of 50 mV, a pulse period of 500 ms, and a quiet time of 2 s.

[0069] (4) The concentration of the HA sample to be detected can be obtained by the result of potential scanning of the electrochemical platform.

[0070] Wherein 1 / 4 SSC refers to a four-fold dilution of the SSC concentration.

[0071] In this embodiment, the SSC is a 20X SSC buffer, and the components thereof are 3 mol / L NaCl and 300 mmol / L sodium citrate, and the pH is 6.9-7.1.

[0072] In this embodiment, the external magnetic field can be realized by a magnet.

[0073] In this embodiment, the electrochemical platform can be an electrochemical biosensor, which has good anti-interference, stability and repeatability.

[0074] It can be understood that, in the present application, in order to capture HA and realize magnetic separation, Au nanoparticles are encapsulated on magnetic beads, and then connected to aptamer one Apt1 through Au-S bond. In addition, the present application also prepares AuPt nanoflower (i.e. AuPtNFs) with catalase properties, which can be connected to aptamer two Apt2 through Pt-S bond, and form a sandwich structure with magnetic beads and HA. The presence of AuPtNFs, H2O2 and hydroquinone (HQ) leads to the generation of an amplified DPV signal of the oxide of HQ. In addition, Apt2 can not only capture the target, but also be connected to single-stranded DNA probe through base complementary pairing, so that the number of AuPtNFs on the sandwich structure is increased, realizing the re-amplification of the detection signal. More importantly, the entire detection can be completed within ten minutes, with good selectivity and high sensitivity. This electrochemical aptamer sensor shows potential as a reliable diagnostic tool for influenza virus, which can effectively control the spread of influenza.

[0075] In the present embodiment, before detecting the concentration of HA, a series of treatments in step (1) are performed on HA. Through this series of treatments, the detection sites in the detection process can be increased, and the detection signal can be double-amplified, thereby improving the detection sensitivity.

[0076] Specifically, in the present embodiment, HA is captured by aptamer one Apt1 in the MB-Au-Apt1 dispersion liquid, and forms a sandwich structure of MB-Au-Apt1@HA@AuPtNFs-Apt2 with aptamer two Apt2 in the AuPtNFs-Apt2 dispersion liquid. The sandwich structure material is dropped on the electrode, and the detection signal of HA can be amplified in the HQ / H2O2 and PBS buffer system by using differential pulse voltammetry. In addition, in the present embodiment, the base complementary pairing principle of aptamer two Apt2 and DNA probe Probe can be used to form an amplification of AuPtNFs, so as to increase the detection sites in the HA detection process, thereby realizing the re-amplification of the HA detection signal. Therefore, by performing the above treatments on the sample HA to be detected, the present application can double-amplify the detection signal of HA when detecting HA, thereby greatly improving the detection sensitivity.

[0077] Please refer to Figure 1In the process of treating the sample HA to be detected, the nucleic acid aptamer Aptl in the MB-Au-Aptl dispersion liquid is used to capture HA and form a sandwich structure of MB-Au-Aptl@HA@AuPtNFs-Apt2 with the nucleic acid aptamer Apt2 in the AuPtNFs-Apt2 dispersion liquid. The material of the sandwich structure is dropped on the electrode, and the detection signal of HA can be amplified in the HQ / H2O2 and PBS buffer system by using differential pulse voltammetry. In this embodiment, the nucleic acid aptamer is used to replace the traditional antibody, which can specifically bind to HA, thereby improving the sensitivity in the detection process of HA.

[0078] In this embodiment, the AuPtNFs-Probe in the AuPtNFs-Probe dispersion liquid can also be connected to the AuPtNFs-Apt2 on the MB-Au-Aptl@HA@AuPtNFs-Apt2 through base complementary pairing, to form MB-Au-Aptl@HA@AuPtNFs-Apt2-AuPtNFs-Probe with multiple AuPtNFs-Probe, thereby increasing the detection site in the detection process of HA, and realizing the re-amplification of the detection signal of HA. Through the re-amplification of the detection signal, the HA protein in the H1N1 influenza virus can be detected sensitively.

[0079] Please refer to Figure 1 , Figure 1 The schematic diagram of treating the sample HA to be detected in this embodiment is shown in FIG. 1. Figure 1 It can be seen that by treating the sample HA to be detected as described above, the detection site of HA during detection can be increased, the detection signal of HA can be double-amplified, and the detection sensitivity of HA can be improved.

[0080] Figure 3 (a) in FIG. 1 is a structural schematic diagram of one-time amplification of the HA detection signal; Figure 3 (b) in FIG. 1 is a structural schematic diagram of two-time amplification of the HA detection signal; Figure 3 (c) in FIG. 1 is a voltage and current curve diagram when the HA detection signal is one-time amplified, the HA signal is two-time amplified, and there is no HA when the electrochemical platform is detected.

[0081] Specifically: Figure 3Part (a) shows the MB-Au-Apt1@HA@AuPtNFs-Apt2 sandwich structure, which uses only aptamer Apt1 to capture HA and forms a sandwich structure with aptamer Apt2 in the AuPtNFs-Apt2 dispersion. This sandwich structure material is dropped onto an electrode, and differential pulse voltammetry is used to amplify the detection signal of HA in an HQ / H2O2 and PBS buffer system.

[0082] Figure 3 Part (b) involves two main processes: first, the detection signal of HA is amplified by using aptamer Apt1 to capture HA and forming a sandwich structure (MB-Au-Apt1@HA@AuPtNFs-Apt2) with aptamer Apt2 in the AuPtNFs-Apt2 dispersion; second, the detection signal of HA is amplified by connecting AuPtNFs-Probes in the AuPtNFs-Probe dispersion to the AuPtNFs-Apt2 in MB-Au-Apt1@HA@AuPtNFs-Apt2 via base pairing.

[0083] pass Figure 3 As shown in the curve in part (c), there is no electrical signal when HA is absent. After secondary amplification of HA, the peak current of the DPV detection signal for HA is significantly greater than that after primary amplification of HA. This indicates that in this embodiment, the DNA probe can amplify AuPtNFs through base complementary pairing, thereby increasing the detection signal.

[0084] Before processing the HA sample to be tested, it is necessary to prepare a nanocomposite material, which includes MB-Au dispersion and AuPtNFs dispersion.

[0085] In this embodiment, the preparation method of MB-Au dispersion includes the following steps:

[0086] (31) Take 5 mL of the prepared Fe3O4 dispersion and disperse it in 40 mL of ethanol and 10 mL of water, and sonicate for 20 min. After sonication, add 1 mL of 25% ammonia water under mechanical stirring, and add 1.2 mL of tetraethyl orthosilicate dropwise. Stir at room temperature for 8 h to obtain suspension one.

[0087] (32) The suspension was separated by a magnet and washed three times with ethanol and deionized water respectively to obtain Fe3O4@SiO2.

[0088] (33) The prepared Fe3O4@SiO2 was dispersed in 48 mL of ethanol and 2 mL of deionized water, and ultrasonically dispersed for 20 min. After ultrasonic, 200 mg of 3-aminopropyl triethoxysilane was added dropwise, and stirred at room temperature for 4 h under nitrogen protection to obtain suspension two.

[0089] (34) The suspension two was separated by magnet, and washed with ethanol and deionized water for three times respectively to obtain Fe3O4@SiO2-NH2, which was dispersed in 50 mL of deionized water to obtain MB dispersion.

[0090] (35) 5 mL of the MB dispersion was taken and ultrasonically treated for 20 min, and then 10 mL of HAuCl4 aqueous solution with a mass concentration of 1 mg / mL and 10 mL of sodium citrate aqueous solution with a mass concentration of 2 mg / mL were added to the ultrasonic-treated MB dispersion, and the mixture was stirred for 10 min. After stirring, 6 mL of sodium borohydride with a mass concentration of 0.144 mg / mL was added at once to react to obtain suspension three.

[0091] (36) After reacting for 1 h, the suspension three was separated by magnet, and washed with ethanol and deionized water for three times to obtain MB-Au, which was dispersed in 50 mL of deionized water to obtain MB-Au dispersion.

[0092] The preparation steps of the Fe3O4 dispersion in step (31) are as follows: 1.35 g of FeCl3·6H2O, 0.22 g of trisodium citrate, and 4.92 g of sodium acetate were dissolved in 40 mL of ethylene glycol, and a uniform solution was formed by magnetic stirring for 30 min. The above reaction complete solution was placed in a polytetrafluoroethylene liner, and sealed in a high-pressure reaction kettle, and reacted at 180°C for 11 h. After the reaction kettle was cooled to room temperature, the material was recovered by magnet, and the recovered material was washed with anhydrous ethanol and deionized water in sequence to obtain Fe3O4 nanoparticles. Finally, the Fe3O4 nanoparticles were dispersed in 20 mL of deionized water to obtain the Fe3O4 dispersion.

[0093] Please refer to Figure 4 , Figure 4 Part (A) of FIG. 1 is a transmission electron microscope (TEM) image of the prepared Fe3O4; Figure 4 Part (B) of FIG. 1 is a transmission electron microscope (TEM) image of the prepared Fe3O4@SiO2; Figure 4 Part (C) of FIG. 1 is a transmission electron microscope (TEM) image of the prepared MB-Au; Figure 4 Part (D) of FIG. 1 is a schematic diagram of the prepared MB-Au before and after magnet separation.

[0094] Reference is made to Section (d) in the Figure 2 In this embodiment, the preparation of the MB-Au-Apt1 dispersion solution comprises the following steps:

[0095] (41) Take 200 μL of the prepared MB-Au dispersion solution in step (36), and take 20 μL of nucleic acid aptamer Apt1 with a volume molar concentration of 10 nM, and add the nucleic acid aptamer Apt1 to the MB-Au dispersion solution, and incubate at 25°C for 2 hours to obtain suspension four. Among them, the obtained suspension four is an intermediate step of the connection of MB-Au and nucleic acid aptamer Apt1, and the nucleic acid aptamer Apt1 is excessive.

[0096] (42) Remove the supernatant of the suspension four with a magnet to obtain a precipitate one; and then wash the precipitate one with PBS buffer solution with a pH of 7.2 and a volume molar concentration of 0.01 M for three times. In this step, the supernatant of the suspension four is removed with a magnet mainly to remove the excessive nucleic acid aptamer Apt1.

[0097] (43) Resuspend the washed precipitate one to 200 μL of PBS buffer solution containing 2% BSA, and oscillate at 25°C for 0.5 h to obtain suspension five.

[0098] (44) Separate the suspension five by a magnet to obtain MB-Au-Apt1, and resuspend it in 200 μL of PBS solution to obtain the MB-Au-Apt1 dispersion solution.

[0099] Among them, the 2% BSA in step (43) refers to 2% bovine serum albumin by mass fraction.

[0100] Among them, the DNA nucleotide sequence of the nucleic acid aptamer Apt1 in step (41) is 5'-HS-(CH2)6-TACTGCACACGACACCGACTGTCACCATCACCTCGGCGCA-3'.

[0101] In this embodiment, the preparation method of the AuPtNFs dispersion solution comprises the following steps:

[0102] (51) Clean the glassware and magnetic stirrer with freshly prepared aqua regia, and then rinse the glassware and magnetic stirrer with deionized water. Add 2 mL of HAuCl4 solution with a mass concentration of 10 mg / mL to the glassware containing 200 mL of water to obtain a mixed solution four, and heat the mixed solution four to boiling.

[0103] (52) Quickly add 3.2 mL of sodium citrate solution with a mass concentration of 10 mg / mL to the boiling mixed solution four, and continue to boil for 10 min.

[0104] (53)Subsequently, 4 mL of ascorbic acid with a mass concentration of 17.6 mg / mL and 5.0 mL of H2PtCl6 with a mass concentration of 10 mg / mL were added into the boiling solution, respectively, and heating was continued for 20 min, and AuPtNFs dispersion was obtained.

[0105] Please refer to Figure 5 , Figure 5 Figure A in the middle is the image of Au nanoparticles in a transmission electron microscope (TEM); Figure 4 Figures B and C in the middle are images of AuPtNFs prepared in a transmission electron microscope (TEM); Figure 4 Figure D in the middle is a color change diagram of the TMB / H2O2 system with or without the addition of AuPtNFs. The left side of the figure is without the addition of AuPtNFs, and the right side of the figure is with the addition of AuPtNFs, because AuPtNFs can make TMB blue.

[0106] Please refer to Figure 2 Figure E in the middle is the preparation method of AuPtNFs-Apt2 dispersion in this embodiment, which includes the following steps:

[0107] (61)Take 200 μL of AuPtNFs dispersion in step (53), and add prepared nucleic acid aptamer two Apt2 into the AuPtNFs dispersion, and incubate at 25°C for 2 h to obtain suspension six. The obtained suspension six is an intermediate step of AuPtNFs and nucleic acid aptamer two Apt2 connection, and the nucleic acid aptamer two Apt2 is excessive.

[0108] (62)Remove the supernatant of the suspension six with a magnet to obtain precipitate two; and then wash the precipitate two with PBS buffer solution with a pH of 7.2 and a volume molar concentration of 0.01 M. In this step, the supernatant of the suspension six is removed with a magnet mainly to remove the excessive nucleic acid aptamer two Apt2.

[0109] (63)Resuspend the washed precipitate two into 200 μL of PBS buffer solution containing 2% BSA, and oscillate at 25°C for 0.5 h to obtain suspension seven.

[0110] (64)Obtain AuPtNFs-Apt2 by magnetically separating the suspension seven, and resuspend it in 200 μL of PBS solution, and AuPtNFs-Apt2 dispersion is obtained.

[0111] The DNA nucleotide sequence of the aptamer two Apt2 in step (61) is 5'-HS-(CH2)6-GGCCTACCGTAGTGTGCGTGGGCACATGTTCGCGCCACCGTGCTACAAC-3'.

[0112] In the embodiment, the preparation of the AuPtNFs-Probe dispersion solution further includes the following steps:

[0113] (71) Take 200 μL of the AuPtNFs dispersion solution in step (53), and add the DNA probe Probe into the AuPtNFs dispersion solution, and incubate at 25°C for 2 h to obtain a suspension eight.

[0114] (72) Remove the supernatant of the suspension eight by a magnet to obtain a precipitate three; and wash the precipitate three with a PBS buffer solution with a pH of 7.2 and a volume molar concentration of 0.01 M.

[0115] (73) Resuspend the washed precipitate three into 200 μL of a PBS buffer solution containing 2% BSA, and oscillate at 25°C for 0.5 h to obtain a suspension nine.

[0116] (74) Separate the suspension nine by a magnet to obtain AuPtNFs-Probe, and resuspend it in 200 μL of a PBS solution to obtain an AuPtNFs-Probe dispersion solution.

[0117] The nucleotide sequence of the DNA probe Probe in step (71) is 5'-HS-(CH2)6-GTTGTAGCACG-3'.

[0118] Embodiment 2

[0119] Before detecting the unknown concentration of HA, a plurality of groups of different known concentrations of HA need to be configured, and the known concentrations of HA are respectively placed in an electrochemical platform for detection to obtain a series of current data. The standard curve of the relationship between the concentration and the current of HA can be drawn through the series of current data. After obtaining the standard curve, if it is necessary to detect the unknown concentration of HA, the HA is only needed to be placed in the electrochemical platform for detection in the manner of embodiment 1, and the electrochemical platform can calculate the concentration of the unknown concentration of HA according to the result of DPV potential scanning and the standard curve, so as to achieve the purpose of detecting the concentration of HA.

[0120] In the present embodiment, two different HA concentration and current relationship between the standard curve of two different groups of experiments. Among them, experiment one is to known concentration of HA by the following way to detect signal amplification, specifically: through MB-Au-Apt1 capture HA and AuPtNFs-Apt2 form sandwich structure, and using differential pulse voltammetry in HQ / H2O2 and PBS system to realize the amplification of HA detection signal. Experiment two is to known concentration of HA by the following way to detect signal amplification twice, specifically: through MB-Au-Apt1 capture HA and AuPtNFs-Apt2 form sandwich structure, and using differential pulse voltammetry in HQ / H2O2 and PBS system to realize the amplification of HA detection signal to realize the amplification of detection signal; AuPtNFs-Probe through base complementary pairing way to connect MB-Au-Apt1@HA@AuPtNFs-Apt2 on AuPtNFs-Apt2, to form with multiple AuPtNFs-Probe MB-Au-Apt1@HA@AuPtNFs-Apt2-AuPtNFs-Probe, so as to realize the amplification of AuPtNFs, thus realize the amplification of detection signal again.

[0121] The specific operation steps of experiment one are as follows:

[0122] (11) a series of known concentration of HA is configured.

[0123] (12) select one of the known concentration of 15 μL of HA to 100 μL of MB-Au-Apt1 dispersion and completely mixed, get mixed liquid a.

[0124] (13) the mixed liquid a in step (12) is separated by external magnetic field to obtain solid a1.

[0125] (14) the solid a1 in step (13) is suspended in 100 μL of PBS buffer, and 15 μL of AuPtNFs-Apt2 is added and mixed to obtain mixed liquid b.

[0126] (15) the mixed liquid b in step (14) is separated by external magnetic field to obtain solid b1, and the solid b1 is MB-Au-Apt1@HA@AuPtNFs-Apt2, and the solid b1 is resuspended in 10 μL of PBS buffer to obtain sample processing liquid one.

[0127] (16) Take 10 μL of the sample treatment fluid one in step (15) and drop it onto the magnetic glassy carbon electrode of the electrochemical platform, and then immerse the magnetic glassy carbon electrode in the PBS buffer solution containing HQ and H2O2 for 6 min. Among them, the pH of the PBS buffer solution is 7.2, the volume molar concentration is 0.01 M, the mass concentration of HQ is 1.0 mg / mL, and the volume molar concentration of H2O2 is 10 mM.

[0128] (17) Then perform DPV potential scanning from -0.1 V to 0.6 V at a pulse amplitude of 50 mV, a pulse period of 500 ms, and a quiet time of 2 s to obtain a DPV signal graph.

[0129] The detection of the above series of HA with known concentrations is performed according to steps (12) to (17) to obtain a series of DPV signal graphs. The specific results are shown in Figure 6

[0130] The peak current in Figure 6 and the concentration are analyzed, and the standard curve between the concentration of HA in Experiment One and the current is plotted, and the standard curve is shown in Figure 7 . The standard curve equation is ΔI=k*lgC+b. Where ΔI is the current increment, C is the concentration of HA, k is the slope of the standard curve, and b is a constant. HA HA

[0131] The specific operation steps of Experiment Two are as follows:

[0132] (21) Configure a series of HA with known concentrations.

[0133] (22) Select 15 μL of one group of HA with known concentrations and add it to 100 μL of MB-Au-Apt1 dispersion and mix completely to obtain a mixed solution a.

[0134] (23) Separate the mixed solution a in step (22) by an external magnetic field to obtain a solid a1.

[0135] (24) Suspend the solid a1 in step (24) in 100 μL of PBS buffer, and then add 15 μL of AuPtNFs-Apt2 and mix to obtain a mixed solution b.

[0136] (25) Separate the mixed solution b in step (14) by an external magnetic field to obtain a solid b1, which is MB-Au-Apt1@HA@AuPtNFs-Apt2, and resuspend the solid b1 in 1 / 4 SSC buffer with a volume of 100 μL, and then add 20 μL of AuPtNFs-Probe dispersion to obtain a mixed solution c. ​​​

[0137] (26) The mixed solution c in step (25) is separated by an external magnetic field to obtain solid c1, which is MB-Au-Apt1@HA@AuPtNFs-Apt2-AuPtNFs-Probe, and the solid c1 is resuspended in 10 μL of PBS buffer to obtain sample processing solution two.

[0138] (27) 10 μL of the sample processing solution two in step (26) is taken and dropped onto the magnetic glassy carbon electrode of the electrochemical platform, and then the magnetic glassy carbon electrode is immersed in a PBS buffer solution containing HQ and H2O2 for 6 min. The PBS buffer solution has a pH of 7.2 and a volume molar concentration of 0.01 M, the mass concentration of HQ is 1.0 mg / mL, and the volume molar concentration of H2O2 is 10 mM.

[0139] (28) Then, DPV potential scanning is performed from -0.1 V to 0.6 V at a pulse amplitude of 50 mV, a pulse period of 500 ms, and a quiet time of 2 s to obtain a DPV signal graph.

[0140] The detection of the above series of known concentrations of HA is performed according to steps (22) to (28) to obtain a series of DPV signal graphs. The specific results are shown in Figure 8 .

[0141] The peak current in Figure 8 and the concentration are analyzed, and a standard curve between the concentration of HA and the current in Experiment Two is plotted. The standard curve is shown in Figure 9 . The standard curve equation is ΔI=k*lgC HA +b. Where ΔI is the current increment, C HA is the concentration of HA, k is the slope of the standard curve, and b is a constant.

[0142] Through the analysis of Figure 8 to Figure 9 , it can be known that Figure 8 the standard equation of the standard curve in Experiment One is ΔI=(13.77±0.71)*lgC HA +(30.82±0.98). Figure 8 the standard equation of the standard curve in Experiment Two is ΔI=(8.55±0.33)*lgC HA +(36.27±0.67).

[0143] The calculation formula of the detection limit (LOD) is LOD=3σ / k, where σ is the standard deviation of the blank sample, and k is the slope of the standard curve. The detection limit in Experiment One and Experiment Two can be calculated by the above calculation formula.

[0144] The detection limit refers to the minimum concentration or content of an element required to produce an analytical signal that can be reliably detected. Therefore, the lower the detection limit, the higher the sensitivity of the method or instrument.

[0145] Through the Figure 6 to Figure 7 Analysis of the data shows that, after amplifying the HA detection signal once in Experiment 1, the electrochemical platform can sensitively detect HA concentrations ranging from 10 pg / mL to 100 ng / mL, with a detection limit of 2.4 pg / mL. Through further analysis... Figure 8 and Figure 9 Analysis of the data shows that, in Experiment 2, after amplifying the HA detection signal twice, the electrochemical platform was able to sensitively detect HA concentrations ranging from 0.5 pg / mL to 10 ng / mL, with a detection limit of 0.3 pg / mL. Analysis of the detection limit reveals that the detection limit after secondary amplification of the HA detection signal is approximately 8 times smaller than that after primary amplification. Therefore, secondary amplification of the HA detection signal enables further sensitive detection of HA.

[0146] Through the Figure 6 and Figure 8 Analysis of these two standard curves shows that... Figure 6 The linear fit R of the standard curve 2 =0.982, Figure 8 The linear fit R of the standard curve 2 =0.991. The closer the goodness-of-fit value is to 1, the better the fit of the standard curve. Therefore, it can be concluded that... Figure 8 A better fit to the standard curve in the standard curve results in a more accurate calculated concentration of HA.

[0147] Analysis of the data from Experiments 1 and 2 shows that, by amplifying the HA detection signal twice, this invention can detect HA at even lower concentrations, and the detection limit is reduced by 8 times. This improves the detection effect and enables more sensitive detection of HA.

[0148] Example 3

[0149] Two groups of HA with a concentration of 50 pg / mL were selected, and experiments one and two of Example 2 were performed on these two groups of HA, respectively. The results were as follows: Figure 10 The experimental results.

[0150] Figure 10 The red curve in the middle represents the DPV signal obtained by HA through Experiment 2; Figure 10 The black curve in the middle represents the DPV signal obtained by HA through Experiment 2. Figure 10It can be seen that the DPV current peak value of the red curve is obviously greater than that of the black curve. The difference between experiment two and experiment one is that AuPtNFs is amplified by the base complementary pairing principle of DNA in experiment two. Therefore, it can be effectively proved that the detection signal of HA can be increased by amplifying AuPtNFs through the base complementary pairing principle of DNA.

[0151] The above describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection required by the present application is defined by the appended claims and their equivalents.

Claims

1. A detection method for detecting H1N1 influenza virus for non-diagnostic purposes, characterized in that, It includes the following steps: The hemagglutinin HA sample to be tested was processed as follows: The HA sample to be tested was added to MB-Au-Apt1 dispersion and mixed completely to obtain mixture one; then the solids in mixture one were separated by an external magnetic field, and the separated solids were resuspended in PBS buffer. AuPtNFs-Apt2 dispersion was added to PBS buffer and mixed to obtain mixture two. The solids in mixture two were separated using an external magnetic field to obtain MB-Au-Apt1@HA@AuPtNFs-Apt2, which was then resuspended in 1 / 4 SSC buffer. AuPtNFs-Probe dispersion was then added to the 1 / 4 SSC buffer and mixed to obtain mixture three. The solids in mixture three were separated using an external magnetic field, and the separated solids were resuspended in PBS buffer to obtain the sample processing solution. The MB-Au-Apt1 dispersion includes MB-Au dispersion and nucleotide aptamer one (Apt1); the nucleotide sequence of nucleotide aptamer one is SEQ ID NO.

1. The AuPtNFs-Apt2 dispersion includes AuPtNFs dispersion and nucleotide aptamer two (Apt2); the nucleotide sequence of nucleotide aptamer two is SEQ ID NO.

1. NO.2; The AuPtNFs-Probe dispersion includes an AuPtNFs dispersion and a DNA probe Probe; the nucleotide sequence of the DNA probe Probe is SEQ ID NO.3; the preparation method of the AuPtNFs dispersion includes the following steps: adding HAuCl4 solution to a container filled with water to obtain mixture four, heating mixture four to boiling; rapidly adding sodium citrate solution to the boiling mixture four, while stirring and boiling, and after boiling for a period of time, adding ascorbic acid and H2PtCl6 respectively, and continuing to heat for 20 minutes to obtain the AuPtNFs dispersion; The prepared sample processing solution was dropped onto the electrode of the electrochemical platform for detecting HA, and then the electrode was immersed in PBS buffer containing hydroquinone HQ and H2O2 for a period of time. Then, a DPV potential scan was performed from -0.1V to 0.6V with a pulse amplitude of 50mV, a pulse period of 500ms, and a quiet time of 2s. The electrochemical platform can determine the concentration of HA in the sample to be tested by using potential scanning results.

2. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 1, characterized in that, During the processing of the sample HA to be tested, the nucleic acid aptamer Apt1 in the MB-Au-Apt1 dispersion is used to capture HA and form a sandwich structure MB-Au-Apt1@HA@AuPtNFs-Apt2 with the nucleic acid aptamer Apt2 in the AuPtNFs-Apt2 dispersion.

3. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 1, characterized in that, The AuPtNFs-Probes in the AuPtNFs-Probe dispersion are linked to the AuPtNFs-Apt2 on MB-Au-Apt1@HA@AuPtNFs-Apt2 through complementary base pairing, thereby forming MB-Au-Apt1@HA@AuPtNFs-Apt2-AuPtNFs-Probe with multiple AuPtNFs-Probes.

4. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 1, characterized in that, Before processing the sample HA to be tested, a nanocomposite material needs to be prepared, which includes MB-Au dispersion and AuPtNFs dispersion.

5. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 4, characterized in that, The preparation method of the MB-Au dispersion includes the following steps: Preparation of Fe3O4 dispersion; The prepared Fe3O4 dispersion was dispersed in a mixture of ethanol and water and ultrasonically dispersed. After ultrasonication, 25% ammonia was added, and tetraethyl orthosilicate was added dropwise. The mixture was then stirred at room temperature to obtain suspension one. The suspension was separated by a magnet and then washed multiple times with ethanol and deionized water to obtain Fe3O4@SiO2. The prepared Fe3O4@SiO2 was dispersed in a mixture of ethanol and deionized water and sonicated. After sonication, 3-aminopropyltriethoxysilane was added dropwise and stirred at room temperature for a period of time under nitrogen protection to obtain suspension II. The suspension was separated by a magnet and washed multiple times with ethanol and deionized water to obtain Fe3O4@SiO2-NH2, which was then dispersed in 50 mL of deionized water to obtain MB dispersion. A certain amount of the MB dispersion was taken and ultrasonically treated. Then, HAuCl4 aqueous solution and sodium citrate aqueous solution were added respectively, and the mixture was stirred. After stirring for a period of time, sodium borohydride was added to react and suspension III was obtained. The suspension was separated by a magnet and washed multiple times with ethanol and deionized water to obtain MB-Au, which was then dispersed in 50 mL of deionized water to obtain the MB-Au dispersion.

6. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 5, characterized in that, The preparation method of the MB-Au-Apt1 dispersion includes the following steps: Take a certain amount of the prepared MB-Au dispersion, add nucleic acid aptamer Apt1 to the MB-Au dispersion, and incubate at 25°C for 2 hours to obtain suspension four; The supernatant of the suspension four was removed using a magnet to obtain precipitate one; then precipitate one was washed three times with PBS buffer. The washed precipitate was resuspended in 200 μL of PBS buffer containing 2% BSA and shaken at 25°C for 0.5 h to obtain suspension V. MB-Au-Apt1 was obtained by separating the suspension five with a magnet and then resuspending it in 200 μL of PBS solution to obtain the MB-Au-Apt1 dispersion.

7. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 6, characterized in that, The preparation of AuPtNFs-Apt2 further includes the following steps: Take a certain amount of the prepared AuPtNFs dispersion, add nucleic acid aptamer II Apt2 to the AuPtNFs dispersion, and incubate at 25°C for 2 hours to obtain suspension VI; The supernatant of suspension six was removed using a magnet to obtain precipitate two; precipitate two was then washed three times with PBS buffer. The washed precipitate was resuspended in 200 μL of PBS buffer containing 2% BSA and shaken at 25 °C for 0.5 h to obtain suspension seven. AuPtNFs-Apt2 was obtained by separating the suspension using a magnet and then resuspending it in 200 μL of PBS solution to prepare the AuPtNFs-Apt2 dispersion.

8. The detection method for detecting H1N1 influenza virus for non-diagnostic purposes as described in claim 7, characterized in that, The preparation of the AuPtNFs-Probe dispersion further includes the following steps: Take a certain amount of the prepared AuPtNFs dispersion, add the DNA probe Probe to the AuPtNFs dispersion, and incubate at 25°C for 2 hours to obtain suspension VIII; The supernatant of the suspension 8 was removed using a magnet to obtain precipitate 3; then the precipitate 3 was washed with PBS buffer. The washed precipitate was triple-suspended in 200 μL of PBS buffer containing 2% BSA and shaken at 25 °C for 0.5 h to obtain suspension nine. The suspension was separated by a magnet to obtain AuPtNFs-Probe, which was then resuspended in 200 μL of PBS solution to obtain the AuPtNFs-Probe dispersion.

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