Preparation method and application of nanopore optical biosensor based on cTnC antibody

Abstract

The invention discloses a preparation method and application of a nanopore optical biosensor based on a cTnC antibody, and relates to the field of optical biosensing detection. The method comprises the following steps: preparing a solid nanopore array AAO through a micro-nano machining technology; and carrying out surface functionalization on the surface of the solid nanopore array, and immobilizing a cTnC antibody for specifically recognizing cTnC to form a biosensing interface with high sensitivity and high specificity. Compared with the prior art, the nanopore optical biosensor prepared by the invention has the advantages of high sensitivity, short detection period, good anti-interference performance and low cost when being used for detecting cTnC.

Description

Technical Field

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

[0002] Cardiac troponin C (cTnC) is a key subunit in the cardiac troponin complex, which is mainly involved in the contraction and relaxation of myocardial cells. The troponin complex is composed of cTnC, cTnI and cTnT, among which cTnC binds to calcium ions and regulates the signal transmission of myocardial contraction. When the myocardium is damaged, cTnC is released from myocardial cells into the blood. Therefore, changes in its serum level can reflect the degree of myocardial damage, which is especially important in the diagnosis of diseases such as myocardial infarction, myocarditis and ischemic heart disease.

[0003] The detection of cardiac troponin C not only helps in the early diagnosis of heart diseases such as myocardial infarction, but also can dynamically monitor the progression of the disease and the effect of treatment. Currently, the common methods used in clinical practice to detect cardiac troponin C include enzyme-linked immunosorbent assay (ELISA), immunochromatography (LFIA), electrochemiluminescence (ECL), fluorescence immunoassay (FIA) and mass spectrometry (MS). These methods use different technical means to detect the concentration of cTnC in serum, but they cannot simultaneously take into account the requirements of sensitivity, detection cycle, anti-interference, economy, etc. Summary of the invention

[0004] In order to overcome the defects of the prior art described above that sensitivity, detection period, anti-interference and economy cannot be taken into account at the same time, the present invention provides a preparation method of a nanopore optical biosensor based on cTnC antibody 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 a nanopore optical biosensor based on cTnC antibody comprises:

[0007] Solid-state nanopore array AAO was prepared by micro-nano processing technology;

[0008] The surface of the solid-state nanopore array is functionalized, and a cTnC antibody for specifically recognizing cTnC is immobilized to form a biosensing interface with high sensitivity and high specificity.

[0009] In the second aspect, a cTnC antibody-based nanopore optical biosensor is obtained by the preparation method described in the first aspect, comprising a solid-state nanopore array AAO, wherein cTnC antibodies for specific recognition of cTnC are fixed on the nanopore surface of the solid-state nanopore array to achieve surface functionalization.

[0010] In a third aspect, a cTnC 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 spectral data;

[0015] The data processing module is used to determine the optical characteristic changes according to the spectral data, thereby realizing the qualitative and / or quantitative detection of cTnC in the Yangping to be detected.

[0016] In a fourth aspect, a cTnC detection method is provided, using the detection platform described in the first aspect, comprising:

[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 through the optical fiber probe;

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

[0020] The optical characteristic change is determined according to the spectral data by a data processing module, and then the qualitative and / or quantitative detection of cTnC on 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 cTnC antibody. Based on the prepared solid-state nanopore array, a higher surface area and a stronger signal amplification effect can be provided when detecting cTnC, thereby greatly improving the sensitivity. Compared with the traditional enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence (ECL), it can capture more subtle changes in biomarkers. At the same time, when the prepared solid-state nanopore array is used to detect cTnC, no chemical markers need to be added, which avoids the influence of markers that may exist in traditional methods, such as non-specific binding or signal interference caused by labeled molecules, and can also achieve real-time monitoring of molecular interactions, provide rapid response and instant results, greatly improve the efficiency and accuracy of detection, and is particularly suitable for early screening and diagnosis of myocardial diseases; in addition, the use of the nanopore optical biosensor for cTnC detection has the advantages of simple operation, no labeling, high sensitivity, strong repeatability, and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation method described in Example 1 of the present application;

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

[0025] Figure 3 This is a schematic diagram of the detection method described 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] As described in the background technology, cTnC, as a biomarker of myocardial injury, has broad application prospects and may provide an important basis for the development of new treatment methods in the future.

[0035] This embodiment provides a method for preparing a nanopore optical biosensor based on cTnC antibody, see Figure 1 ,include:

[0036] Solid-state nanopore array AAO was prepared by micro-nano processing technology;

[0037] The surface of the solid-state nanopore array is functionalized, and a cTnC antibody for specifically recognizing cTnC is immobilized to form a biosensing interface with high sensitivity and high specificity.

[0038] In some preferred embodiments, the surface functionalization comprises:

[0039] Modifying the nanopore surface of the solid-state nanopore array with streptavidin molecules to form a streptavidin-modified surface;

[0040] Based on the high affinity interaction between biotin and streptavidin, the biotinylated cTnC antibody was immobilized on the streptavidin-modified surface.

[0041] It should be noted that in this embodiment, the specific antibody of cardiac troponin C (i.e., the cTnC antibody) can be accurately fixed on the surface of the AAO membrane using the biotin-streptavidin system. This surface functionalization treatment ensures the stability and efficiency of the antibody on the sensor surface, while retaining the specificity and activity of the antibody, reducing the nonspecific binding effect, and effectively capturing the target molecule cTnC. Compared with traditional immunoassay methods, this surface functionalization technology can ensure higher detection accuracy, especially in complex biological samples.

[0042] In some optional embodiments, the modifying the nanopore surface of the solid-state nanopore array with streptavidin molecules comprises:

[0043] Introducing amino reactive groups on the silica surface of the nanopores by silanization, or modifying the polymer surface by plasma oxidation to form carboxyl or hydroxyl reactive groups;

[0044] Covalently couple the amine groups of streptavidin to the surface active groups using a cross-linking agent (e.g., EDC / NHS) in a specified pH range (e.g., 6.0-7.4) and a buffer (e.g., MES buffer);

[0045] After the reaction, the surface is washed to remove unbound streptavidin, thereby obtaining a functionalized sensor surface, namely the streptavidin-modified surface.

[0046] In some optional embodiments, the biotinylation of the cTnC antibody comprises:

[0047] Under a buffer condition of pH 7-9, the cTnC antibody is mixed with a biotin labeling reagent NHS-biotin for coupling reaction, incubated for a preset time to form a stable biotinylated molecule, and then the unreacted biotin reagent is removed by dialysis or filtration.

[0048] In some embodiments, incubation for 1-2 hours can form stable biotinylated molecules.

[0049] In some optional embodiments, the immobilizing the biotinylated cTnC antibody to the streptavidin-modified surface comprises:

[0050] Dissolving the biotinylated cTnC antibody in a buffer (such as PBS buffer), immersing the streptavidin-modified surface in the solution, and incubating at a specified temperature for a specified time (such as 30 minutes to 2 hours at room temperature or 4° C.) to ensure specific binding of biotin to streptavidin;

[0051] After incubation, the surface is washed with buffer to remove unbound biotinylated cTnC antibody and reduce nonspecific adsorption.

[0052] Those skilled in the art will appreciate that the binding of biotin to streptavidin has an extremely high affinity (Kd≈10-15M), which can ensure the stability and functionality of the immobilization.

[0053] In some preferred embodiments, the method of preparing a solid-state nanopore array AAO by micro-nano processing technology comprises:

[0054] Nanoimprinting a polished high-purity aluminum substrate using a mold with a regular nanopore array (such as a silicon mold or a polymer mold) to form a regular nanopattern on the surface to improve the orderliness and regularity of the pore array during anodization;

[0055] The imprinted aluminum substrate is placed in an acidic electrolyte for anodization treatment. The electrochemical reaction occurs preferentially at the pre-imprinted pattern position, thereby gradually forming a regular nanopore array structure.

[0056] In the above preferred embodiment, AAO is prepared by nanoimprinting combined with anodization. By nanoimprinting the aluminum substrate before anodization, a regular nanopattern is pre-constructed to improve the orderliness and regularity of the pore array during the anodization process.

[0057] It should be noted that by adjusting the pore size, spacing and oxidation process parameters of the mold, the pore structure of the final AAO membrane can be precisely controlled to provide an ideal microenvironment for the binding of biomolecules.

[0058] It should be emphasized that the nanopore optical biosensor can be expanded into a multiplex detection platform, capable of simultaneously detecting multiple biomarkers or different types of molecules. The nanopore array can be optimized and designed as needed, thereby achieving efficient detection of multiple target molecules on a single sensor platform, improving the detection throughput and application flexibility.

[0059] Example 2

[0060] This embodiment provides a cTnC antibody-based nanopore optical biosensor, which is obtained by the preparation method described in Example 1 and includes a solid-state nanopore array AAO, wherein cTnC antibodies for specifically recognizing cTnC are fixed on the nanopore surface of the solid-state nanopore array to achieve surface functionalization.

[0061] In some preferred embodiments, the solid-state nanopore array has a pore size of 100 nm-200 nm, and / or a pore depth of 10 μm-25 μm.

[0062] This embodiment also provides a cTnC detection platform, 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 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;

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

[0067] The data processing module is used to determine the optical characteristic changes according to the spectral data, thereby realizing the qualitative and / or quantitative detection of cTnC in the Yangping 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 cTnC detection method, using the aforementioned detection platform, see 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 through the optical fiber probe;

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

[0076] The optical characteristic change is determined according to the spectral data by a data processing module, and then the qualitative and / or quantitative detection of cTnC on the sample to be detected is determined according to the optical characteristic change.

[0077] In some specific implementations, the sample to be detected is injected into the sensor chip via a syringe to complete the detection.

[0078] It should be noted that in the above embodiments, reflection interference spectroscopy (RIfS) is used for real-time detection, and the changes in molecular interactions or surface properties are analyzed by monitoring the changes in the interference spectrum generated after the light is reflected at the sensor film interface. Multi-wavelength light emitted by a broadband light source (such as white light) is incident on the sensor film surface vertically or at a certain angle. Part of the light is reflected at the air-film interface, and part of the light is transmitted into the film and reflected again at the film-substrate interface. These two parts of reflected light are superimposed and interfered to form an interference spectrum. When molecular adsorption, desorption or other physical and chemical changes occur on the film surface, its optical thickness (the product of the actual thickness of the film and the refractive index) changes accordingly, resulting in changes in the phase or intensity of the interference spectrum. By real-time monitoring and analysis of these spectral changes by a high-precision spectrometer, qualitative / quantitative analysis of molecular interactions, concentration changes and interface reaction processes can be achieved.

[0079] In the above embodiment, the binding reaction between the antibody and cTnC is monitored by the change in the reflectance spectrum. The RIfS technology can highly sensitively detect the change in membrane thickness caused by the binding of the antibody to the target molecule cTnC, resulting in a change in the optical signal. The signal intensity is recorded in real time and qualitative / quantitative analysis can be achieved through the standard curve. Reflection interference spectroscopy can provide label-free, real-time and high-resolution signals, accurately capture subtle molecular interactions, and provide a new detection method for the quantitative analysis of cTnC. The detection method based on the nanopore optical biosensor can provide a higher surface area and a stronger signal amplification effect when detecting cTnC (see Figure 4 ), not only has high sensitivity and high specificity, with a detection limit as low as pg / mL level (minimum detection limit 0.25pg / mL), but also can achieve rapid and accurate detection of cardiac troponin C, with a response time of less than 10 minutes. It has the advantages of simple operation, no labeling, high sensitivity, and strong repeatability. It has broad clinical application prospects, especially for the early diagnosis and prognosis evaluation of cardiovascular diseases.

[0080] In some optional embodiments, the interference spectrum can be converted into a Fourier spectrum suitable for observation by fast Fourier transformation (FFT) of the reflection spectrum, which is more suitable for optical biosensor recognition and detection.

[0081] In some specific implementations, qualitative and quantitative detection of cardiac troponin C is achieved by analyzing the spectral red shift.

[0082] It can be understood that the options in the above-mentioned embodiment 1 are also applicable to this embodiment, so they will not be described again here.

[0083] The same or similar reference numerals correspond to the same or similar components;

[0084] 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;

[0085] 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.

[0086] 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.

[0087] 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 a nanopore optical biosensor based on cTnC antibody, characterized in that: include: Solid-state nanopore array AAO was prepared by micro-nano processing technology; The surface of the solid-state nanopore array is functionalized, and a cTnC antibody for specifically recognizing cTnC is immobilized to form a biosensing interface with high sensitivity and high specificity.

2. The method for preparing an antibody-based nanopore optical biosensor according to claim 1, characterized in that: The surface functionalization includes: Modifying the nanopore surface of the solid-state nanopore array with streptavidin molecules to form a streptavidin-modified surface; Based on the high affinity interaction between biotin and streptavidin, the biotinylated cTnC antibody was immobilized on the streptavidin-modified surface.

3. The method for preparing an antibody-based nanopore optical biosensor according to claim 2, characterized in that: The step of modifying the nanopore surface of the solid-state nanopore array by using streptavidin molecules comprises: Introducing amino reactive groups on the silica surface of the nanopores by silanization, or modifying the polymer surface by plasma oxidation to form carboxyl or hydroxyl reactive groups; The amine groups of streptavidin are covalently coupled to the surface active groups using a cross-linking agent in a specified pH range and buffer; After the reaction, the surface is washed to remove unbound streptavidin, thereby obtaining a functionalized sensor surface, namely the streptavidin-modified surface.

4. The method for preparing an antibody-based nanopore optical biosensor according to claim 2, characterized in that: The biotinylation of the cTnC antibody comprises: Under a buffer condition of pH 7-9, the cTnC antibody is mixed with a biotin labeling reagent NHS-biotin for coupling reaction, incubated for a preset time to form a stable biotinylated molecule, and then the unreacted biotin reagent is removed by dialysis or filtration.

5. The method for preparing an antibody-based nanopore optical biosensor according to claim 2, characterized in that: The step of fixing the biotinylated cTnC antibody to the streptavidin-modified surface comprises: Dissolving the biotinylated cTnC antibody in a buffer, immersing the streptavidin-modified surface in the solution, and incubating at a specified temperature for a specified time to ensure specific binding of biotin to streptavidin; After incubation, the surface is washed with buffer to remove unbound biotinylated cTnC antibody and reduce nonspecific adsorption.

6. A method for preparing an antibody-based nanopore optical biosensor according to any one of claims 1 to 5, characterized in that: The method of preparing a solid-state nanopore array AAO by micro-nano processing technology comprises: A mold with a regular nanopore array is used to perform nanoimprinting on a polished high-purity aluminum substrate to form a regular nanopattern on its surface to improve the orderliness and regularity of the pore array during anodization. The imprinted aluminum substrate is placed in an acidic electrolyte for anodization treatment. The electrochemical reaction occurs preferentially at the pre-imprinted pattern position, thereby gradually forming a regular nanopore array structure.

7. A nanopore optical biosensor based on cTnC antibody, obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The invention comprises a solid-state nanopore array AAO, wherein a cTnC antibody for specifically recognizing cTnC is fixed on the nanopore surface of the solid-state nanopore array to realize surface functionalization.

8. The cTnC antibody-based nanopore optical biosensor according to claim 7, characterized in that: The solid-state nanopore array has a pore size of 100 nm-200 nm, and / or a pore depth of 10 μm-25 μm.

9. A cTnC detection platform, characterized in that: include: Tungsten halogen lamp or white light source to provide broadband light source; The nanopore optical biosensor according to claim 7 or 8, 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 spectral data; The data processing module is used to determine the optical characteristic changes according to the spectral data, thereby realizing the qualitative and / or quantitative detection of cTnC in the Yangping to be detected.

10. A cTnC detection method, using the detection platform of claim 9, 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 through the optical fiber probe; Performing spectrum detection according to the reflected light signal by an optical spectrometer to determine spectrum data; The optical characteristic change is determined according to the spectral data by a data processing module, and then the qualitative and / or quantitative detection of cTnC on the sample to be detected is determined according to the optical characteristic change.

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