Nanopore biosensor, system and method for detecting tumor markers

Abstract

The invention relates to the technical field of electrochemical biosensing detection, and discloses a nanopore biosensor, system and method for detecting tumor markers. The carbon fiber paper is used as a substrate, so that excellent mechanical strength and conductivity are provided; a silicon-based sensitive layer with a nano-scale pore structure is constructed on the surface of the substrate by a sol-gel method, so that the comprehensive regulation and control of the pore diameter are realized; click chemistry is introduced to realize specific capture of target molecules, and the stability and tolerance of a sensing system are improved. The method for detecting the tumor marker by combining the nanopore membrane with click chemistry has universality and universality, is simple and convenient to operate, greatly shortens the detection time, is strong in specificity, high in sensitivity, high in precision, good in reproducibility and low in cost, can be widely applied to detection of the tumor marker, and has a wide application prospect. And the application of commercial carbon fiber paper in the field of life analysis is expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensor detection, and in particular to a nanopore biosensor, system and method for detecting tumor markers. Background Art

[0002] Malignant tumors have become a major global public health issue. Many cancers have no obvious symptoms in their early stages, and abnormally elevated tumor markers may be the only clue. Their detection has significant clinical value and social significance. The development of new detection technologies can drive continuous innovation and advancement in tumor marker testing, and is a concrete manifestation of new productivity in healthcare and other fields.

[0003] Tumor marker detection methods require high specificity and sensitivity. Compared to traditional methods such as immunoassays, molecular biology, mass spectrometry, and cytology and histology, nanopore technology, with its outstanding advantages such as high sensitivity, high resolution, labeling-free, and rapid detection, has attracted increasing attention from researchers. This technology enables real-time detection at the single-molecule level by exploiting changes in electrochemical signals as biomolecules pass through nanopores, making it one of the most advanced detection methods currently available.

[0004] Nanopore technology originates from the transmembrane molecular transport of single molecules in organisms through nanoscale pores on cell membranes. Different from natural nanopores, solid-state nanopores refer to nanopores prepared on substrates such as glass, silicon nitride, polyethylene terephthalate, and molybdenum disulfide, and functionalized and modified through chemical and advanced micro-nanotechnology. Although the reported work has achieved remarkable research results in the development of tumor marker sensors and the construction of functionalized nanopore detection platforms, and has been widely used in tumor marker detection, there are still certain limitations and challenges, such as poor substrate conductivity, uncontrollable pore size, low mechanical strength, and complex chemical modification, which affect the accuracy and precision of detection. Therefore, there is an urgent need to develop a new generation of solid-state nanopores with good conductivity, controllable pore size and shape, and stable solid-state membrane structure.

[0005] Carbon paper based on polyacrylonitrile (PAN)-based carbon fibers can meet the above structural requirements. As a core component in hydrogen fuel cells, carbon paper has a highly conductive, porous, high-strength, and corrosion-resistant skeleton, and commercial carbon paper is easy to obtain and has stable performance. However, the surface of carbon paper is chemically inert and difficult to modify directly. Although chemical modification methods include surface oxidation, surface grafting (chemical grafting, radiation grafting, and plasma grafting), chemical vapor deposition, and chemical plating, which can be used to improve the surface inertness of carbon fibers, increase active groups, and optimize surface morphology, each method has certain disadvantages, such as difficulty in controlling reaction conditions, environmental pollution, reduced fiber strength, difficulty in ensuring grafting efficiency and uniformity, and complex processing and high cost. In addition, the pore size of commercial carbon paper is mostly in the micron level and is determined by its respective brand model and cannot be arbitrarily controlled. These two points limit its further application as a solid-state nanopore substrate.

[0006] The use of sol-gel is expected to overcome the two major shortcomings of carbon paper as a nanopore. The sol-gel method is a widely used and effective method in the chemical industry for preparing nanopore films. The silica sol-gel process is a relatively complex system, and each step of hydrolysis, polycondensation, and aging can affect the pore size. Compared with other methods, the sol-gel method is simple, has high coating efficiency, and can adjust the pore size. In addition, by introducing different chemical groups or functionalizing agents during the film formation process, functional modification can be achieved, which can be said to kill two birds with one stone.

[0007] At present, the process of capturing targets in solid-state nanopores is often time-consuming and inefficient. This is because the functionalization of solid-state nanopores is limited to the modification of biological molecules such as nucleic acid aptamers, specific antibodies or proteins. Although biological molecules can serve as both recognition units and capture units, responsible for binding to targets and enriching them in nanopores, the modification of such molecules requires mild reaction conditions. Heating, acid-base, and even vigorous stirring and shaking can easily lead to denaturation, inactivation or degradation, which to a certain extent limits the functionalization of nanopores. Moreover, their specific binding to targets relies on non-covalent forces including hydrogen bonds, hydrophobic interactions, and ionic bonds, all of which are weak interaction forces, resulting in time-consuming recognition and capture processes and difficulty in efficiently enriching targets in the pores, thus affecting the stability and accuracy of the method. Therefore, improving the reaction kinetics of the capture process is also key to improving the detection performance of nanopores.

[0008] Click chemistry holds promise for overcoming this dilemma. Proposed by Nobel Laureate in Chemistry K. Barry Sharpless, it is a class of highly efficient and selective chemical reactions encompassing cycloadditions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, and carbon-carbon multiple bond additions. Click chemistry offers rapid reaction times, mild conditions, high yields, few byproducts, and excellent biocompatibility. In biosensors, click chemistry has been widely applied in areas such as biomolecule labeling and immobilization, signal amplification, multifunctional design, nanomaterial functionalization, in situ detection, and microfluidic chip integration. Click chemistry can be used to immobilize antibodies, aptamers, or enzymes on sensor surfaces, achieving functional modification. Using click chemistry as a capture unit may yield promising results because the covalent bonds formed by click chemistry have higher bond energies than weak interactions, representing strong interactions. Molecules or atomic groups linked by covalent bonds are more stable, and their application in molecular capture is expected to significantly improve capture efficiency and stability. This application has not yet been reported. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention provides a solid-state nanopore biosensor, system and method for detecting tumor markers.

[0010] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A nanopore biosensor for detecting tumor markers, the biosensor comprising: A nanoporous membrane, wherein the nanoporous membrane uses carbon fiber paper as a substrate, and a functionalized silicon-based sensitive layer is constructed on the carbon fiber paper substrate. The silicon-based sensitive layer is formed in situ by hydrolysis-condensation of a silane coupling agent containing click chemistry molecule-1 through sol-gel hydrolysis and polycondensation, and nanopores are formed; The silane coupling agent has a hydrolyzed alkoxy group at one end and a click chemical molecule I or a reactive group for introducing a click chemical molecule I at the other end; The target recognition unit is an aptamer modified with a click chemistry molecule II at one end. The aptamer can specifically recognize the target tumor marker. The target recognition unit passes through the nanopore, and the click chemistry molecule I and the click chemistry molecule II undergo a click reaction to covalently fix the aptamer.

[0011] Preferably, the silane coupling agent is selected from 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropylmethyldiethoxysilane, 3-mercaptopropyltrimethoxysilane, vinyltriethoxysilane, 3-azidopropyltriethoxysilane, 5-norbornene-2-methyltrimethoxysilane, 6-(1,2,4,5-tetrazine-3-yl)hexyltriethoxysilane, and 3-isocyanatepropyltriethoxysilane.

[0012] Preferably, the click chemistry molecule I and click chemistry molecule II are respectively selected from one reactant in the click chemistry reaction of azide and terminal alkyne, azide and ring-strained alkyne, tetrazine and trans-cyclooctene, tetrazine and norbornene, thiol and alkene, thiol and alkyne, tetrazole and alkene, thioester and primary amine; preferably, the click chemistry molecule I is norbornene, and the click chemistry molecule II is tetrazine.

[0013] Preferably, the method for preparing the biosensor comprises the following steps: 1) The silane coupling agent sol-gel is hydrolyzed and polycondensed, and then placed on a carbon fiber paper substrate, and then aged to obtain a nanoporous membrane; 2) The aptamer reacts with the tumor marker to obtain a target recognition unit, which is then passed through the nanopore membrane.

[0014] Preferably, the volume of the silane coupling agent sol-gel modified carbon fiber paper is 1-5 mL / cm 2 ; and / or, the reaction time of the aptamer and the tumor marker is 15 to 60 minutes.

[0015] Preferably, the hydrolysis-polycondensation conditions in step (1) are as follows: the concentration of the silane coupling agent is 5% to 20%, the solvent is anhydrous ethanol, the reaction time is 2 to 5 hours, and the reaction temperature is 40 to 60°C.

[0016] Preferably, the carbon fiber paper is one of Japan Toray carbon paper, Germany SGL carbon paper, United States Avcarb carbon paper, and Carbon Energy Technology carbon paper.

[0017] Furthermore, the present invention also provides a tumor marker detection system, which comprises: The biosensor; two electrolyte chambers, with the biosensor disposed between the two electrolyte chambers; a voltage supply device for supplying voltage to the two electrolyte chambers; The data processing unit is used to detect and record the steady-state current changes of the biosensor under applied voltage, process the data, and output the detection results.

[0018] As preferred, the concentration of the electrolyte in the electrolyte chamber is 1M; and / or, the voltage providing device provides a voltage of -1V~1V to the two electrolyte chambers.

[0019] As preferred, the electrolyte is KCl; and / or, the voltage providing device is an electrochemical workstation.

[0020] Further, the present application also provides a method for detecting tumor markers by using the tumor marker detection system, comprising the following steps: 1) The voltage providing device applies a controllable external voltage to the electrolyte chamber, and the data processing unit records the steady-state current change of the biosensor under the applied voltage; 2) Different concentrations of tumor marker standard solutions are configured, and after the tumor marker standard solutions react with the aptamers, a plurality of biosensors are obtained through the nanopore membrane. The concentration of the tumor marker is taken as the independent variable X, and the current change value is taken as the dependent variable Y. A standard curve between the concentration of the tumor marker and the current size is established by data processing to obtain a fitting equation; (3) The tumor marker in the unknown serum is reacted with the aptamer, and the target biosensor is obtained through the nanopore membrane. The obtained current value is substituted into the fitting equation to obtain the concentration value.

[0021] Aptamer (Aptamer) is a single-stranded oligonucleotide (DNA or RNA) or polypeptide molecule that can specifically bind to target molecules. Aptamer has many advantages. First, compared with traditional microbial culture, the detection period is short, the detection limit is low, and it has high affinity and strong specificity, which is due to the large surface area and a large number of receptor binding sites of the aptamer, and the three-dimensional structure is easy to form helix, hairpin, stem loop, convex ring, clover and pseudoknot structure, which can be tightly combined with target substances based on van der Waals force, hydrogen bond and hydrophobic interaction, and can distinguish similar substances; secondly, compared with antibody detection, the obtained aptamer is easy to synthesize in vitro, has good repeatability, high stability and is easy to store; thirdly, compared with molecular biology, the operation is simple and easy to operate, and the detection cost is low; at the same time, the target of the aptamer is wide, including pesticides, tissues, cells, viruses, proteins, toxins, vitamins and allergens; the aptamer is easy to label fluorescence and the activity is not affected, so it is easy to combine with other detection technologies. Therefore, the present application uses aptamer as a recognition element to construct a nanopore sensor to realize rapid and quantitative detection of tumor markers.

[0022] Tumor markers, also known as tumor markers, refer to substances that are characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or produced in response to tumor stimulation by the host. They can reflect tumor development and progression and monitor tumor response to treatment. Tumor markers are present in the tissues, body fluids, and excretions of cancer patients. Tumor markers can be proteins, enzymes, hormones, metabolites, or non-coding RNA. Preferred tumor markers in this application are thyroid-stimulating hormone (TSH).

[0023] This invention uses carbon fiber paper as a substrate, providing excellent mechanical strength and electrical conductivity. A sol-gel method is used to construct a silicon-based sensitive layer with a nanoscale porous structure on the substrate surface, enabling comprehensive pore size control. APTES stably bonds to the carbon paper surface through a silanization reaction, further enhancing the carbon paper's hydrophilicity. Click chemistry is introduced to achieve specific capture of target molecules, improving the stability and tolerance of the sensing system. This new detection strategy, based on the combination of novel solid-state nanopores and click chemistry, is universal and versatile, capable of rapidly and accurately detecting tumor markers, breaking through the bottlenecks and difficulties of existing tumor marker detection and providing strong support for precision medicine.

[0024] The detection method of the present invention is simple to operate, greatly shortens the detection time, has strong specificity, high sensitivity, high precision, good reproducibility, and low cost. It can be widely used in tumor marker detection and expands the application of commercial carbon fiber paper in the field of life analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Fiber morphology and element distribution of carbon paper before and after modification according to Example 1 of the present invention.

[0026] Figure 2 This is an AFM image of the surface morphology of the original carbon paper before modification in Example 1 of the present invention.

[0027] Figure 3 This is an AFM image of the surface morphology of the original carbon paper after modification in Example 1 of the present invention.

[0028] Figure 4 This is a graph showing the water contact angle test results of the original carbon paper before and after modification in Example 1 of the present invention.

[0029] Figure 5 This is a graph showing the relationship between thyroid stimulating hormone (TSH) concentration and current value in Example 2 of the present invention.

[0030] Figure 6 This is a comparison chart of the results of measuring tumor markers in unknown serum using the method of the present application and the standard method (electrochemiluminescence method) in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0032] Example 1 Preparation of nanoporous membranes: 1) 3-Aminopropyltriethoxysilane (APTES) was modified with norbornene molecules via organic synthesis to obtain functionalized APTES. This APTES was then used as a silane monomer for hydrolysis-condensation. A 5% functionalized APTES solution was prepared in anhydrous ethanol and hydrolyzed at 60°C for 4 hours to obtain a functionalized APTES-norbornene sol-gel. 2) APTES-norbornene sol-gel was absorbed by Toray hydrophilic carbon paper (Toray, Japan), and the APTES-norbornene sol-gel was modified on the carbon paper surface. After being placed in the dark, nanopores were constructed to obtain a solid nanoporous membrane.

[0033] Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used together. Figure 1 shown. Figure 1 (a) and Figure 1 (b) is the unmodified bare carbon paper. Under the scanning electron microscope, Figure 1 (a) Bare carbon paper exhibits a fibrous network structure. The carbon fibers have uniform diameters and varying lengths, interwoven into a dense network. Magnification reveals a relatively smooth surface. Figure 1 (b) No obvious defects or modifications were found.

[0034] Figure 1 (c) Figure 1 (d) Figure 1 (e) is an electron microscopy image after modification with APTES-norbornene sol-gel. Figure 1 In (c), obvious traces of chemical modification are visible, but the original morphology of the carbon fibers remains intact, remaining slender and straight. The fibers are interwoven, maintaining an intricate network structure. The sol-gel adheres tightly to the carbon fibers and evenly coats them. This result indicates that the introduction of APTES-norbornene does not alter the basic arrangement of the carbon fibers, but rather forms a good interfacial bond through a weak interaction force. Figure 1 (d) It can be seen that the modified nanopores were successfully constructed. Their formation is a direct result of the self-assembly and solidification of the APTES-norbornene sol-gel during the reaction process, demonstrating the ability of the sol-gel method to precisely control the material structure at the microscale.

[0035] To further verify the successful APTES-norbornene modification, combined with the results of SEM observations, EDS technology was used to accurately measure the elemental distribution on the carbon fiber surface. EDS detects the characteristic X-ray energy emitted by different elements on the sample surface, enabling rapid identification and quantification of element types and contents. Figure 1 As shown in Figures (f), (g), and (h), EDS results clearly reveal the uniform distribution of N, O, and Si on the carbon fiber surface, with no apparent agglomeration or localized enrichment. This uniform distribution of the three elements indicates better surface coverage and a more stable chemical state, confirming the effectiveness of the APTES-norbornene modification process.

[0036] Figure 2 、 3 The original carbon paper ( Figure 2 ) and APTES-norbornene modified surfaces ( Figure 3 ). Compared with the morphology of the original carbon paper, the surface peak height is significantly reduced after modification. The root mean square roughness (Rq) and average surface roughness (Ra) of the carbon paper before and after modification were obtained from the morphology images. After modification with APTES-norbornene, Rq decreased from 599 nm to 366 nm, and Ra decreased from 487 nm to 317 nm, indicating that the modification layer significantly reduced the surface roughness and formed a uniform coverage. This smoothing effect is due to the stable binding of APTES to the carbon paper surface through a silanization reaction. The synergistic effect of the siloxane network formed by the hydrolysis-condensation of APTES and the hydrophobic groups of norbornene fills the microscopic pores or cracks on the carbon fiber surface while forming a continuous and dense covering layer during the sol-gel process. This reduces the vertical fluctuation range of the surface structure, thereby weakening the original peak-to-valley difference.

[0037] The water contact angle (CA) test showed that the contact angle of the untreated original carbon paper was 133.4°, indicating strong surface hydrophobicity and poor water spreading ( Figure 4 a). After modification with APTES-norbornene sol-gel, the water absorption capacity of the sample was significantly enhanced, and the droplets were slowly absorbed ( Figure 4 b). Therefore, screenshot recording was used during the test, capturing an image every 62 milliseconds to precisely capture the moment of droplet-sample contact. The contact angle measured at this critical moment was 115.3°. The modified carbon paper surface is more hydrophilic, facilitating water spreading. This indicates that the Si-O-Si network contains pores that allow water molecules to penetrate via capillary action, consistent with the SEM results. The introduction of amide groups (C=O-NH) and silanol groups (Si-OH) formed by partial hydrolysis of silane further enhances the carbon paper's surface hydrophilicity, allowing the solution containing the tumor marker and aptamer to more easily pass through the carbon paper surface.

[0038] Example 2 The tumor marker is detected by using the tumor marker detection system, and the steps are as follows: Step (1): Synthesize aptamer with high affinity for thyroid stimulating hormone (TSH), and modify tetrazine molecule at one end of the aptamer; Step (2): Prepare 8 TSH standard solution samples with concentration ranging from 0.3 to 625 pg mL -1 After the target is fully combined with the aptamer solution, the solution is vertically passed through the nanopore membrane prepared in Example 1 to obtain the treated solid-state nanopore membrane (i.e. biosensor); Step (3): Place the treated solid-state nanopore membrane between two chambers filled with KCl electrolyte in turn, apply voltage (-1V~1V) and perform linear scan through electrochemical workstation, record the steady-state current change of the treated solid-state nanopore membrane under linear scan voltage (-1V~1V) respectively, take TSH concentration as independent variable X and current value corresponding to 1V voltage as dependent variable Y, establish standard curve between tumor marker concentration and current size, and obtain fitting equation; Step (4): For tumor markers in unknown serum, react with aptamer solution for 15 min, fully combine the target with the aptamer solution, then pass the solution vertically through the nanopore membrane prepared in Example 1 to obtain the treated solid-state nanopore membrane, place the treated solid-state nanopore membrane between two chambers filled with KCl electrolyte, apply voltage (-1V~1V) and perform linear scan through electrochemical workstation, measure the current value, and substitute the current value corresponding to 1V voltage into the fitting equation to obtain the concentration value.

[0039] This example utilizes the high reaction rate characteristics of click chemistry to capture the target, and in this process, the nanopore channel serves as an ion conduction channel to record the ion current change under linear scan voltage (-1V~1V). As shown in Figure 5 With the gradual increase of TSH concentration, the observed ion current shows a decreasing trend, and this change trend can be fitted by logarithmic equation, R2 is 0.9975, indicating that the fitting effect of the relationship between TSH concentration and steady-state current is very good, with high prediction accuracy and reliability, and the quantitative detection limit can reach 0.3 pg mL⁻¹, indicating that the method has high sensitivity.

[0040] Figure 6The results of the tumor markers in 5 unknown sera detected by the method of the application and the standard method (WS / T404.10-2022 electrochemiluminescence method) are compared in the following figure. It can be seen that the results of the two methods are very similar, indicating that the detection method of the application has certain reliability.

[0041] The above describes the embodiments of the present application, and through the above description of the disclosed embodiments, those skilled in the art can implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nanopore biosensor for detecting tumor markers, characterized in that: The biosensor contains: A nanoporous membrane, wherein the nanoporous membrane uses carbon fiber paper as a substrate, and a functionalized silicon-based sensitive layer is constructed on the carbon fiber paper substrate. The silicon-based sensitive layer is formed in situ by hydrolysis-condensation of a silane coupling agent containing click chemistry molecule-1 through sol-gel hydrolysis and polycondensation, and nanopores are formed; The silane coupling agent has a hydrolyzed alkoxy group at one end and a click chemical molecule I or a reactive group for introducing a click chemical molecule I at the other end; The target recognition unit is an aptamer modified with a click chemistry molecule II at one end. The aptamer can specifically recognize the target tumor marker. The target recognition unit passes through the nanopore, and the click chemistry molecule I and the click chemistry molecule II undergo a click reaction to covalently fix the aptamer.

2. The nanopore biosensor for detecting tumor markers according to claim 1, characterized in that: The silane coupling agent is selected from one of 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropylmethyldiethoxysilane, 3-mercaptopropyltrimethoxysilane, vinyltriethoxysilane, 3-azidopropyltriethoxysilane, 5-norbornene-2-methyltrimethoxysilane, 6-(1,2,4,5-tetrazine-3-yl)hexyltriethoxysilane, and 3-isocyanatepropyltriethoxysilane.

3. The nanopore biosensor for detecting tumor markers according to claim 1, characterized in that: The click chemistry molecule I and click chemistry molecule II are respectively selected from one reactant in the click chemistry reaction of azide and terminal alkyne, azide and ring-strained alkyne, tetrazine and trans-cyclooctene, tetrazine and norbornene, thiol and alkene, thiol and alkyne, tetrazole and alkene, thioester and primary amine; preferably, the click chemistry molecule I is norbornene, and the click chemistry molecule II is tetrazine.

4. The nanopore biosensor for detecting tumor markers according to claim 1, characterized in that: The method for preparing the biosensor comprises the following steps: 1) The silane coupling agent sol-gel is hydrolyzed and polycondensed, and then placed on a carbon fiber paper substrate, and then aged to obtain a nanoporous membrane; 2) The aptamer reacts with the tumor marker to obtain a target recognition unit, which is then passed through the nanopore membrane.

5. The nanopore biosensor for detecting tumor markers according to claim 4, characterized in that: The volume of the silane coupling agent sol-gel modified carbon fiber paper is 1~5mL / cm 2 ; and / or, the reaction time of the aptamer and the tumor marker is 15 to 60 minutes.

6. The nanopore biosensor for detecting tumor markers according to claim 1, characterized in that: The carbon fiber paper is one of Japan's Toray carbon paper, Germany's SGL carbon paper, the United States' Avcarb carbon paper, and Carbon Energy Technology carbon paper.

7. A tumor marker detection system, characterized in that: The detection system includes: The biosensor according to any one of claims 1 to 6; two electrolyte chambers, with the biosensor disposed between the two electrolyte chambers; a voltage supply device for supplying voltage to the two electrolyte chambers; The data processing unit is used to detect and record the steady-state current changes of the biosensor under applied voltage, process the data, and output the detection results.

8. A tumor marker detection system according to claim 7, characterized in that: The concentration of the electrolyte in the electrolyte chamber is 1M; and / or, the voltage providing device provides a voltage of -1V to 1V for the two electrolyte chambers.

9. The tumor marker detection system according to claim 7, characterized in that: The electrolyte is KCl; and / or the voltage providing device is an electrochemical workstation.

10. A method for detecting tumor markers using a tumor marker detection system according to any one of claims 7 to 9, characterized in that: The following steps are involved: 1) The voltage supply device applies a controllable external voltage to the electrolyte chamber, and the data processing unit records the steady-state current changes of the biosensor under the applied voltage; 2) Prepare standard solutions of tumor markers at different concentrations. After reacting with the aptamer, the solutions are passed through a nanopore membrane to form multiple biosensors. Using tumor marker concentration as the independent variable (X) and current change as the dependent variable (Y), a standard curve between tumor marker concentration and current is established through data processing to obtain a fitting equation. 3) The tumor marker in the unknown serum reacts with the aptamer and passes through the nanopore membrane to obtain the target biosensor. The obtained current value is substituted into the fitting equation to obtain the concentration value.

Images