Fluorescent biosensor, system and method for detecting tumor markers

Abstract

The invention relates to the technical field of photochemical biosensing detection, and discloses a fluorescent biosensor, system and method for detecting tumor markers, commercialized filter paper is used as a substrate, a silica-based sensitive layer is constructed on the surface of the substrate by a sol-gel method to obtain a nanopore membrane, and specific capture of target molecules is realized by introducing click chemistry. The stability and the tolerance of a sensing system are improved. The method is simple and convenient to operate, high in result visualization degree, capable of greatly shortening the detection time, high in specificity, high in sensitivity, high in precision, good in reproducibility and low in cost, and can be widely applied to tumor marker detection.

Description

Technical Field

[0001] The present invention relates to the field of photochemical biosensor detection technology, and in particular to a fluorescent 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 important clinical value and social significance. The development of new detection technologies can drive continuous innovation and advancement in tumor marker detection.

[0003] Detection methods for tumor markers 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 of high sensitivity, high resolution, no labeling, and rapid detection, has attracted increasing attention from researchers. Nanopore technology originates from the transmembrane molecular transport of single molecules in organisms through nanoscale pores in cell membranes. Different from natural nanopores, solid-state nanopores are nanopores prepared on substrates such as glass, silicon nitride, polyethylene terephthalate, and molybdenum disulfide, and functionalized using chemical and advanced micro-nanotechnology techniques.

[0004] The sol-gel method is a widely used and effective method in the chemical industry for preparing nanopore thin films. The silica sol-gel process is a relatively complex system, and each step, including hydrolysis, polycondensation, and aging, can affect pore size. Compared with other methods, the sol-gel method offers simplicity, high coating efficiency, and adjustable pore size. Furthermore, functional modification can be achieved by introducing different chemical groups or functionalizing agents during the film formation process, achieving two goals at once.

[0005] Portable colorimetric sensors, with their high sensitivity, ease of use, multimodal cross-validation, and low cost, are driving the expansion of tumor marker testing from the laboratory to the clinic and at home. Future integration with artificial intelligence (e.g., machine learning to optimize signal analysis) and the Internet of Things (IoT) (real-time data transmission) will further personalize and intelligentize cancer screening. However, the detection limits of existing colorimetric sensors currently fall short of clinical needs. For example, traditional methods for detecting tumor-derived exosomes (e.g., flow cytometry) lack sensitivity, while nanozyme platforms based on single-target identification are susceptible to heterogeneous interference. Furthermore, some sensors suffer from reduced sensitivity in complex biological samples (e.g., serum) due to background interference. Therefore, the development of portable, sensitive, accurate, and low-cost colorimetric sensors is urgently needed. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides a fluorescent biosensor, system and method for detecting tumor markers.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A fluorescent biosensor for detecting tumor markers, the biosensor comprising: A nanoporous membrane, wherein the nanoporous membrane uses filter paper as a substrate, and a functionalized silicon-based sensitive layer is constructed on the filter 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 a mixture of aptamer-1 modified with a click chemistry molecule II at one end and aptamer-2 modified with a fluorescent molecule 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.

[0008] Click chemistry, proposed by Nobel Prize winner in Chemistry K. Barry Sharpless, is a class of highly efficient and selective chemical reactions. These reactions include cycloadditions, nucleophilic ring-opening reactions, non-aldol carbonyl chemistry, and addition reactions of carbon-carbon multiple bonds. Click chemistry is characterized by rapid reaction speed, mild conditions, high yields, few byproducts, and excellent biocompatibility. Click chemistry reaction combinations include azides and terminal alkynes, azides and ring-strained alkynes, tetrazines and trans-cyclooctenes, tetrazines and norbornenes, thiols and alkenes, thiols and alkynes, tetrazoles and alkenes, and thioesters and primary amines. This application prefers the reaction between tetrazines and norbornenes to assist in the capture and detection of tumor markers.

[0009] 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.

[0010] 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.

[0011] Preferably, the fluorescent molecule is one of FAM, VIC, HEX, TET, JOE, CY3, CY5, ROX, and Texas Red.

[0012] Filter paper is a porous paper filter medium made from fibrous materials (such as cotton and wood pulp). It separates solids from liquids or liquids from liquids through physical barriers. Most filter paper is composed of cotton fibers and is manufactured using different methods depending on its intended use. Because it is made of fiber, the surface of filter paper is covered with tiny pores. The size and distribution of these pores determine the filter paper's filtration accuracy and flow rate. Filter paper is a common laboratory and industrial product, making it readily available, inexpensive, and a ubiquitous raw material.

[0013] Preferably, the filter paper is Sartorius filter paper, Whatman filter paper or Pall Corporation filter paper.

[0014] 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 filter paper substrate, and 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.

[0015] Preferably, the volume of the silane coupling agent sol-gel modified filter paper is 0.5-1.5 mL / cm 2 ; and / or, the reaction time of the aptamer and the tumor marker is 8-12 minutes.

[0016] 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.

[0017] Furthermore, the present invention also provides a tumor marker detection system, which comprises: The biosensor; A fluorescence observation device for obtaining a color value of the biosensor; The data processing unit is used to process data and output detection results.

[0018] Preferably, the fluorescence observation device is a fluorescence observation dark box.

[0019] Furthermore, the present invention also provides a method for detecting tumor markers using the tumor marker detection system, comprising the following steps: 1) Prepare tumor marker standard solutions of different concentrations, react with aptamers, and pass through nanopore membranes to obtain multiple biosensors; 2) Place the biosensor in a fluorescence observation device, take a picture under excitation light, and extract the color value; 3) Using tumor marker concentration as the independent variable X and color value as the dependent variable Y, a standard curve between tumor marker concentration and color value was established to obtain the fitting equation; 4) The tumor marker in the unknown serum is reacted with the aptamer to obtain a tumor marker-aptamer solution. After the reaction, the target biosensor is obtained through a nanoporous membrane. Step (2) is repeated and the obtained color value is substituted into the fitting equation to obtain the concentration value.

[0020] Aptamers are single-stranded oligonucleotides (DNA or RNA) or polypeptides that specifically bind to target molecules. Aptamers offer numerous advantages. First, compared to traditional microbial culture, they offer shorter detection cycles, lower detection limits, and high affinity and specificity. This is due to their large surface area and numerous receptor-binding sites. Aptamers also possess a three-dimensional structure that easily forms helices, hairpins, stem-loops, convex loops, trefoil structures, and pseudoknots. Aptamers bind tightly to their targets through van der Waals forces, hydrogen bonds, and hydrophobic interactions, and can distinguish between structurally similar substances. Second, compared to antibody-based assays, screened aptamers can be readily synthesized in large quantities in vitro, offering excellent reproducibility, high stability, and easy storage. Third, compared to molecular biology, aptamers are simple to perform and have lower detection costs. Furthermore, aptamers target a wide range of targets, including pesticides, tissues, cells, viruses, proteins, toxins, vitamins, and allergens. Aptamers can be easily labeled with fluorescent markers without compromising their activity, making them easy to combine with other detection technologies. Therefore, the present application uses aptamers as recognition elements to construct colorimetric sensors to achieve rapid and quantitative detection of tumor markers.

[0021] 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).

[0022] This invention uses commercially available filter paper as a substrate and employs a sol-gel method to construct a silicon-based sensitive layer on the substrate surface to create a nanoporous membrane. Click chemistry is introduced to achieve specific capture of target molecules, improving the stability and tolerance of the sensing system. The immobilization and enrichment of fluorescent molecules significantly enhances detection sensitivity. This new detection strategy, combined with click chemistry, is universal and versatile, enabling rapid and accurate detection of tumor markers, breaking through existing bottlenecks and difficulties in tumor marker detection and providing strong support for precision medicine.

[0023] The method of the present invention is simple to operate, has a high degree of visualization of the results, greatly shortens the detection time, has strong specificity, high sensitivity, high precision, good reproducibility, and low cost, and can be widely used in tumor marker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the XPS graph of the nanoporous membrane in Example 1.

[0025] Figure 2 This is the laser confocal scanning microscope result of the positive sample in Example 2.

[0026] Figure 3 This is the laser confocal scanning microscopy result of the negative sample in Example 2.

[0027] Figure 4 This is a graph showing the relationship between thyroid stimulating hormone (TSH) concentration and color value in Example 3.

[0028] Figure 5 The figure is a comparison of the results of measuring tumor markers in 5 unknown sera using the method of the present application and the standard method (electrochemiluminescence method). DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1 Prepare the nanoporous membrane. The specific steps are as follows: 1) Functionalized APTES was obtained by modifying norbornene on 3-aminopropyltriethoxysilane molecules. A 5% functionalized APTES solution was prepared in anhydrous ethanol as a silane monomer and hydrolyzed at 60°C for 4 hours to obtain a functionalized APTES-norbornene sol-gel. 2) Use a pipette to draw up 1 mL of the APTES-norbornene sol-gel and drop it onto the surface of a filter paper cut into 1 cm × 1 cm pieces for modification. After being placed in the dark, a silicon-based sensitive layer is constructed and nanopores are formed, resulting in a nanoporous membrane.

[0031] Figure 1 X-ray photoelectron spectroscopy (XPS) results of the modified filter paper are presented. Two new peaks appeared near 101.08 eV and 399.48 eV, corresponding to the Si2p and N1s peaks, respectively. The N1s peak originates from the amide group, confirming the effectiveness of the APTES-norbornene modification process.

[0032] Example 2 The tumor marker detection system was used to qualitatively identify TSH-positive and TSH-negative samples and to obtain fluorescence images. The specific steps are as follows: 1) Targeting TSH, aptamers-1 and -2 with high affinity were synthesized. One end of aptamer-1 was modified with a FAM fluorescent molecule, and one end of aptamer-2 was modified with a tetrazine, resulting in an aptamer solution. 2) The positive and negative samples were reacted with the aptamer solution, respectively. After 10 minutes of reaction, the solution was passed vertically through the nanoporous membrane prepared in Example 1. Norbornene and tetrazine underwent a click chemistry reaction, successfully capturing the target molecule and enriching the FAM-labeled aptamer within the nanochannel. 3) Place the nanoporous membranes of the positive and negative samples after the reaction in a fluorescence observation dark box respectively. Under irradiation with excitation light of 490 nm wavelength, take photos to obtain fluorescence images of the positive and negative samples after passing through the solid-state nanochannel.

[0033] like Figure 2 、 3 The figure shows fluorescence images of positive and negative samples after they pass through the solid-state nanochannel. The positive sample shows a strong green fluorescence signal. Through comparative analysis, it can be clearly observed that compared with the almost invisible fluorescence background in the negative sample channel, the positive sample channel shows a significant green fluorescence signal.

[0034] Example 3 The specific steps for establishing the TSH standard curve are as follows: Step (1): Prepare 6 samples with a concentration range of 0.5-20 pg mL -1 The TSH standard solution was reacted with the aptamer solution for 10 minutes to allow the target and the aptamer solution to fully bind. The solution was then passed vertically through the nanoporous membrane prepared in Example 1 to cause a click chemical reaction between norbornene and tetrazine. Step (2): Place the reacted nanopore membrane (i.e., biosensor) in a fluorescence observation dark box, take photos under 490 nm excitation light, extract the green value, use the tumor marker concentration as the independent variable X, and the color value as the dependent variable Y, establish a standard curve between the tumor marker concentration and the green value, and obtain the fitting equation.

[0035] like Figure 4 As shown in Figure 2, with the gradual increase of TSH concentration, the fluorescence intensity gradually increased, and this change trend can be fitted by a linear equation, R 2 The value of the green value was 0.9989, indicating that the fitting effect of the relationship between TSH concentration and green value was very good, with high prediction accuracy and reliability, and the quantitative detection limit could reach 0.5 pg mL⁻¹, indicating that the method had high sensitivity.

[0036] To detect target tumor markers, the steps are as follows: For unknown tumor markers in serum, the target was reacted with the aptamer solution for 10 minutes to allow for full binding between the target and the aptamer solution. The solution was then passed vertically through the nanoporous membrane prepared in Example 1. The reacted nanoporous membrane (i.e., the target biosensor) was placed in a fluorescence observation dark box. Under irradiation with 490 nm wavelength excitation light, a photograph was taken, and the green value was extracted and substituted into the fitting equation to obtain the concentration value.

[0037] Figure 5 The following chart compares the results of tumor markers measured in five unknown serum samples using the present method and the standard method (electrochemiluminescence method according to WS / T404.10-2022). As can be seen, the results of the two methods are very similar, indicating that the detection method of the present application has a certain degree of reliability.

[0038] The above is a description of the embodiments of the present invention. The above description of the disclosed embodiments will enable professionals in the field to implement or use the present invention. Various modifications to these embodiments will be apparent to professionals in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescent biosensor for detecting tumor markers, characterized in that: The biosensor contains: A nanoporous membrane, wherein the nanoporous membrane uses filter paper as a substrate, and a functionalized silicon-based sensitive layer is constructed on the filter 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 a mixture of aptamer-1 modified with a click chemistry molecule II at one end and aptamer-2 modified with a fluorescent molecule 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. A fluorescent 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 fluorescent 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 fluorescent biosensor for detecting tumor markers according to claim 1, characterized in that: The fluorescent molecule is one of FAM, VIC, HEX, TET, JOE, CY3, CY5, ROX, and Texas Red.

5. The fluorescent biosensor for detecting tumor markers according to claim 1, characterized in that: The filter paper is Sartorius filter paper, Whatman filter paper or Pall Corporation filter paper.

6. The fluorescent 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 filter paper substrate, and 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.

7. The fluorescent biosensor for detecting tumor markers according to claim 6, characterized in that: The volume of the silane coupling agent sol-gel modified filter paper is 0.5~1.5mL / cm 2 ; and / or, the reaction time of the aptamer and the tumor marker is 8-12 minutes.

8. A tumor marker detection system, characterized in that: The detection system includes: The biosensor according to any one of claims 1 to 7; A fluorescence observation device for obtaining a color value of the biosensor; The data processing unit is used to process data and output detection results.

9. A tumor marker detection system according to claim 8, characterized in that: The fluorescence observation device is a fluorescence observation dark box.

10. A method for detecting tumor markers using the tumor marker detection system according to any one of claims 8 or 9, characterized in that: The following steps are involved: 1) Prepare tumor marker standard solutions of different concentrations, react with aptamers, and pass through nanopore membranes to obtain multiple biosensors; 2) Place the biosensor in a fluorescence observation device, take a picture under excitation light, and extract the color value; 3) Using tumor marker concentration as the independent variable X and color value as the dependent variable Y, a standard curve between tumor marker concentration and color value was established to obtain the fitting equation; 4) The tumor marker in the unknown serum is reacted with the aptamer to obtain a tumor marker-aptamer solution. After the reaction, the target biosensor is obtained through a nanoporous membrane. Step (2) is repeated and the obtained color value is substituted into the fitting equation to obtain the concentration value.

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