A method for detecting avian influenza virus H9 using a self-driven and self-sensing cantilever beam sensor

Abstract

The present invention relates to a method for detecting avian influenza virus H9 by a self-driven and self-sensing cantilever beam sensor in the field of detection technology. The present invention includes a microcantilever sensor modified with a nanomaterial with an amino functional group, an electrical signal amplifier, and an analog-to-digital converter. The nanomaterial with an amino functional group is a metal-organic framework of UiO-66-NH2, and the metal-organic framework of UiO-66-NH2 is a UiO-66-NH2 / AuNPs nanomaterial. The preparation method is as follows: the microcantilever sensor is modified with the UiO-66-NH2 / AuNPs nanomaterial; the modified microcantilever sensor is connected to a self-made circuit board, an analog-to-digital converter, and a development board to form a detection device. And the device is connected to a computer for signal processing; an H9 standard solution is dropped at the detection end of the cantilever sensor, and the results are analyzed by collecting electrical signals. The present invention has rapid detection and convenient operation.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for detecting avian influenza virus H9 using a self-driven self-sensing cantilever beam sensor. Background Art

[0002] The avian influenza H9 virus was first discovered in sick turkeys in 1996. It has attracted widespread attention from the world due to its high infectivity and mortality rate. Every year, the outbreak of influenza virus poses a huge threat to people's economy and health. According to previous reports, H9 can be transmitted to mammals and humans, indicating that H9 has potential hazards. Traditional virus diagnosis methods usually include virus isolation, serological tests, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), etc. Although these methods are highly sensitive, they are not suitable for real-time detection of viruses due to their long analysis time, high detection cost, and the need for professional operators. Therefore, it is of great practical value to develop a detection method that can achieve quantitative, highly sensitive, easy to operate, and low-cost detection of viruses.

[0003] In the prior art, biosensors for viral antigen detection all use antibodies as biological recognition elements, which are costly, difficult to modify, and inconvenient to use, thus limiting their scope of use.

[0004] In recent years, biosensors have been widely used in biological detection. Microcantilever-based biosensors have attracted much attention due to their miniaturization, label-free detection and high sensitivity. Generally speaking, microcantilever can be used as either a microbalance or a surface stress sensor. The former detects by measuring the shift in the resonant frequency caused by the change in mass before and after the adsorption of the target molecule on the surface; while the latter detects by detecting the bending deformation of the cantilever during the biochemical reaction. All sensors based on microcantilever are equipped with a readout device that can measure the mechanical response of the system. The piezoelectric method in the electrical method (i.e., a layer of piezoelectric material is attached to the surface of the cantilever. When the cantilever is bent and deformed, the piezoelectric layer will generate induced charges, and the bending deformation of the microcantilever will be reflected by measuring the amount of induced charges) has become a new field of microcantilever sensor research due to its high sensitivity, fast response, low cost and easy circuit integration.

[0005] PVDF (polyvinylidene fluoride) is a piezoelectric polymer with the advantages of large piezoelectric constant (under the same external load conditions, the voltage is more than ten times that of piezoelectric ceramics), light, thin, soft, and easy to process into any shape. Therefore, we chose PVDF piezoelectric film as the sensing element, and established a sensor device with self-driven and self-sensing characteristics through self-made circuits, analog-to-digital converters, development boards, and personal computer detection programs. When the stress on the cantilever surface changes, a voltage signal can be obtained. The antigen-antibody specific force causes the cantilever surface stress to change, driving the constructed detection device, and no additional excitation conditions are required to drive it, which is the self-driving part; at the same time, due to the positive piezoelectric effect of the piezoelectric film, the sensor surface generates self-induced charges, and the charge signal is converted into a voltage signal output through the analog-to-digital converter and the development board, completing the sensing part, which is the self-sensing part; in addition, nanomaterials have the advantages of large specific surface area and easy surface modification, which helps to fix more signal probes. In recent years, it has become a common method to improve the sensitivity of biosensors. UiO-66-NH2 is a classic zirconium-based metal organic framework (MOF) with rich amino and carboxyl groups on its surface, which can covalently immobilize antibodies. Gold nanoparticles (AuNPs) can be used as a good substrate material for immobilizing biomolecules and amplifying detection signals because of their large specific surface area and good biocompatibility. Summary of the invention

[0006] The present invention solves the problems of high cost, difficulty in modification and limited application scope of the technology during the detection process, and designs and prepares a method for detecting avian influenza H9 subtype virus with rapid detection, convenient operation and wide application scope.

[0007] The object of the present invention is achieved by a method for detecting avian influenza virus H9 using a self-driven self-sensing cantilever beam sensor, comprising a microcantilever sensor modified with a nanomaterial having an amino functional group, an electrical signal amplifier, and an analog-to-digital converter, wherein the nanomaterial having an amino functional group is a metal organic framework of UiO-66-NH2, and the metal organic framework of UiO-66-NH2 is a UiO-66-NH2 / AuNPs nanomaterial, comprising the following steps:

[0008] (1) Modification of microcantilever sensor: clean the microcantilever sensor, drop UiO-66-NH2 / AuNPs nanomaterial dispersion on the surface of the sensor detection end to obtain a pretreated microcantilever sensor, dry it at room temperature, and drop a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide on the surface of the pretreated microcantilever sensor;

[0009] (2) Assembly: Connect the modified microcantilever sensor to the homemade circuit board, analog-to-digital converter, and development board to form a detection device. Connect the device to a computer for signal processing;

[0010] (3) Detection and data analysis: Add H9 standard solution to the detection end of the cantilever sensor and collect electrical signals to analyze the results.

[0011] Furthermore, step (1) specifically includes the following sub-steps:

[0012] (1.1) Clean the detection end of the microcantilever sensor with anhydrous ethanol and deionized water in turn, blow dry with nitrogen, and seal for storage;

[0013] (1.2) Dropping UiO-66-NH2 / AuNPs nanomaterial dispersion on the surface of the sensor detection end and drying at room temperature to obtain a pretreated microcantilever sensor;

[0014] (1.3) dripping a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide onto the surface of the pretreated microcantilever sensor obtained in step (1.2), and then washing with a phosphate buffer solution;

[0015] (1.4) Then drop H9 antibody on the surface of the sensor detection end treated in step (1.3) and let it stand at 4°C for 12 hours;

[0016] (1.5) Rinse the surface of the sensor detection end obtained in step (1.4) with a phosphate buffer solution;

[0017] (1.6) Then, 1% bovine serum albumin was added dropwise to the surface of the detection end of the sensor obtained in step (1.5) for blocking, and finally, the sensor was stored at 4°C.

[0018] Furthermore, the preparation of the UiO-66-NH2 / AuNPs nanomaterial in step (2) includes the following steps:

[0019] (2.1) Dissolve zirconium chloride, acetic acid and deionized water in N,N-dimethylformamide and stir to obtain a mixed solution. Add 2-aminoterephthalic acid to the mixed solution and continue stirring.

[0020] (2.2) subjecting the solution obtained in step (2.1) to a hydrothermal reaction at 120° C. for 24 hours, centrifuging, washing, and drying to obtain UiO-66-NH2;

[0021] (2.3) Using trisodium citrate to reduce gold chloride, spherical gold nanoparticles AuNPs were obtained;

[0022] (2.4) The UiO-66-NH2 obtained in step (2.2) is mixed with the spherical gold nanoparticles AuNPs obtained in step (2.4), and after vigorous shaking, the UiO-66-NH2 / AuNPs nanomaterial can be obtained.

[0023] Furthermore, in the step (1.3), a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide is added dropwise to the surface of the sensor detection end, maintained for 1 hour, and then rinsed with a phosphate buffer solution.

[0024] Furthermore, the concentration of the UiO-66-NH2 / AuNPs nanomaterial dispersion is 5 to 10 mg / mL.

[0025] Furthermore, in step (3), the sensor detection end adopts a PVDF film.

[0026] Furthermore, in step (1.4), the concentration of H9 is 100 ng / mL-1000 ng / mL.

[0027] Furthermore, in step (2.2), the vacuum drying conditions are 70-90° C. and a pressure of 10-100 Pa for 12-24 h.

[0028] The present invention modifies the detection end of the micro-cantilever sensor by UiO-66-NH2 / AuNPs nanomaterials, so that the antibody can be tightly combined with the substrate and the signal is amplified. At the same time, the specific reaction of the antigen and antibody causes the surface stress of the cantilever to change, thereby changing the output voltage of the sensor, and establishing the relationship between the H9 concentration and the sensor output voltage. The micro-cantilever sensor constructed based on electrical principles realizes self-driven self-sensing detection of H9. The present invention has many advantages such as low cost, simple operation, and no need for external power supply. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a transmission electron microscopy image of UiO-66-NH2 / AuNPs of the present invention.

[0030] Figure 2 It is a structural schematic diagram of the PVDF piezoelectric film of the present invention and a physical diagram of the sensor.

[0031] Figure 3 This is a schematic diagram of the sensor of the present invention, a left: sealing ring, a right: base; (b) combination diagram of sealing ring and PVDF piezoelectric film, (c) schematic diagram of final assembly.

[0032] Figure 4 It is an integrated schematic diagram of the virus detection device of the present invention.

[0033] Figure 5 It is the voltage response of the micro-cantilever sensor of the present invention to H9.

[0034] Figure 6It is a comparison of the sensor signal amplification effects of different materials of the present invention.

[0035] Figure 7 It is a schematic diagram of the optimization of the UiO-66-NH2 concentration of the present invention.

[0036] Figure 8 It is a schematic diagram of the optimization of the activation time of the present invention. DETAILED DESCRIPTION

[0037] The present invention is further analyzed, explained and compared by means of specific embodiments and comparative examples.

[0038] 1. First, prepare UiO-66-NH2 nanomaterials, the steps are as follows:

[0039] (1) First, zirconium chloride (0.1498 g), acetic acid (5 mL) and deionized water (148 μL) were dissolved in 40 mL of N,N-dimethylformamide and stirred to obtain a mixed solution. Then, 2-aminoterephthalic acid (141.1 mg) was added to the mixed solution and stirred continuously.

[0040] (2) After stirring for 2 h, it was added to a 100 mL reactor for hydrothermal reaction and heated at 120 °C for 24 h. After cooling to room temperature, the product was collected by centrifugation at 6000 rpm and repeatedly washed with N,N-dimethylformamide. Finally, the white solid precipitate was dried in a vacuum oven at 90 °C for 24 h to obtain UiO-66-NH2 nanomaterials.

[0041] Second, spherical AuNPs were prepared, and the steps were as follows:

[0042] (1) First, add 55 μL of 9 wt % gold chloride to 49.95 mL of ultrapure water, boil and reflux, then add 0.75 mL (10 mg / mL) of trisodium citrate as a reducing agent, stir continuously for 15 min to obtain a wine-red dispersed solution containing AuNPs, and then stop heating;

[0043] (2) Allow the solution to cool naturally to room temperature, centrifuge, redisperse in 10 mL of deionized water, and store at 4 °C in the dark for future use to obtain AuNPs.

[0044] 3. Synthesis of UiO-66-NH2 / AuNPs: The amino groups in UiO-66-NH2 can be used to effectively adsorb and capture AuNPs. AuNPs are mixed with UiO-66-NH2 and shaken vigorously to obtain an aqueous solution of UiO-66-NH2 / AuNPs composite nanomaterials ( Figure 1 ).

[0045] 4. The steps for preparing the sensor are as follows:

[0046] The structural diagram of PVDF piezoelectric film is shown in Figure 1 , from top to bottom are the upper surface silver layer, PVDF piezoelectric layer, lower surface silver layer, and PET polyester sheet (used to protect the lower surface silver layer).

[0047] 5. The processing steps of the experimental sensor are as follows:

[0048] The upper surface of the sensor is treated with gold spraying, which can be used to protect the electrode and modify the surface functionally;

[0049] Take a 5 cm RVVP double-core shielded wire (to prevent external static electricity from interfering with the sensor signal), remove 5 mm of the outer skin at both ends, and weld the two inner core wires at one end to the pins on the upper and lower surfaces of the sensor, and weld the other end to the PH 2.0 terminal wire. To facilitate subsequent experiments, the upper surface is connected to the black wire and the lower surface is connected to the red wire by default;

[0050] Finally, wrap the Parafilm sealing film around the welding point to fix it and prevent the electrodes from contacting each other. Figure 2 .

[0051] 6. Assemble the sensor. The sensor consists of three parts: sealing ring, PVDF piezoelectric film and base ( Figure 3 );

[0052] (1) First, apply epoxy resin glue evenly on the surface of the sealing ring;

[0053] (2) Cover the sensor detection area on top ( Figure 3 b, the hollow position is the sensing part);

[0054] (3) After the glue has solidified, apply epoxy resin glue around the sealing ring and push the sensor and the sealing ring into the base;

[0055] (4) Apply epoxy resin glue to the interface for sealing ( Figure 3 c).

[0056] 7. Modify the microcantilever sensor using UiO-66-NH2 / AuNPs, the steps are as follows:

[0057] (1) The previously prepared unmodified microcantilever sensor was cleaned with anhydrous ethanol and deionized water in sequence, blown dry with nitrogen, and then stored in a sealed bag;

[0058] (2) Drop UiO-66-NH2 / AuNPs nanomaterial dispersion (7 mg / mL) on the sensor surface and dry at room temperature;

[0059] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide mixture dropwise, wait for 1 h, and then rinse the sensor surface with phosphate buffer solution;

[0060] (4) Drop H9 antibody on the sensor surface and place it at 4 °C for 12 h;

[0061] (5) Rinse the modified microcantilever sensor with phosphate buffer solution;

[0062] (6) Blocking was performed with 1% bovine serum albumin, and the modified microcantilever sensor was stored at 4 °C to eliminate nonspecific adsorption.

[0063] (7) Finally, connect the modified micro-cantilever sensor to the homemade circuit, analog-to-digital converter, and development board, and connect it to the computer via a USB data cable. The assembly is complete. Figure 4 ).

[0064] 8. Test part:

[0065] (1) Add 100 μL of H9 standard solution of different concentrations to the detection area of ​​the microcantilever sensor;

[0066] (2) When an immune reaction occurs between the antigen and the antibody, a specific force is generated, causing the stress on the upper and lower surfaces of the microcantilever to change.

[0067] The above changes lead to an increase in the output voltage. By recording the voltage changes produced by antigen solutions of different concentrations through software, the relationship between the concentration of the antigen solution and the output voltage can be established.

[0068] The present invention constructs a new type of micro-cantilever sensor device with self-driven and self-sensing characteristics for virus detection by combining PVDF piezoelectric film with integrated circuits. Under optimal conditions, the concentration of H9 is in the range of 100 ng / mL-1000 ng / mL, and its logarithmic value has a good linear relationship with the voltage output peak of the micro-cantilever sensor. The linear equation is: y=-122.738+36.986lgC, the correlation coefficient is 0.991, and the detection limit is 50 ng / mL. The micro-cantilever sensor proposed by the present invention based on the piezoelectric principle also provides a platform for other analytes. It does not require an external power supply, but relies on the voltage generated by the stress change of the piezoelectric film itself to achieve self-driven and self-sensing detection of H9, which has broad application prospects ( Figure 5-8 ).

Claims

1. A method for detecting avian influenza virus H9 using a self-driven self-sensing cantilever beam sensor, the method being used for non-disease diagnosis and / or disease treatment purposes, characterized in that: The invention comprises a micro-cantilever sensor modified with a nano material having an amino functional group, an electric signal amplifier, and an analog-to-digital converter, wherein the nano material having an amino functional group is a metal organic framework of UiO-66-NH2, and the metal organic framework of UiO-66-NH2 is a UiO-66-NH2 / AuNPs nano material. The preparation method thereof comprises the following steps: (1) Preparation of UiO-66-NH2 / AuNPs: zirconium chloride, acetic acid and deionized water are dissolved in N,N-dimethylformamide and stirred to obtain a mixed solution. 2-aminoterephthalic acid is added to the mixed solution and stirred continuously for hydrothermal reaction at 120°C for 24 hours. The mixture is centrifuged, washed with N,N-dimethylformamide and dried under vacuum to obtain UiO-66-NH2, i.e., a zirconium-based metal organic framework (MOF). Gold chloride is reduced with trisodium citrate to obtain spherical gold nanoparticles AuNPs, i.e., gold nanoparticles. UiO-66-NH2 is mixed with spherical gold nanoparticles AuNPs and shaken vigorously to obtain UiO-66-NH2 / AuNPs nanomaterial, i.e., a nanomaterial having an amino functional group and a metal organic framework composite gold nanoparticle of UiO-66-NH2. (2) Modifying the microcantilever sensor: clean the microcantilever sensor, drop the UiO-66-NH2 / AuNPs nanomaterial dispersion on the surface of the sensor detection end to obtain a pretreated microcantilever sensor, dry it at room temperature, mix 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, drop it on the surface of the pretreated microcantilever sensor, and then drop the H9 antibody on the surface of the microcantilever sensor; (3) Assembly: Connect the modified microcantilever sensor to a homemade circuit board, an analog-to-digital converter, and a development board to form a detection device, and connect the device to a computer for signal processing. The sensor detection end uses a PVDF film; (4) Detection and data analysis: H9 standard solution was added to the detection end of the cantilever sensor, and the results were analyzed by collecting electrical signals.

2. The method for detecting avian influenza virus H9 by a self-driven self-sensing cantilever beam sensor according to claim 1, characterized in that: Step (2) specifically includes the following steps: (2.1) The detection end of the microcantilever sensor was cleaned with anhydrous ethanol and deionized water in turn, dried with nitrogen, and sealed for storage; (2.2) dropping UiO-66-NH2 / AuNPs nanomaterial dispersion on the surface of the sensor detection end and drying at room temperature to obtain a pretreated microcantilever sensor; (2.3) mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and dripping the mixture onto the surface of the pretreated microcantilever sensor obtained in step (2.2), and then washing the mixture with a phosphate buffer solution; (2.4) drop the H9 antibody on the surface of the sensor detection end treated in step (2.3) and let it stand at 4°C for 12 hours; (2.5) Rinse the surface of the sensor detection end obtained in step (2.4) with a phosphate buffer solution; (2.6) Then, 1% bovine serum albumin was added dropwise to the surface of the detection end of the sensor obtained in step (2.5) for blocking, and finally, the sensor was stored at 4°C.

3. The method for detecting avian influenza virus H9 by a self-driven self-sensing cantilever beam sensor according to claim 2, characterized in that: In the step (2.3), 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are mixed, added dropwise to the surface of the sensor detection end, maintained for 1 hour, and then rinsed with a phosphate buffer solution.

4. The method for detecting avian influenza virus H9 by a self-driven self-sensing cantilever beam sensor according to claim 2, characterized in that: The concentration of the UiO-66-NH2 / AuNPs nanomaterial dispersion is 5-10 mg / mL.

5. The method for detecting avian influenza virus H9 by a self-driven self-sensing cantilever beam sensor according to claim 1, characterized in that: In step (4), the concentration of H9 is 100 ng / mL-1000 ng / mL.

6. The method for detecting avian influenza virus H9 by a self-driven self-sensing cantilever beam sensor according to claim 5, characterized in that: In step (1), the vacuum drying conditions are 70-90° C. and a pressure of 10-100 Pa for 12-24 hours.

Images