Method for detecting biomolecules in dry state environment based on resonant SAW sensor

Abstract

The present application relates to a kind of biological molecule detection method under dry state environment based on resonant SAW sensor, it solves the technical problem of low detection sensitivity, higher detection limit, poor stability of existing liquid-phase environment biological detection system based on SAW sensor, it is with resonant SAW sensor as core detection unit, biological molecule binding function layer is constructed on the surface of resonant SAW sensor, biological recognition molecule is fixed in biological molecule binding function layer, the resonant frequency change caused by monitoring target biological molecule and biological recognition molecule combination, realize high sensitivity detection to target biological molecule.The present application is suitable for antibody drug screening, antigen-antibody and nucleic acid recognition of disease diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biomolecule detection technology, and more specifically, to a method for detecting biomolecules in a dry environment based on a resonant SAW sensor. Background Technology

[0002] In fields such as disease diagnosis, food safety, and environmental monitoring, biomolecular detection is gradually shifting from traditional laboratory analysis to point-of-care testing (POCT). This necessitates that detection technologies not only possess the ability to identify low concentrations of target molecules but also meet requirements such as rapid response and ease of operation. However, while existing detection technologies achieve high sensitivity, they often heavily rely on experimental conditions and operational procedures, limiting their practical application, especially in resource-constrained areas and large-scale testing scenarios, where these shortcomings are further highlighted. Therefore, there is an urgent need to develop a biodetection method that balances high sensitivity with portability.

[0003] Among existing detection methods, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and chemiluminescence detection can achieve high detection sensitivity and specificity, but they require multi-step reaction processes and sophisticated optical or fluid control systems, resulting in complex and time-consuming detection procedures and strong dependence on experimental environment and equipment. Furthermore, while fluorescence detection offers high sensitivity, its labeling process may affect the natural activity of biomolecules, limiting its application in real-time dynamic monitoring and high-throughput screening. Additionally, rapid detection technologies such as lateral flow assay strips (LFA) offer advantages such as ease of operation, rapid response, and no need for complex equipment, but their detection sensitivity and quantitative capabilities are insufficient, making it difficult to meet the detection needs of low-concentration target molecules.

[0004] Surface Acoustic Wave (SAW) sensors, a piezoelectric effect-based sensing technology, consist of piezoelectric materials, interdigital transducers (IDTs), and reflective gratings. This technology utilizes the propagation characteristics of surface acoustic waves along the surface of the piezoelectric material. The working principle is as follows: after receiving an electrical signal from the reader, the interdigital transducer converts it into a mechanical vibration wave (i.e., a surface acoustic wave) propagating along the substrate surface. Upon encountering the reflective grating, a portion of the surface acoustic wave is reflected back to the interdigital transducer, where it is converted into an echo electrical signal that returns to the reader. By detecting and analyzing the echo signal, the change in the measured physical quantity can be obtained, thus achieving the sensing function. SAW sensors mainly have two structural types: delayed linear and resonant. The acoustic wave modes of SAW sensors include shear-mode surface acoustic waves, Love surface acoustic waves, and Rayleigh surface acoustic waves. SAW sensors have advantages such as fast response speed, real-time monitoring, and miniaturization and integration, making them valuable for portable detection and rapid analysis. Based on the above characteristics, SAW sensors can be applied to the field of biosensing. For example, patent application CN108593765A discloses a biosensor and its detection method based on surface acoustic wave (SAW) mode. This biosensor is used to detect liquid samples by constructing a biofunctional membrane in the acoustic wave propagation region. The biofunctional membrane adsorbs the liquid sample to be detected, allowing target biomolecules to bind to the membrane in the liquid phase environment for detection and analysis. However, this technical solution suffers from low detection sensitivity and a high detection limit, and its stability also needs improvement. Summary of the Invention

[0005] This application aims to address the technical problems of low detection sensitivity, high detection limit, and poor stability in existing SAW sensor-based liquid-phase biological detection systems, and provides a method for detecting biomolecules in a dry environment based on a resonant SAW sensor.

[0006] This application provides a method for detecting biomolecules in a dry environment based on a resonant SAW sensor, comprising the following steps:

[0007] Step S1, Sensor surface functionalization treatment:

[0008] Prepare a resonant SAW sensor, which includes a piezoelectric substrate with an interdigital transducer and two reflective gratings connected to it.

[0009] The resonant SAW sensor was cleaned and then dried. Oxygen plasma treatment was applied to the surface of the resonant SAW sensor to activate it and generate a large number of hydroxyl groups. The activated resonant SAW sensor was then immersed in an APTES-anhydrous ethanol solution to react and form an aminated modification layer on its surface. The resonant SAW sensor surface was cleaned, dried again, and then subjected to heat treatment. The resonant SAW sensor was then immersed in a glutaraldehyde-PB buffer solution and reacted at room temperature to form an aldehyde-activated layer on its surface, which serves as a biomolecule binding functional layer. Finally, the resonant SAW sensor was cleaned and then dried.

[0010] Step S2, antibody fixation and blocking:

[0011] A solution containing biorecognition molecules is dropped onto the region corresponding to the interdigitated transducer on the biomolecule binding functional layer and incubated. After incubation, the surface of the resonant SAW sensor is cleaned and then dried. A sealing liquid is dropped onto the surface of the resonant SAW sensor for sealing. After sealing, the surface of the resonant SAW sensor is cleaned and then dried.

[0012] Step S3: In the dry state, an excitation signal is applied to the interdigital transducer of the resonant SAW sensor through a signal generator, and then the initial frequency f0 of the output signal of the resonant SAW sensor is obtained.

[0013] Step S4, detect the target analyte:

[0014] The analyte is introduced into the region corresponding to the interdigital transducer of the biomolecule binding functional layer, so that the target biomolecule in the analyte undergoes a specific binding reaction with the biorecognition molecules on the surface of the biomolecule binding functional layer. Then, the resonant SAW sensor is cleaned and dried.

[0015] Step S5: In the dry state, an excitation signal is applied to the interdigital transducer of the resonant SAW sensor through a signal generator, and then the frequency f1 of the output signal of the resonant SAW sensor is obtained; next, the frequency offset Δf=f1-f0 is calculated; then the target biomolecule is detected or the concentration of the target biomolecule is detected based on the frequency offset.

[0016] Preferably, step S4 involves sandwich detection, where the analyte is premixed with a gold nanoparticle-labeled secondary antibody to form a sandwich complex as the test sample, and then the test sample is introduced into the region corresponding to the interdigital transducer of the biomolecule binding functional layer.

[0017] Preferably, the acoustic mode of the resonant SAW sensor is shear horizontal surface acoustic wave.

[0018] Preferably, in step S1, the APTES-anhydrous ethanol solution has an APTES volume fraction of 2%; the glutaraldehyde PB buffer solution has a glutaraldehyde volume fraction of 5%.

[0019] Preferably, the analyte is a trastuzumab monoclonal antibody solution, which is prepared by mixing a gold nanoparticle-labeled mouse anti-human IgG kappa antibody with the trastuzumab monoclonal antibody solution in equal proportions to obtain the sample to be tested.

[0020] Preferably, the operating frequency of the resonant SAW sensor is greater than 2 GHz.

[0021] The beneficial effects of this application are that it enables label-free detection of biomolecules with high sensitivity, low detection limit, and strong stability. The sensitivity can reach 6370 Hz / (ng / mL) (9.43 × 10⁻⁶). 5 (Hz / nM), detection limit as low as 67 pM.

[0022] The detection process is carried out in a dry environment, which can significantly reduce energy attenuation during sound wave propagation, improve detection stability, and increase detection sensitivity compared to existing liquid phase detection methods. At the same time, this method has low dependence on the fluid control system, which can simplify the detection system structure and thus improve the system's environmental adaptability.

[0023] The detection system features a miniaturized and integrated design, making it suitable for on-site, real-time detection scenarios.

[0024] It can be used to detect low-abundance protein biomarkers in biological samples such as serum and plasma, and is suitable for early screening and dynamic monitoring of tumor biomarkers. In the field of antibody drug screening, it can be used for rapid evaluation of the binding behavior between antibodies and target molecules, thereby supporting high-throughput screening and performance evaluation of antibody drugs. In addition, it can be used for real-time detection of biomolecular interaction processes such as protein-protein, antigen-antibody, and nucleic acid recognition, providing technical support for disease diagnosis, personalized medicine, and drug development.

[0025] Further features and aspects of this application will be clearly described in the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of a detection system based on a resonant SAW sensor;

[0027] Figure 2 This is a schematic diagram illustrating the principle of the sandwich method detection process;

[0028] Figure 3The comparison is made between the frequency offset values ​​of the output signals of the SAW sensor when the target antibody labeled with gold nanoparticles and the target antibody without gold nanoparticles are detected separately.

[0029] Figure 4 This is a graph showing the frequency shift as a function of trastuzumab monoclonal antibody drug concentration;

[0030] Figure 5 It is the frequency shift before and after detecting an analyte at a concentration of 67 pM. Detailed Implementation

[0031] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] The specific embodiments described below are merely preferred embodiments of this application, and the scope of protection of this application is not limited thereto. Those skilled in the art can make modifications or variations based on the principles, concepts, and spirit of this application, and the resulting technical solutions should all be covered within the scope of protection of this application.

[0033] The method for detecting biomolecules in a dry environment based on a resonant SAW sensor mainly includes the following steps:

[0034] The first step is to functionalize the sensor surface.

[0035] Prepare a resonant SAW sensor, which includes a piezoelectric substrate with an interdigital transducer and two reflective gratings connected to it, the interdigital transducer being located between the two reflective gratings. The acoustic wave generation of this resonant SAW sensor utilizes sheared horizontal surface acoustic waves.

[0036] The resonant SAW sensor was immersed in isopropanol solution for 15 min to remove surface organic contaminants, followed by drying with nitrogen. Based on the material properties of the piezoelectric substrate, the sensor surface was treated with oxygen plasma for 5 min to activate the surface and generate a large number of hydroxyl groups. The treated sensor was immediately immersed in a 2% (v / v) APTES-anhydrous ethanol solution and reacted at 37°C for 3 h to form an aminated modification layer on the sensor surface. After the reaction, the sensor surface was cleaned three times each with anhydrous ethanol and ultrapure water, then dried with nitrogen and heated in a 110°C oven for 1 h to enhance the stability of the aminated modification layer. Subsequently, the sensor was immersed in a 5% (v / v) glutaraldehyde PB buffer solution and reacted at room temperature for 1.5 h. h, so that the surface amino groups can further form an aldehyde activation layer. The aldehyde activation layer serves as a biomolecule binding functional layer. Thus, a biomolecule binding functional layer is formed on the piezoelectric substrate. The biomolecule binding functional layer covers the interdigital transducer and two reflective grids. After the reaction, the substrate is washed twice with PB buffer solution and ultrapure water, and then dried with nitrogen gas for later use.

[0037] The second step is antibody fixation and blocking.

[0038] The following steps were taken to immobilize biorecognition molecules and block non-specific sites on the sensor surface: A PB buffer solution containing 10 μg / mL Mouse anti-human IgG Fc monoclonal antibody was added to the sensor surface. This PB buffer solution was added to the region corresponding to the interdigital transducers on the aldehyde activation layer, immobilizing the antibody molecules on the aldehyde activation layer (i.e., immobilizing them on the biomolecule binding functional layer). The surface was incubated overnight at 4 °C, with the antibody molecules serving as recognition molecules. After incubation, the sensor surface was washed twice with PBT buffer solution and dried with nitrogen. Casein blocking buffer was added to the sensor surface, and the surface was blocked at 37 °C for 1 h to reduce non-specific adsorption during subsequent detection. After blocking, the surface was washed twice each with PBT buffer solution and ultrapure water, and dried with nitrogen.

[0039] The third step is to apply an excitation signal to the interdigital transducer of the sensor through a signal generator, and then use a vector network analyzer to obtain the frequency of the sensor output signal. This frequency is the initial frequency f0.

[0040] The fourth step is to use the sandwich method to detect the target analyte.

[0041] A sandwich immunoassay was used to detect the target analyte. The specific steps are as follows: A trastuzumab monoclonal antibody solution of a certain concentration was mixed with a gold-labeled Mouse anti-human IgG kappa monoclonal antibody solution in equal volume proportions and reacted in a centrifuge tube for 2 hours to obtain the test sample, in which the trastuzumab monoclonal antibody was used as the analyte. The gold-labeled Mouse anti-human IgG kappa monoclonal antibody was used as the secondary antibody to specifically bind the analyte and form a sandwich structure. By introducing the gold label, the surface mass loading of the sensor can be increased, thereby reducing the sound wave propagation speed and ultimately causing a larger resonant frequency shift, which amplifies the signal. During the detection process, the test sample was introduced into the region corresponding to the interdigital transducer on the aldehyde activation layer, so that the trastuzumab monoclonal antibody could specifically bind to the recognition molecules on the surface of the functional layer for 3 hours. Subsequently, the sensor was immersed in PBT buffer solution and ultrapure water twice each to remove unbound molecules, and then dried with nitrogen gas to ensure that the detection system was in a dry detection environment. Trastuzumab monoclonal antibody binds to recognition molecules, causing changes in the surface quality of the interdigital transducer region.

[0042] The fifth step is to apply an excitation signal to the interdigital transducer of the sensor through a signal generator, and use a vector network analyzer to obtain the frequency of the sensor output signal. This frequency is the detection frequency f1. Next, the detection of the target biomolecule or the quantitative detection of its concentration can be achieved based on the frequency offset Δf=f1-f0.

[0043] A standard curve can be established by mixing trastuzumab monoclonal antibody solutions of different concentrations with a mixed solution of mouse anti-human IgG kappa monoclonal antibody labeled with gold nanoparticles in equal volumes to obtain test samples of different concentrations. These test samples of different concentrations are then introduced into the corresponding regions of the interdigital transducers on the aldehyde activation layer. An excitation signal is then applied to the interdigital transducers of the sensor via a signal generator to obtain the frequency of the sensor's output signal. The frequency offset Δf is calculated, and a standard curve is fitted based on multiple frequency offsets and the corresponding concentrations of the trastuzumab monoclonal antibody solution. After establishing the standard curve, during actual detection, the calculated frequency offsets are used to determine the concentration of the target biomolecule based on the standard curve.

[0044] The detection sensitivity can reach 6370 Hz / (ng / mL) (9.43 × 10⁻⁶). 5 The detection limit is 67 pM, which is a significant improvement over the commonly used technology (0.93–1709 Hz / (ng / mL)) and can meet the detection requirements of low concentration biomolecules.

[0045] Figure 4The frequency shift is shown as a function of trastuzumab monoclonal antibody drug concentration.

[0046] Figure 5 The frequency shift before and after detecting an analyte at a concentration of 67 pM is shown, indicating that the present invention can also generate a signal when detecting an analyte at a concentration of 67 pM.

[0047] The detection process is carried out in a dry environment, thereby reducing the impact of liquid viscosity damping and fluid load on sound wave propagation in the traditional liquid environment, improving the quality factor (Q value) and frequency resolution of the detection system, improving detection stability, increasing detection sensitivity, and reducing the detection limit.

[0048] The operating frequency of a resonant SAW sensor can be set to a high frequency greater than 2 GHz, which is beneficial for improving detection sensitivity.

[0049] Those skilled in the art will understand that in the fourth step described above, the mixture of gold-labeled Mouse antihuman IgG kappa monoclonal antibody may not be used. Figure 3 As can be seen, under the same concentration of target antibody, the frequency shift of the antibody labeled with gold nanoparticles is much greater than that of the antibody without gold nanoparticle labeling. This demonstrates that gold nanoparticle labeling can amplify changes in the propagation characteristics of surface acoustic waves, increase the intensity of the detection signal, and thus lower the detection limit to the pM level.

[0050] The acoustic mode of the resonant SAW sensor adopts sheared horizontal surface acoustic waves, which gives the sensor a high Q value and a higher resonant frequency, thus improving detection sensitivity.

[0051] It should be noted that the acoustic wave mode of a resonant SAW sensor can also be Rayleigh wave or Love wave.

[0052] Those skilled in the art will understand that the identifying molecule is not limited to a specific antibody or antigen, but can also be a nucleic acid aptamer, polypeptide or protein, or enzyme molecule.

Claims

1. A method for detecting biomolecules in a dry environment based on a resonant SAW sensor, characterized in that, Includes the following steps: Step S1, Sensor surface functionalization treatment: Prepare a resonant SAW sensor, which includes a piezoelectric substrate on which an interdigital transducer and two reflective gratings are connected; The resonant SAW sensor is cleaned and then dried. The surface of a resonant SAW sensor was activated by oxygen plasma treatment to generate a large number of hydroxyl groups. The activated resonant SAW sensor was then immersed in an APTES-anhydrous ethanol solution to react and form an aminated modification layer on its surface. The sensor surface was then cleaned, dried, and subjected to heat treatment. Next, the sensor was immersed in a glutaraldehyde-PB buffer solution and reacted at room temperature to form an aldehyde-activated layer on its surface, which serves as a biomolecule-binding functional layer. Finally, the sensor was cleaned and dried. Step S2, antibody fixation and blocking: A solution containing biorecognition molecules is dropped onto the region corresponding to the interdigital transducer on the biomolecule binding functional layer and incubated. After incubation, the surface of the resonant SAW sensor is cleaned and then dried. A sealing liquid is dropped onto the surface of the resonant SAW sensor for sealing treatment; after sealing, the surface of the resonant SAW sensor is cleaned and then dried. Step S3: In the dry state, an excitation signal is applied to the interdigital transducer of the resonant SAW sensor through a signal generator, and then the initial frequency f0 of the output signal of the resonant SAW sensor is obtained. Step S4, detect the target analyte: The analyte is introduced into the region corresponding to the interdigital transducer of the biomolecule binding functional layer, so that the target biomolecule in the analyte undergoes a specific binding reaction with the biorecognition molecules on the surface of the biomolecule binding functional layer. Then, the resonant SAW sensor is cleaned and dried. Step S5: In the dry state, an excitation signal is applied to the interdigital transducer of the resonant SAW sensor through a signal generator, and then the frequency f1 of the output signal of the resonant SAW sensor is obtained; next, the frequency offset Δf=f1-f0 is calculated; then the target biomolecule is detected or the concentration of the target biomolecule is detected based on the frequency offset.

2. The method for detecting biomolecules in a dry environment based on a resonant SAW sensor according to claim 1, characterized in that, In step S4, the sandwich method is used for detection. The analyte is premixed with the secondary antibody labeled with gold nanoparticles to form a sandwich complex as the test sample. The test sample is then introduced into the region corresponding to the interdigital transducer of the biomolecule binding functional layer.

3. The method for detecting biomolecules in a dry environment based on a resonant SAW sensor according to claim 1 or 2, characterized in that, The acoustic mode of the resonant SAW sensor is shear horizontal surface acoustic wave.

4. The method for detecting biomolecules in a dry environment based on a resonant SAW sensor according to claim 1, characterized in that, In step S1, the APTES-anhydrous ethanol solution has an APTES concentration of 2% by volume; the glutaraldehyde-PB buffer solution has a glutaraldehyde concentration of 5% by volume.

5. The method for detecting biomolecules in a dry environment based on a resonant SAW sensor according to claim 2, characterized in that, The analyte is a trastuzumab monoclonal antibody solution. The sample to be tested is obtained by mixing the gold nanoparticle-labeled mouse anti-human IgG kappa antibody with the trastuzumab monoclonal antibody solution in equal proportions.

6. The method for detecting biomolecules in a dry environment based on a resonant SAW sensor according to claim 2, characterized in that, The resonant SAW sensor operates at a frequency greater than 2 GHz.

Images