Preparation method and application of nanopore optical biosensor based on aptamer

Abstract

The invention discloses a preparation method and application of a nanopore optical biosensor based on an aptamer, and relates to the field of optical biosensing detection. The preparation method comprises the following steps: constructing a solid nanopore array based on an AAO film; and carrying out surface functionalization on the solid-state nanopore array by using the aptamer, so that the functionalized solid-state nanopore array can generate optical characteristic change for observation after being specifically combined with tropomyosin. Compared with the prior art, the prepared nanopore optical biosensor is low in cost, high in detection sensitivity, high in detection speed and capable of rapidly detecting and identifying tropomyosin.

Description

Technical Field

[0001] The present invention relates to the technical field of optical biosensing detection, and more specifically, to a preparation method of an aptamer-based nanopore optical biosensor and its application. Background Art

[0002] Tropomyosin is a fibrous protein that is widely present in muscle and non-muscle cells and is an important regulator of muscle contraction and cytoskeletal stability. In muscle cells, tropomyosin binds to actin and cooperates with troponin to control muscle contraction and relaxation by regulating changes in calcium ion concentration. When the calcium ion concentration increases, tropomyosin moves, exposing the binding site of actin, allowing myosin to interact with actin and initiate the contraction process. In non-muscle cells, tropomyosin participates in the dynamic regulation and stability of the cytoskeleton, which is essential for cell shape maintenance, movement, division and intercellular connection. Its double-stranded helical structure gives it the ability to bind to actin efficiently, and it is divided into multiple subtypes according to its source and function. Abnormal expression or gene mutation of tropomyosin is associated with a variety of diseases, such as hereditary cardiomyopathy and certain types of cancer, so its detection is of great significance in disease diagnosis and research. Detecting tropomyosin levels can help study pathological processes such as muscle diseases, cardiovascular diseases and cancer, and provide important clues for revealing the molecular mechanisms and treatment strategies of these diseases.

[0003] The detection range of tropomyosin is usually 1ng / mL to 500ng / mL, and high concentrations may indicate myocardial damage or muscle disease. At present, the clinical methods for detecting tropomyosin mainly include immunological methods, mass spectrometry analysis, and rapid immunoassay (POCT). Immunological methods such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunoturbidimetry use antibodies to specifically identify tropomyosin, which have high sensitivity and high specificity and are suitable for high-throughput detection. The detection range is usually 1ng / mL to 500ng / mL, but they are easily interfered by cross-reactions and have high costs. Mass spectrometry provides extremely high accuracy and resolution through high-performance liquid chromatography (HPLC) combined with mass spectrometry technology. It can detect tropomyosin as low as pg / mL level, and can also detect multiple biomarkers at the same time, but the equipment is expensive and the detection cycle is long. Rapid immunoassay is suitable for rapid diagnosis in the emergency room or on-site, and results can be obtained in 10-20 minutes, but the sensitivity is slightly lower. Summary of the invention

[0004] In order to overcome the defects of the prior art described above that the cost, sensitivity and detection speed cannot be taken into account at the same time, the present invention provides a preparation method of an aptamer-based nanopore optical biosensor and its application.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] In a first aspect, a method for preparing an aptamer-based nanopore optical biosensor comprises:

[0007] Construction of solid-state nanopore arrays based on AAO membrane;

[0008] The solid-state nanopore array is surface functionalized by using an aptamer, so that the functionalized solid-state nanopore array can generate optical characteristic changes for observation after specific binding with tropomyosin.

[0009] In the second aspect, an aptamer-based nanopore optical biosensor includes a solid-state nanopore array constructed based on an AAO membrane; wherein an aptamer is fixed to the solid-state nanopore array to achieve surface functionalization, and the aptamer is used to specifically bind to tropomyosin to produce optical characteristic changes for observation.

[0010] In a third aspect, a tropomyosin detection platform comprises:

[0011] Tungsten halogen lamp or white light source to provide broadband light source;

[0012] The nanopore optical biosensor as described in the second aspect is used to receive a sample to be detected;

[0013] An optical fiber probe, used to receive the broadband light source and project the broadband light source onto the surface of the nanopore optical biosensor; and also used to collect reflected light signals;

[0014] An optical spectrometer, used for performing spectral detection on the reflected light signal to determine reflected spectrum data;

[0015] The data processing module is used to determine the optical characteristic changes according to the reflection spectrum data, so as to achieve the qualitative and / or quantitative detection of the Yangpingzhong tropomyosin to be detected.

[0016] In a fourth aspect, a method for detecting tropomyosin, using the detection platform described in the third aspect, comprises:

[0017] A halogen tungsten lamp or a white light source is used to provide a broadband light source, and the broadband light source is projected onto the surface of the nanopore optical biosensor at a certain angle through an optical fiber probe;

[0018] Injecting a sample to be detected into the nanopore optical biosensor, and collecting a reflected light signal from the nanopore optical biosensor during the sample binding reaction through the optical fiber probe;

[0019] Performing spectrum detection according to the reflected light signal by an optical spectrometer to determine reflected spectrum data;

[0020] The optical characteristic change is determined according to the reflection spectrum data by a data processing module, and then the qualitative and / or quantitative detection of tropomyosin of the sample to be detected is determined according to the optical characteristic change.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] The present invention discloses a preparation method and application of a nanopore optical biosensor based on an aptamer. The preparation method completes functional modification by fixing an aptamer that can bind to tropomyosin with high specificity to the surface of the nanopore optical biosensor. Compared with the prior art, the prepared nanopore optical biosensor has low cost, high detection sensitivity, and fast detection speed, can quickly detect and identify tropomyosin, has the advantages of high sensitivity, high specificity, strong convenience, and reusability, and is suitable for a wide range of detection scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the process of preparing the aptamer-based nanopore optical biosensor in Example 1 of the present application;

[0024] Figure 2 This is a schematic diagram of the structure of the detection platform in Example 2 of the present application;

[0025] Figure 3 This is a schematic diagram of the detection method in Example 2 of the present application;

[0026] Figure 4 This is a schematic diagram of the detection results in Example 2 of the present application. DETAILED DESCRIPTION

[0027] The terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way can be interchanged in appropriate cases, which is only to describe the distinction mode adopted by the objects of the same attributes in the embodiments of the present application when describing. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, so that the process, method, system, product or equipment containing a series of units need not be limited to those units, but may include other units that are not clearly listed or inherent to these processes, methods, products or equipment. The term "determine" widely covers various actions, and may include acquisition, calculation, calculation, processing, derivation, investigation, search (for example, search in a table, database or other data structure), ascertainment, and similar actions, and may also include reception (for example, receiving information), access (for example, accessing data in a memory) and similar actions, and may also include generation, creation, establishment and similar actions, as well as parsing, selection, selection and similar actions, etc. The relevant definitions of other terms will be given in the following description.

[0028] It should be noted that when an element is considered to be "connected" to another element, it can be directly connected to the other element, or connected to the other element through an intermediate element. In addition, the "connection" in the following embodiments should be understood as "electrical connection", "communication connection", etc. if there is transmission of electrical signals or data between the connected objects.

[0029] The drawings are for illustrative purposes only and should not be construed as limiting the present patent;

[0030] In order to better illustrate the present embodiment, some parts in the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product;

[0031] It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0033] Example 1

[0034] This embodiment provides a method for preparing a nanopore optical biosensor based on an aptamer. Figure 1 ,include:

[0035] Constructing solid-state nanopore arrays based on AAO (porous anodic aluminum oxide) membranes;

[0036] The solid-state nanopore array is surface functionalized by using an aptamer, so that the functionalized solid-state nanopore array can generate optical characteristic changes for observation after specific binding with tropomyosin.

[0037] It should be noted that aptamers are artificially screened single-stranded DNA or RNA molecules with high specificity and high affinity, which can replace traditional antibodies for the detection of tropomyosin. Compared with traditional antibody detection, aptamer detection has many advantages. First, the chemical properties of aptamers are stable, they can tolerate a large pH range and temperature changes, are not easily inactivated, and are suitable for detection in complex environments; while antibodies are prone to lose activity under extreme conditions; secondly, the screening and synthesis costs of aptamers are low, and they can be quickly obtained through in vitro screening (SELEX), which does not rely on biological preparation and is suitable for large-scale production; thirdly, aptamers have a small molecular weight and can bind to the hidden binding sites of target molecules to improve detection sensitivity; in addition, aptamers can be chemically modified to enhance their detection performance or combined with sensors to improve the flexibility and reusability of detection. Therefore, aptamer-based detection is superior to traditional antibody detection in terms of stability, economy, and flexibility.

[0038] It should be understood that the aptamer is an aptamer that can specifically bind to tropomyosin and can be obtained by a person skilled in the art through SELEX.

[0039] It should be emphasized that the existing nanopore optical biosensor or electrochemical sensor technology has high development costs and may face background interference problems in complex samples. The preparation method provided in this embodiment is easy to operate and has low production costs. The prepared nanopore optical biosensor has good stability, can be reused, has high detection sensitivity and specificity, is not limited by the detection site, has a short detection time, and can monitor tropomyosin in real time.

[0040] In some preferred embodiments, the solid-state nanopore array is a SiO2 nanopore array.

[0041] In some optional embodiments, the SiO2 nanopore array is prepared based on an array template method, comprising:

[0042] An AAO membrane is used as a template; wherein the pore size and pore spacing of the AAO membrane are regulated by electrochemical anodization conditions;

[0043] Mixing the silicon precursor solution and the solvent in proportion to prepare a uniform sol;

[0044] Immersing the AAO template in the sol, allowing the sol to fill the template pores through capillary action;

[0045] After the solvent evaporates, the filled AAO template is heat treated to solidify the SiO2 film and enhance its mechanical stability;

[0046] The AAO template is dissolved by chemical etching to obtain the SiO2 nanopore array with high uniformity.

[0047] It should be noted that the method can accurately control the pore size and pore spacing and achieve high-throughput preparation, providing a reliable structural basis for the development of optical biosensors.

[0048] In some specific implementations, the silicon precursor solution is a tetraethoxysilane (TEOS) solution.

[0049] In some specific implementations, the heat treatment is performed at a temperature of 500°C.

[0050] In some specific implementations, NaOH solution is used to dissolve the AAO template.

[0051] In some preferred embodiments, the surface functionalization comprises:

[0052] A hydroxylation treatment is performed on the Au layer of the nanopore wall of the solid-state nanopore array to modify the active hydroxyl groups, and the introduction of active hydroxyl groups provides reaction sites for subsequent chemical modification;

[0053] APTES (3-aminopropyltriethoxysilane) was used to silanize the Au surface in the middle of the nanopore wall. The siloxane groups of APTES reacted with the hydroxyl groups to generate amino functional groups on the Au surface, laying the foundation for further chemical bonding.

[0054] Glutaraldehyde is added to combine with the amino functional group (-NH2 bond), and the aldehyde group is modified on the Au surface through the aldimine condensation reaction between the amino group and the dialdehyde group to form a stable aldehyde modification layer, which provides active sites for the fixation of the aptamer;

[0055] A tropomyosin aptamer with an amino group at the end was added as the aptamer introduction system. The tropomyosin aptamer was fixed to the Au surface through the ammonia aldehyde condensation reaction (Schiff base formation) of the amino group and the aldehyde group, which ensured the directionality of the antibody and the exposure of the active site, thereby improving the specificity and sensitivity of the detection.

[0056] The addition of ethanolamine solution blocked the groups not bound to the tropomyosin aptamer to prevent nonspecific adsorption.

[0057] It should be noted that the above preferred embodiment ensures the specific functionalization of the surface of the nanopore optical biosensor based on step-by-step chemical modification and reaction, and achieves efficient fixation and detection preparation for tropomyosin.

[0058] Example 2

[0059] This embodiment provides an aptamer-based nanopore optical biosensor, including a solid-state nanopore array constructed based on an AAO membrane; wherein an aptamer is fixed to the solid-state nanopore array to achieve surface functionalization, and the aptamer is used to specifically bind to tropomyosin to produce optical characteristic changes for observation.

[0060] In some preferred embodiments, the solid-state nanopore array is a SiO2 nanopore array.

[0061] In some preferred embodiments, the pore size of the solid-state nanopore array is 80 nm-100 nm, and / or the pore depth is 5 μm-10 μm.

[0062] This embodiment also provides a detection platform for tropomyosin, see Figure 2 ,include:

[0063] Tungsten halogen lamp or white light source to provide broadband light source;

[0064] The nanopore optical biosensor as described in the above embodiment is used to receive a sample to be detected;

[0065] An optical fiber probe, used for coupling the broadband light source and projecting the broadband light source onto the surface of the nanopore optical biosensor; and also for collecting reflected light signals;

[0066] An optical spectrometer, used for performing spectral detection on the reflected light signal to determine reflected spectrum data;

[0067] The data processing module is used to determine the optical characteristic changes according to the reflection spectrum data, so as to achieve the qualitative and / or quantitative detection of the Yangpingzhong tropomyosin to be detected.

[0068] In some specific implementations, the optical spectrometer covers a wavelength range from 350 nm to 1050 nm.

[0069] In some examples, a hardware entity of the data processing module is provided, including: a processor, a memory and a communication interface; wherein the processor generally controls the overall operation of the electronic device; the communication interface is used to enable the electronic device to communicate with other terminals (such as an optical spectrometer) or a server through a network; the memory is configured to store instructions and applications executable by the processor, and can also cache data to be processed or processed by the processor and various modules in the electronic device (including but not limited to image data, audio data, voice communication data and video communication data), which can be implemented by flash memory (FLASH), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM) or random access memory (RAM).

[0070] The processor may include one or more processing elements. Therefore, the processor may include one or more integrated circuits (ICs) configured to perform the functions of the processor. In addition, each integrated circuit may include a circuit (e.g., a first circuit, a second circuit, and other circuits, etc.) configured to perform the functions of the processor. Exemplarily, the processor may be a central processing unit (CPU), a microprocessor (MPU), a digital signal processor (DSP), or a field programmable gate array (FPGA), etc.

[0071] Furthermore, data may be transmitted between the processor, the communication interface and the memory via a bus, which may include any number of interconnected buses and bridges, and the bus connects various circuits of one or more processors and memories together.

[0072] This embodiment also provides a method for detecting tropomyosin, using the aforementioned detection platform, referring to Figure 3 ,include:

[0073] A halogen tungsten lamp or a white light source is used to provide a broadband light source, and the broadband light source is projected onto the surface of the nanopore optical biosensor at a certain angle through an optical fiber probe;

[0074] Injecting a sample to be detected into the nanopore optical biosensor, and collecting a reflected light signal from the nanopore optical biosensor during the sample binding reaction through the optical fiber probe;

[0075] Performing spectrum detection according to the reflected light signal by an optical spectrometer to determine reflected spectrum data (interference spectrum);

[0076] The optical characteristic change is determined according to the reflection spectrum data by a data processing module, and then the qualitative and / or quantitative detection result of the tropomyosin of the sample to be detected is determined according to the optical characteristic change (spectral change).

[0077] It should be noted that the above-mentioned embodiment adopts reflection interference spectroscopy (RIFS), which realizes detection based on the interaction between white light and thin film, and measures the target object tropomyosin by the change of interference pattern caused by the difference in optical length reflected from the two interfaces. Specifically, the optical lengths of the two interfaces are different, and the light will be reflected at the two interfaces, resulting in the replacement of the maximum and minimum values ​​of the reflection pattern. The measured value of the optical pattern can be calculated by the interference equation. The solid-state nanopore array sensor has a high-resolution reflection spectrum (i.e., it has good resolution peaks). When the specific aptamer binds to the analyte, the change in local refractive index causes the interference spectrum to red-shift, significantly improving the detection sensitivity. Compared with planar films, nanoporous films have a larger surface area and can bind more receptor molecules. The refractive index changes are greater, which further enhances the spectral changes and effectively improves the detection sensitivity. For example, the detection results are as follows: Figure 4 In addition, RIFS technology can monitor the mechanism and kinetics of binding reactions in real time, providing an efficient and sensitive solution for analyte screening and detection.

[0078] In some specific implementations, the sample to be detected is injected into the nanopore optical biosensor via a syringe to complete the detection.

[0079] It should be noted that the detection method described in the above embodiment can be used to detect Staphylococcus aureus, Salmonella, Listeria and Escherichia coli O157:H7, with a minimum detection limit of 10 CFU / mL, and is not limited by the detection site, thus ensuring food hygiene and safety.

[0080] In some preferred embodiments, before determining the change in optical features, the data processing module performs fast Fourier transformation (FFT) on the reflection spectrum data to convert the reflection spectrum data (interference spectrum) into Fourier spectrum data suitable for observation, which is more suitable for identification and detection of optical biosensors.

[0081] It can be understood that the device of this embodiment corresponds to the method of the above-mentioned embodiment 1, and the options in the above-mentioned embodiment 1 are also applicable to this embodiment, so they will not be described repeatedly here.

[0082] The same or similar reference numerals correspond to the same or similar components;

[0083] The terms used to describe the positional relationship in the drawings are only used for illustrative purposes and should not be construed as limiting the present application;

[0084] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0085] In different specific implementations, the method or system described in the present application can be implemented in software, hardware or a combination thereof. In addition, the order of the steps of the method can be changed, and various elements can be added, reordered, combined, omitted, modified, etc.

[0086] Obviously, the above-mentioned embodiments of the present application are merely examples for clearly illustrating the present application, and are not intended to limit the implementation methods of the present application, and are not intended to limit the present application. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. Each discrete structure / function module or unit can be integrated together to form an independent part, or each module can exist separately, or two or more modules can be integrated to form an independent part, and the structure and function of the discrete components can be implemented as a combined structure or component. It is not necessary and impossible to enumerate all the implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the claims of the present application.

Claims

1. A method for preparing an aptamer-based nanopore optical biosensor, characterized in that: include: Construction of solid-state nanopore arrays based on AAO membrane; The solid-state nanopore array is surface functionalized by using an aptamer, so that the functionalized solid-state nanopore array can generate optical characteristic changes for observation after specific binding with tropomyosin.

2. The method for preparing an aptamer-based nanopore optical biosensor according to claim 1, characterized in that: The solid-state nanopore array is a SiO2 nanopore array.

3. The method for preparing an aptamer-based nanopore optical biosensor according to claim 2, characterized in that: The SiO2 nanopore array is prepared based on an array template method, comprising: An AAO membrane is used as a template; wherein the pore size and pore spacing of the AAO membrane are regulated by electrochemical anodization conditions; Mixing the silicon precursor solution and the solvent in proportion to prepare a uniform sol; Immersing the AAO template in the sol, allowing the sol to fill the template pores through capillary action; After the solvent evaporates, the filled AAO template is heat treated to solidify the SiO2 film and enhance its mechanical stability; The AAO template is dissolved by chemical etching to obtain the SiO2 nanopore array.

4. The method for preparing an aptamer-based nanopore optical biosensor according to any one of claims 1 to 3, characterized in that: The surface functionalization includes: Performing a hydroxylation treatment on the Au layer of the nanopore wall of the solid-state nanopore array to modify the active hydroxyl groups; APTES was used to silanize the Au surface in the middle of the nanopore wall. The siloxane groups of APTES reacted with the hydroxyl groups to generate amino functional groups on the Au surface. Adding glutaraldehyde to combine with the amino functional group, and modifying the aldehyde group on the Au surface through the aldehyde amine condensation reaction of the amino group and the dialdehyde group; A tropomyosin aptamer with an amino group at the end is added, and the tropomyosin aptamer is attached to the Au surface through an amine-aldehyde condensation reaction; The addition of ethanolamine solution blocked the groups not bound to the tropomyosin aptamer to prevent nonspecific adsorption.

5. An aptamer-based nanopore optical biosensor, characterized in that: It includes a solid-state nanopore array constructed based on an AAO membrane; wherein an aptamer is fixed on the solid-state nanopore array to achieve surface functionalization, and the aptamer is used to specifically bind to tropomyosin to produce optical characteristic changes for observation.

6. The aptamer-based nanopore optical biosensor according to claim 5, characterized in that: The solid-state nanopore array is a SiO2 nanopore array.

7. The aptamer-based nanopore optical biosensor according to claim 5 or 6, characterized in that: The solid-state nanopore array has a pore size of 80 nm-100 nm, and / or a pore depth of 5 μm-10 μm.

8. A tropomyosin detection platform, characterized in that: include: Tungsten halogen lamp or white light source to provide broadband light source; The nanopore optical biosensor according to any one of claims 5 to 7, used to receive a sample to be detected; An optical fiber probe, used to receive the broadband light source and project the broadband light source onto the surface of the nanopore optical biosensor; and also used to collect reflected light signals; An optical spectrometer, used for performing spectral detection on the reflected light signal to determine reflected spectrum data; The data processing module is used to determine the optical characteristic changes according to the reflection spectrum data, so as to achieve the qualitative and / or quantitative detection of the Yangpingzhong tropomyosin to be detected.

9. A method for detecting tropomyosin, using the detection platform of claim 8, characterized in that: include: A halogen tungsten lamp or a white light source is used to provide a broadband light source, and the broadband light source is projected onto the surface of the nanopore optical biosensor at a certain angle through an optical fiber probe; Injecting a sample to be detected into the nanopore optical biosensor, and collecting a reflected light signal from the nanopore optical biosensor during the sample binding reaction through the optical fiber probe; Performing spectrum detection according to the reflected light signal by an optical spectrometer to determine reflected spectrum data; The optical characteristic change is determined according to the reflection spectrum data by a data processing module, and then the qualitative and / or quantitative detection result of the tropomyosin of the sample to be detected is determined according to the optical characteristic change.

10. The method for detecting tropomyosin according to claim 9, characterized in that: Before determining the change of the optical characteristics, the data processing module performs fast Fourier transformation on the reflection spectrum data to convert the reflection spectrum data into Fourier spectrum data.

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