An ultrahigh sensitive lyophilized surface acoustic wave immunosensor based on double-antibody sandwich method of dmsn@au nps and gold staining solution

Abstract

The application discloses an ultrahigh sensitive Love wave surface acoustic wave immunosensor based on a double-antibody sandwich method of DMSN@AuNPs and gold staining solution. The immunoelectrode coated with a good capture antibody and non-specifically blocked can specifically capture an antigen to be measured, and then a DMSN@AuNPs@ detection antibody is combined with a gold staining solution to realize signal amplification, and real-time quantitative detection is realized by calculating the phase change of the sensor. The DMSN loaded with a large amount of AuNPs by utilizing the advantages of rich channels, large specific surface area, uniform particle size, easy functionalization and adjustable structure can greatly improve the signal amplification effect of gold staining, and greatly reduce the linear range and detection lower limit of the Love wave surface acoustic wave sensor for detecting antigens based on the traditional colloidal gold combined with the gold staining solution. The sensor and the nanomaterials can be mass-produced and long-term stored, and have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanobiosensing and biodetection technology, and particularly relates to an ultrasensitive Lep-type surface acoustic wave immunosensor based on the double-antibody sandwich method of DMSN@AuNPs and gold staining solution, specifically a Lep-type surface acoustic wave immunosensor for detecting procalcitonin (PCT), a pneumonia marker, in human exhaled gas condensate. Background Technology

[0002] Pneumonia and other lower respiratory tract infections are the fourth leading cause of death worldwide. Diagnosing pneumonia currently presents several challenges, particularly in differentiating between viral, bacterial, and fungal pneumonia. Different types of pneumonia often present with similar symptoms, leading to misdiagnosis and delayed treatment. Patients may also be infected with multiple pathogens simultaneously, further complicating diagnosis. Preliminary examinations (such as chest X-rays and routine blood tests) have low sensitivity and specificity for differentiating types and rely on sophisticated and expensive optical equipment. Bacterial cultures are time-consuming, and manual detection of viruses and fungi is even more complex.

[0003] Ultrasensitive biomarker detection is a hot research topic in the global biomedical field, providing a foundation for the early detection of respiratory diseases. Exhaled breath condensate (EBC) is a non-invasive sample, particularly suitable for the elderly and children. Compared to other body fluids, EBC contains fewer interfering substances and is more closely associated with the airways, containing many markers related to airway inflammation. Procalcitonin (PCT) is the most commonly used indicator for assessing the type and severity of infection, often used to differentiate between bacterial and non-bacterial pneumonia. PCT is higher in patients with bacterial pneumonia, usually not elevated in viral infections, and lower in mycoplasma pneumonia.

[0004] The challenge in detecting EBC samples lies in the extremely low concentrations of biomarkers. For example, the optimal cutoff value for PCT in plasma is 1.09 ng / mL, while the PCT concentration in healthy individuals with EBC is 0.009 ± 0.005 ng / mL, rising to 0.096 ± 0.042 ng / mL in patients with community-acquired pneumonia (CAP). To more accurately and rapidly detect the extremely low concentrations of pneumonia biomarkers in EBC, the development of ultrasensitive integrated sensors with micro / nano structures is crucial for advancing next-generation detection technologies.

[0005] The Lep-type surface acoustic wave (SAW) sensor boasts numerous advantages, including high sensitivity, resistance to liquid corrosion, real-time detection, and mass production capability, making it suitable for the quantitative detection of disease biomarkers. Currently, immunogold immunoassay remains the primary method for enhancing the sensitivity of Lep-type SAW sensors. However, traditional immunogold immunoassay-based SAW biosensors have detection limits for protein biomarkers in the range of 0.1-1 ng / mL, and their linear range (on the order of ng / mL) is insufficient for detecting low-concentration disease biomarkers in end-stage bronchopulmonary angina (EBC). To achieve even lower biomarker detection, it is necessary to further improve the detection sensitivity of Lep-type SAW immunoassay sensors and reduce their linear range and detection limit. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing an ultrasensitive Lep-type surface acoustic wave immunosensor based on a sandwich method of DMSN@AuNPs and gold staining solution.

[0007] The present invention is achieved through the following technical solution: an ultrasensitive Lep-type surface acoustic wave immunosensor based on the double antibody sandwich method of DMSN@AuNPs and gold staining solution, the sensor comprising a Lep-type surface acoustic wave immunoelectrode, DMSN@AuNPs@detection antibody solution, gold staining solution, sample dilution solution, PBS buffer, electrode cleaning solution and fluid detection chamber;

[0008] A Leff type surface acoustic wave immunoelectrode coated with antibody and non-specifically blocked is installed in a fluid detection chamber. The Leff type surface acoustic wave immunoelectrode is a Leff type surface acoustic wave electrode coated with capture antibody, which can specifically capture the antigen biomarker to be detected. The DMSN@AuNPs@ detection antibody is obtained by mixing thiolized DMSNs and AuNPs solution, sonicating, and then modifying the detection antibody corresponding to the antigen to be tested. The signal is amplified by using the DMSN@AuNPs@ detection antibody that can bind to the antigen in combination with gold staining solution. Real-time quantitative detection is performed by calculating the amplitude, phase, and resonant frequency changes of the output signal caused by the surface mass deposition of the Leff type surface acoustic wave sensor.

[0009] Furthermore, the capture antibody-coated Leff type surface acoustic wave electrode is a Leff type surface acoustic wave electrode modified with the capture antibody Ab1 corresponding to the antigen to be tested. The fabrication process of the Leff type surface acoustic wave electrode is as follows: First, input and output split-finger gold interdigitated electrodes (IDTs) are fabricated on the surface of an ST-Z cut quartz wafer substrate using MEMS micromachining technology. The IDTs can excite horizontal shear-type surface acoustic waves in the ST-Z cut quartz piezoelectric material. Then, a silica waveguide layer is fabricated on top of the IDTs using plasma vapor deposition to focus the sound waves while preventing liquid interference with the operation of the IDTs and thus preventing signal attenuation. A gold layer is deposited on the delay line region above the waveguide layer, and the surface of the gold layer is modified with the capture antibody corresponding to the antigen to be tested.

[0010] Further, the method for modifying the capture antibody corresponding to the antigen to be tested is as follows: First, the gold layer of the Lep-type surface acoustic wave electrode is washed sequentially with NaOH and HCl solutions, and then activated with piranha solution; after washing and drying, Staphylococcus aureus protein A (SPA) solution is added to each electrode and incubated for a period of time to promote the directional fixation of the antibody; then, the capture antibody Ab1 of the antigen to be tested and 10% BSA are added to the surfaces of the working and reference electrodes, respectively, and incubated overnight at 4°C; finally, the free binding sites of the working and reference channels are blocked for a period of time with 10% BSA solution at 37°C to reduce non-specific binding; at the end of each of the above steps, the electrodes are washed with PBS and dried with nitrogen.

[0011] Further, the DMSN@AuNPs@detection antibody solution is a DMSN@AuNPs solution modified with the detection antibody Ab2 corresponding to the antigen to be tested. The specific modification method is to adjust the pH of the DMSN@AuNPs solution to 8-9 with K2CO3 solution, then add the detection antibody, and incubate on a shaker at 37°C for 1 hour; then centrifuge at 14000 rpm at 4°C to wash away excess detection antibody, and take the precipitate and redissolve it in 0.01M PBS at pH 7.4 with 2% BSA (g / ml).

[0012] Furthermore, the DMSN@AuNPs solution is a DMSN solution loaded with a large amount of AuNPs. The specific preparation process involves mixing the thiolized DMSNs with the AuNPs solution and sonicating for 10 minutes.

[0013] Further, the thiolized DMSNs solution is: a thiolized DMSN solution; the preparation method is as follows: first, in a clean glass bottle, TEA is added to deionized water and gently stirred in an oil bath at 80°C for 30 minutes; then CTAB and NaSal are added and stirred for 1 hour; next, a mixture of TEOS and ethanol is added and slowly stirred for 2 hours, then the solid particles are collected by vacuum filtration and washed with ethanol to remove residual reactants; subsequently, the solid particles are dispersed in an ethanol solution, sonicated for 2 hours, filtered using an acidic methanol solution (100 mL of absolute methanol contains 6 mL of 37% HCl by mass), washed with ethanol, and the obtained DMSN is dispersed in ethanol; ammonia and MPTMS are added to the DMSNs ethanol solution, and the mixture is vigorously stirred in an oil bath at 24°C for 12 hours or more to obtain thiolized DMSNs.

[0014] Further, the AuNPs solution is a colloidal gold solution; the preparation method is as follows: first, add chloroauric acid solution to a clean flask, and boil the flask while stirring it on a magnetic stirrer; after boiling, quickly add sodium citrate solution to the chloroauric acid solution, and continue to boil while stirring; during the boiling process, the solution color will first change from grayish-black to purple, and when the solution color finally turns wine red, stop heating and cool to room temperature, and store it in the refrigerator's cold storage area away from light.

[0015] Furthermore, the gold staining solution is a freshly prepared solution consisting of equal volumes of a reducing hydroxylamine hydrochloride solution and an oxidizing chloroauric acid solution. The [AuCl4]− in the gold staining solution is fully reduced to gold under the catalysis of AuNPs and continuously coats the surface of AuNPs, thereby uniformly increasing the surface mass deposition and achieving signal amplification. The higher the concentration of the antigen to be tested, the more DMSN@AuNPs@detection antibodies are captured. The large amount of AuNPs loaded on the DMSN significantly enhances the signal amplification effect of gold staining.

[0016] The beneficial effects of this invention are that it is the first to propose a Leff-type surface acoustic wave (SAW) immunosensor based on DMSN@AuNPs combined with gold staining solution to amplify signals, and applies it to the detection of extremely low concentrations of PCT in EBC for early screening of pneumonia. The Leff-type SAW sensor has many advantages, including high sensitivity, small size and easy integration, passive low power consumption, mass production capability, real-time and rapid detection, label-free operation, and resistance to liquid corrosion. This invention utilizes DMSN, which has advantages such as abundant pores, large specific surface area, uniform particle size, easy functionalization, and tunable structure, to load a large number of AuNPs. This not only effectively prevents AuNP aggregation but also greatly enhances the signal amplification capability of a single tag, significantly reducing the detection limit of the SAW sensor for antigen biomarkers. Based on this method, multiple disease biomarkers can be detected jointly by simply changing the capture antibody and the detection antibody labeled on DMSN@AuNPs, thus broadening its application range. Furthermore, both the sensor and nanomaterials of this invention can be mass-produced and stored long-term, showing promising application prospects. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the Loft-type surface acoustic wave immunosensor and its detection method of the present invention; in the figure: Loft-type surface acoustic wave immunosensor 0, ST-Z cut quartz substrate 1, input interdigitated electrode 2, output interdigitated electrode 3, waveguide layer 4, top gold layer 5, PCB circuit board 6, input signal line 7, output signal line 8, input ground line 9, output ground line 10, DMSN@AuNPs@ detection antibody solution 11, gold staining solution 12, sample diluent 13.

[0018] Figure 2 These are transmission electron microscopy (TEM) characterization images and EDS elemental maps of the DMSN@AuNPs@ detection antibodies of this invention;

[0019] Figure 3 The present invention is a Lefu-type surface acoustic wave immunosensor based on DMSN@AuNPs to detect typical responses of different concentrations of PCT;

[0020] Figure 4 This invention provides a standard curve for the detection of PCT antigen using the Lefu-type surface acoustic wave immunosensor based on DMSN@AuNPs. Detailed Implementation

[0021] To better understand the technical solution of this application, the embodiments of this application will be described in detail below with reference to the accompanying drawings.

[0022] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0023] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0024] The present invention proposes a Lep-type surface acoustic wave immunosensor and a double-antibody sandwich method for antigen detection based on DMSN@AuNPs combined with gold staining solution to amplify the signal. Figure 1 Description. A Leff type surface acoustic wave immunosensor for detecting PCT in EBC using a double-antibody sandwich method based on DMSN@AuNPs combined with gold staining solution to amplify the signal, specifically comprises: an Anti-PCT coated Leff type surface acoustic wave immunoelectrode 0, a DMSN@AuNPs@detection antibody solution 11, a gold staining solution 12, a sample diluent 13, PBS buffer, an electrode cleaning solution, a fluid detection flow chamber, a system for generating and detecting radio frequency signals, and a syringe pump.

[0025] The Lep-type surface acoustic wave immunoelectrode 0 consists of an ST-Z cut quartz substrate 1, an input interdigitated electrode 2, an output interdigitated electrode 3, a waveguide layer 4, a top gold layer 5, a PCB circuit board 6, input signal lines 7, output signal lines 8, an input ground line 9, and an output ground line 10. The top gold layer 5 is modified with an Anti-PCT capture antibody and specifically blocked with BSA. The Lep-type surface acoustic wave immunoelectrode 0 can specifically capture the antigen marker to be detected in the sample diluent 13, and then use the DMSN@AuNPs@detection antibody 11, which can bind to the antigen, in combination with gold staining solution 12 to achieve signal amplification. The Lep-type surface acoustic wave immunoelectrode 0 is connected to the radio frequency signal generation and detection system via an SMA. The input radio frequency electrical signal excites the Lep-type surface acoustic wave immunoelectrode to generate a horizontal shear-type surface acoustic wave, and the frequency, amplitude, and phase of the output signal are measured by the detection system and stored in the PC.

[0026] The Loft-type surface acoustic wave immunoelectrode 0 is a Loft-type surface acoustic wave electrode modified with the capture antibody (Ab1) corresponding to PCT. The fabrication process of the Loft-type surface acoustic wave electrode is as follows: First, using MEMS micromachining technology, 50 pairs of split-finger input gold interdigitated electrodes 1 and 2 are fabricated on the surface of an ST-Z-cut quartz wafer substrate 1, with a resonant frequency of approximately 220MHz. Then, a 2μm thick silica waveguide layer 4 is fabricated above the split-finger gold interdigitated IDTs using plasma vapor deposition, focusing the acoustic waves while preventing liquid interference with the IDTs and thus preventing signal attenuation. A 50nm thick top gold layer 5 is deposited on the delay line region above the waveguide layer 4. The surface of the top gold layer 5 is modified with the capture antibody corresponding to the antigen to be tested and non-specifically blocked by BSA.

[0027] The modification method for the capture antibody corresponding to PCT is as follows: The top gold layer 5 of the Lep-type surface acoustic wave electrode is soaked in 1M NaOH for 30 minutes, then in 1M HCl solution for 5 minutes, and then soaked in freshly prepared piranha solution (a mixture of concentrated sulfuric acid and 30% hydrogen peroxide in a 7:3 volume ratio) for 1 minute to activate the electrode. After cleaning and drying, 10 μL of 1 mg / mL Staphylococcus aureus protein A (SPA) solution is added to each electrode and incubated for 0.5 h to promote targeted antibody fixation. Then, 10 μL of 100 μg / mL PCT capture antibody (Ab1) and 10 μL of 10% BSA are added to the surfaces of the working and reference electrodes, respectively, and incubated overnight at 4°C. Finally, the free binding sites of the working and reference channels are blocked for 1 h at 37°C with 10 μL of 10% BSA solution to reduce non-specific binding. At the end of each step, the electrodes are washed with PBS and dried with nitrogen.

[0028] The DNSN@AuNPs@detection antibody solution 11 is a DMSN@AuNPs solution modified with the detection antibody (Ab2) corresponding to PCT. The specific modification method involves adjusting the pH of the DMSN@AuNPs solution to 8-9 using K2CO3 solution, then adding 50 μL of 1 mg / mL PCT detection antibody (Ab2) to each 1 mL of solution, and incubating on a shaker at 37°C for 1 hour. Afterwards, it is centrifuged at 14,000 rpm at 4°C for 10 min to wash away excess detection antibody. The precipitate is then redissolved in 2% BSA (g / mL) (in 0.01 M PBS, pH 7.4).

[0029] The DMSN@AuNPs solution is a DMSN solution loaded with a large amount of AuNPs. The specific preparation process is as follows: 50 μL of 10 mg / mL thiolized DMSNs (T-DMSNs) is mixed with 1 mL of 1 mg / mL AuNPs solution and sonicated for 10 minutes.

[0030] The thiolized DMSNs solution is a thiolized DMSN solution with a particle size of approximately 220 nm. The preparation method involves first adding 136 mg of TEA to 50 mL of deionized water in a clean glass bottle and gently stirring for 30 minutes in an oil bath at 80°C. Then, 760 mg of CTAB and 538 mg of NaSal are added and stirred for 1 hour. Next, a mixture of 8 mL of TEOS and 0.8 mL of ethanol is added, and the mixture is slowly stirred for 2 hours. The solid particles are then collected by vacuum filtration and washed with ethanol to remove residual reactants. Subsequently, the solid particles are dispersed in an ethanol solution and sonicated for 2 hours. The solution is then filtered using an acidic methanol solution (100 mL of absolute methanol containing 6 mL of 37% HCl by mass), washed with ethanol, and the resulting DMSN is dispersed in ethanol, with approximately 100 mg of DMSN per 10 mL of ethanol. Thiolated DMSNs (T-DMSNs) can be obtained by adding 250 μL of ammonia and 120 μL of LMPTMS to 10 mL of ethanol solution of 10 mg / mL DMSNs and stirring vigorously in an oil bath at 24 °C for 18 hours.

[0031] The AuNPs solution is a colloidal gold solution with a particle size of approximately 15 nanometers. The preparation method involves first adding 100 mL of 0.01% chloroauric acid solution to a clean flask and then boiling the flask while stirring at high speed on a magnetic stirrer. After boiling, 5 mL of 1% sodium citrate solution is quickly added to the chloroauric acid solution, and boiling continues while stirring for 15 to 30 minutes. During boiling, the solution color will initially change from grayish-black to purple, and when the solution color finally turns wine-red, heating is stopped and the solution is cooled to room temperature and stored in the refrigerator away from light.

[0032] The gold staining solution 12 is a freshly prepared solution consisting of equal volumes of 20 mM reducing hydroxylamine hydrochloride solution and 10 mM oxidizing chloroauric acid solution. Under the catalysis of AuNPs, the [AuCl4]− in the gold staining solution can be fully reduced to gold and continuously coated on the surface of AuNPs, thereby uniformly increasing the surface mass deposition and achieving signal amplification. The higher the concentration of the antigen to be tested, the more DMSN@AuNPs@detection antibodies are captured. The DMSN, loaded with a large number of AuNPs, significantly enhances the signal amplification effect of gold staining.

[0033] The sample diluent 13 is: PCT solution diluted with PBS to a concentration of 0.01 ng / mL-100 ng / mL.

[0034] A method for detecting PCT in EBC using a Leffe surface acoustic wave immunosensor based on a double-antibody sandwich method using DMSN@AuNPs combined with gold staining solution to amplify the signal is as follows: A Leffe surface acoustic wave immunoelectrode 0 coated with PCT capture antibody and non-specifically blocked is installed in a fluid detection chamber. PBS buffer is injected into the chamber at a flow rate of 50 μL / min until the detection signal stabilizes. Then, 40 μL of sample dilution solution 13 is injected onto the surface of the Leffe surface acoustic wave immunoelectrode 0 at the same flow rate. After 10 minutes, PBS buffer is injected for washing. Then, 40 μL of DMSN@AuNPs@detection antibody solution 11 is injected at the same flow rate. After standing for 20 minutes, PBS buffer is injected. After the signal stabilizes, 100 μL of freshly prepared gold staining solution 13 is injected at the same flow rate. Then, PBS buffer is immediately injected for washing until the signal stabilizes. Real-time quantitative detection is performed by calculating the phase change caused by the change in the surface mass of the Leffe surface acoustic wave sensor.

[0035] Example 1:

[0036] Prepare the DMSN@AuNPs@detection antibody solution as described above: Mix 50 μL of 10 mg / mL thiolized DMSNs (T-DMSNs) with 1 mL of 1 mg / mL AuNPs solution and sonicate for 10 minutes to obtain the DMSN@AuNPs solution. Then, adjust the pH of the DMSN@AuNPs solution to 8-9 using K2CO3 solution. Next, add 50 μL of 1 mg / mL PCT detection antibody (Ab2) to each 1 mL of pH-adjusted DMSN@AuNPs solution and incubate on a shaker at 37°C for 1 hour. Afterward, centrifuge at 14,000 rpm at 4°C for 10 min to wash away excess detection antibody. Redissolve the precipitate in 2% BSA (g / mL) (in 0.01 M PBS, pH 7.4). Characterize the prepared DMSN@AuNPs@detection antibody using TEM and EDS. The results are as follows: Figure 2 As shown.

[0037] Example 2:

[0038] PCT antigen sample dilutions were tested sequentially from smallest to largest: 0, 0.01, 0.1, 1, 10, and 100 ng / mL. Figure 3 The results showed that after injecting 40 μL of DMSN@AuNPs@detection antibody solution 11, the phase of the Rough-type surface acoustic wave sensor changed slightly, and the higher the antigen concentration, the greater the phase change. After adding gold staining solution 13, the phase change increased further, and the higher the antigen concentration, the more significant the decrease. Example 2 preliminarily verified that DMSN@AuNPs can improve the sensitivity of Rough-type surface acoustic wave detection of antigens. The standard curve obtained from repeating the experiment three times is shown below. Figure 4As shown, the results indicate that the Lep-type surface acoustic wave sensor based on DMSN@AuNPs combined with gold staining solution amplifies the signal, exhibiting good linearity for PCT standard samples in the concentration range of 0.01 ng / L to 100 ng / L. The linear relationship is Y = 19.85X + 51.83, with a linearity of 0.9881. Based on the 3σ principle, the detection limit is calculated to be 6.28 pg / mL, which meets the concentration requirements for PCT in exhaled breath condensate from clinical patients. This sensor and detection method can be subsequently used for the detection of disease biomarkers in EBC clinical samples.

[0039] It should be stated that the content and specific embodiments of this invention are intended to demonstrate the practical application of the technical solutions provided by this invention, and should not be construed as limiting the scope of protection of this invention. Any modifications and changes made to this invention within the spirit and scope of the claims fall within the protection scope of this invention.

Claims

1. A highly sensitive Lep-type surface acoustic wave immunosensor based on a double-antibody sandwich method using DMSN@AuNPs and gold staining solution, characterized in that, The sensor includes a Lep-type surface acoustic wave immunoelectrode, DMSN@AuNPs@ detection antibody solution, gold staining solution, sample dilution solution, PBS buffer, electrode cleaning solution, and a fluid detection chamber; A Loft-type surface acoustic wave immunoelectrode coated with antibody and non-specifically blocked is installed in the fluid detection chamber. This Loft-type surface acoustic wave immunoelectrode is a capture antibody-coated Loft-type surface acoustic wave electrode capable of specifically capturing the antigen biomarker to be detected. The DMSN@AuNPs@detection antibody solution is a DMSN@AuNPs solution modified with the detection antibody Ab2 corresponding to the antigen to be tested. The DMSN@AuNPs@detection antibody is obtained by mixing thiolized DMSNs with AuNPs solution, sonicating, and then modifying with the detection antibody corresponding to the antigen to be tested. Specifically, the modification method involves adjusting the pH of the DMSN@AuNPs solution to 8-9 with K2CO3 solution, adding the detection antibody, and incubating on a shaker at 37°C for 1 hour. Then, centrifugation at 14000 rpm at 4°C for 10 min is performed to wash away excess detection antibody. The precipitate is then redissolved with 2% BSA (g / ml) in a 0.01 mL solution at pH 7.

4. In PBS of M; the DMSN@AuNPs solution is a DMSN solution loaded with a large amount of AuNPs; the signal amplification is achieved by using DMSN@AuNPs@detection antibody that can bind to the antigen in combination with gold staining solution. The [AuCl4]﹣ in the gold staining solution is fully reduced to gold under the catalysis of AuNPs and continuously coats the surface of AuNPs, thereby uniformly increasing the mass deposition on the surface. The higher the concentration of the antigen to be tested, the more DMSN@AuNPs@detection antibody is captured. The large amount of AuNPs loaded on the DMSN significantly improves the signal amplification effect of gold staining. Real-time quantitative detection is achieved by calculating the amplitude, phase, and resonant frequency changes of the output signal caused by surface quality deposition in a Lep-type surface acoustic wave sensor.

2. The ultrasensitive Lep-type surface acoustic wave immunosensor based on the DMSN@AuNPs and gold staining solution double antibody sandwich method according to claim 1, characterized in that, The capture antibody-coated Leff type surface acoustic wave electrode is a Leff type surface acoustic wave electrode modified with the capture antibody Ab1 corresponding to the antigen to be tested. The fabrication process of the Leff type surface acoustic wave electrode is as follows: First, input and output split-finger gold interdigitated electrodes (IDTs) are fabricated on the surface of an ST-Z cut quartz wafer substrate using MEMS micromachining technology. The IDTs can excite horizontal shear-type surface acoustic waves in the ST-Z cut quartz piezoelectric material. Then, a silica waveguide layer is fabricated on top of the IDTs using plasma vapor deposition to focus the sound waves while preventing liquid interference with the operation of the IDTs and thus preventing signal attenuation. A gold layer is deposited on the delay line region above the waveguide layer, and the surface of the gold layer is modified with the capture antibody corresponding to the antigen to be tested.

3. The ultrasensitive Lep-type surface acoustic wave immunosensor based on the DMSN@AuNPs and gold staining solution double antibody sandwich method according to claim 2, characterized in that, The method for modifying the capture antibody corresponding to the antigen to be tested is as follows: First, the gold layer of the Lep-type surface acoustic wave electrode is washed sequentially with NaOH and HCl solutions, and then activated with piranha solution; after washing and drying, Staphylococcus aureus protein A solution is added to each electrode and incubated for a period of time to promote the directional fixation of the antibody; then, the capture antibody Ab1 of the antigen to be tested and 10% BSA are added to the surfaces of the working and reference electrodes, respectively, and incubated overnight at 4°C; finally, the free binding sites of the working and reference channels are blocked for a period of time with 10% BSA solution at 37°C to reduce non-specific binding; at the end of each of the above steps, the electrodes are washed with PBS and dried with nitrogen.

4. The ultrasensitive Lep-type surface acoustic wave immunosensor based on the DMSN@AuNPs and gold staining solution double antibody sandwich method according to claim 1, characterized in that, The specific preparation process of the DMSN@AuNPs solution involves mixing thiolized DMSNs with AuNPs solution and then sonicating for a period of time.

5. The ultrasensitive Lep-type surface acoustic wave immunosensor based on the DMSN@AuNPs and gold staining solution double antibody sandwich method according to claim 4, characterized in that, The thiolized DMSNs are: a thiolized DMSN solution with a particle size of 220 nm. The preparation method is as follows: First, in a clean glass bottle, 136 mg of TEA is added to 50 mL of deionized water and gently stirred in an oil bath at 80°C for 30 minutes. Then, 760 mg of CTAB and 538 mg of NaSal are added and stirred for 1 hour. Next, a mixture of 8 mL of TEOS and 0.8 mL of ethanol is added and slowly stirred for 2 hours. The solid particles are collected by vacuum filtration and washed with ethanol to remove residual reactants. Subsequently, the solid particles are dispersed in an ethanol solution, sonicated for 2 hours, and filtered using an acidic methanol solution (100 mL of absolute methanol containing 6 mL of 37% HCl by mass). The solution is washed with ethanol, and the resulting DMSNs are dispersed in ethanol. 250 μL of ammonia and 120 μL of MPTMS are added to 10 mL of the DMSNs ethanol solution, and the mixture is vigorously stirred in an oil bath at 24°C for 18 hours to obtain the thiolized DMSNs.

6. The ultrasensitive Lep-type surface acoustic wave immunosensor based on the DMSN@AuNPs and gold staining solution double antibody sandwich method according to claim 4, characterized in that, The AuNPs solution is a colloidal gold solution with a particle size of 15 nanometers. The preparation method is as follows: First, add 100 mL of 0.01% chloroauric acid solution to a clean flask and boil the flask while stirring it with a magnetic stirrer. After boiling, quickly add 5 mL of 1% sodium citrate solution to the chloroauric acid solution and continue to boil while stirring for 15 to 30 minutes. During the boiling process, the solution color will first change from grayish-black to purple. When the solution color finally turns wine red, stop heating and cool to room temperature. Store in the refrigerator in the dark.

7. The ultrasensitive Lep-type surface acoustic wave immunosensor based on the DMSN@AuNPs and gold staining solution double antibody sandwich method according to claim 1, characterized in that, The gold staining solution is a freshly prepared solution consisting of equal volumes of 20 mM hydroxylamine hydrochloride solution (which has reducing properties) and 10 mM chloroauric acid solution (which has oxidizing properties).

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