Method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy

Abstract

The present invention belongs to the technical field of tumor marker detection, and specifically relates to a method for detecting tumor markers by SERS based on magnetic separation and superhydrophobic aggregation strategy. The present invention adopts the strategy of "magnetic separation capture → probe separation → probe aggregation", adds magnetic beads capable of capturing tumor markers in the sample, quickly extracts tumor markers from the sample through magnetic separation, and has simple operation; then adds SERS probes that can specifically bind to the markers, and then adopts a desorption process for the sample after magnetic separation to separate the Raman probes on the surface of the tumor markers; finally, the desorbed Raman probes are aggregated by a superhydrophobic SERS substrate, and Raman signals are collected. This method has the advantages of strong specificity, high sensitivity, good reproducibility, etc., improves the sensitivity and reproducibility of SERS detection of tumor markers, and solves the bottleneck problem that existing detection methods restrict the practical application of SERS in the early screening of tumor markers.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid detection of tumor markers, and particularly relates to a method for detecting tumor markers by surface-enhanced Raman scattering (SERS) based on magnetic separation and superhydrophobic convergence strategy. Background Art

[0002] Cancer is one of the major diseases that plague human survival and is the second leading cause of death globally, causing great physical, emotional, and economic harm to individuals and their families, and also imposing a huge burden on society. Currently, cancer diagnosis mainly relies on a series of imaging and histocytological techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), fluorescence molecular imaging, histopathology, cytology, etc. However, these methods also have problems such as high cost, radiation damage, high professional skill requirements, and long time consumption. Moreover, they cannot detect lesion tissues and cell abnormalities in the initial stage and are difficult to make a rapid and sensitive diagnosis of early local small lesions. Early cancer diagnosis is very important clinically as it can prevent the metastasis of cancer cells and provide patients with more treatment opportunities and higher cure rates. Therefore, developing highly sensitive and rapid early cancer diagnosis methods, especially non-invasive or minimally invasive examination methods, is very important for popularizing early cancer screening and regular cancer physical examinations.

[0003] Tumor cells can directly produce or induce some specific proteins, enzymes, gene products, metabolites, etc. to be produced by non-tumor cells. Substances that can reflect the occurrence and development of tumors are called tumor markers and are widely present in body fluids such as blood, urine, and saliva. Currently, the main three targeted objectives for tumor marker detection are circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and exosomes (Exosomes). However, in the early stage of cancer, the concentration of tumor markers is extremely low, posing a huge challenge to the sensitivity and accuracy of detection methods. Enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), fluorescence method, electrochemical method, mass spectrometry, etc., which are commonly used for cancer marker detection, are not suitable for tumor marker detection or have problems such as insufficient detection sensitivity and accuracy, long detection cycle, and cumbersome preparation.

[0004] In recent years, surface-enhanced Raman scattering (SERS) technology based on noble metal nanostructures has the advantages of rapidity, accuracy, reliability, sensitivity, etc. and has gradually been introduced into the field of cancer diagnosis. Compared with other methods, the SERS detection method has a lower detection limit and is not interfered by water in body fluids, showing significant advantages in the diagnosis of early stages of tumor markers with extremely low concentrations such as exosomes.

[0005] Currently, typical SERS tumor marker detection methods modify antibodies that can specifically capture tumor markers on the substrate. After capture, Raman probes that can specifically recognize tumor markers are added to form a typical sandwich structure, and then Raman testing is directly performed. However, the Raman probes are dispersed throughout the SERS substrate (centimeter scale), with a large and uneven dispersion area, while the laser spot is very small (micrometer scale), resulting in the disadvantages of low sensitivity and poor signal repeatability during the SERS detection process. Summary of the Invention

[0006] The present invention discloses a SERS method for highly sensitive detection of tumor markers. The present invention adopts a "magnetic separation capture → probe separation → probe convergence" detection strategy for SERS screening of tumor markers. First, magnetic beads that can capture tumor markers in the sample are added, and the tumor markers in the sample are purified by magnetic separation. Secondly, SERS probes are added to label the cancerous tumor markers. Then, a desorbing agent is added to separate the Raman probes on the surface of the cancerous tumor markers. Finally, the separated Raman probes are converged and detected through a superhydrophobic substrate. This detection strategy indirectly detects the converged Raman probes, and there is no interference from other impurities during the detection process, so it has the advantages of strong specificity, high sensitivity, and good repeatability. In addition, magnetic separation can be used in the detection to quickly extract tumor markers from complex matrix samples such as serum or even whole blood, with fast and simple operation, avoiding the complex and time-consuming pre-treatment process of tumor marker purification.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategies, comprising the following steps:

[0008] Step S1: Use composite nanomagnetic beads as a capture agent, add them to a sample containing tumor markers and incubate together to capture the tumor markers in the sample; then separate the composite nanomagnetic beads captured with tumor markers from the solution to obtain a "capture agent - tumor marker" enrichment solution;

[0009] Step S2: Add Raman probes that can specifically bind to tumor markers to the "capture agent - tumor marker" enrichment solution and incubate, and magnetically separate the Raman probes that are not specifically bound to obtain a "capture agent - tumor marker - Raman probe" composite structure;

[0010] Step S3: Adopt a desorption process to strip the Raman probes in the "capture agent - tumor marker - Raman probe" composite structure and make them free in the solution to obtain desorbed Raman probes, magnetically separate the desorbed "capture agent - tumor marker", and then add an Ag nanoparticle solution to the solution and mix evenly to obtain a mixed solution containing Ag nanoparticles and desorbed Raman probes;

[0011] Step S4: Drop the mixture in Step S3 onto the superhydrophobic SERS substrate, and collect the Raman signal after drying, thereby achieving rapid measurement of the content of tumor markers.

[0012] As a further improvement to the method for SERS detection of tumor markers based on magnetic separation and superhydrophobic aggregation strategy:

[0013] Preferably, when the tumor marker is exosome, the composite nanomagnetic sphere in Step S1 is a core-shell structure Fe3O4@TiO2. The core of the core-shell structure Fe3O4@TiO2 is a Fe3O4 nanosphere with a diameter of 200 - 400 nm, and is coated with a TiO2 shell layer with a thickness of 20 - 100 nm.

[0014] Preferably, when the tumor marker is other proteins except exosome, the composite nanomagnetic sphere in Step S1 is a core-shell structure Fe3O4@SiO2-Abs. The core of the core-shell structure Fe3O4@SiO2-Abs is a Fe3O4 nanosphere with a diameter of 200 - 400 nm, and is coated with a SiO2 shell layer with a thickness of 20 - 100 nm, and the surface of the SiO2 shell layer is modified with an antibody Abs that can specifically bind to the tumor marker to be detected.

[0015] Preferably, the composite nanomagnetic sphere is a core-shell structure Fe3O4@TiO2, and the preparation method is as follows: Disperse the Fe3O4 nanospheres in ethanol, slowly add ammonia water and tetrabutyl titanate, tetrabutyl titanate hydrolyzes, and a TiO2 layer is formed on the surface of the Fe3O4 nanospheres to obtain the core-shell structure Fe3O4@TiO2;

[0016] Preferably, the composite nanomagnetic sphere is a core-shell structure Fe3O4@TiO2, and the preparation method is as follows: Disperse Fe3O4 nanospheres with a particle size of 300 - 500 nm in 100 ml of ethanol, with a dispersion concentration of 0.5 - 0.75 mg / ml, add 200 - 400 μL of ammonia water and mix well, then gradually add tetrabutyl titanate to ethanol, with the concentration of tetrabutyl titanate in ethanol being 10 - 30 mM, heat and stir for 16 - 24 h, collect the black product for washing and vacuum drying to obtain the core-shell structure Fe3O4@TiO 2。

[0017] Preferably, the composite nanomagnetic sphere is a core-shell structure Fe3O4@SiO2-Abs, and the preparation method is as follows:

[0018] 1) Disperse the Fe3O4 nanospheres in ethanol, slowly add ammonia water and tetraethyl orthosilicate, tetraethyl orthosilicate hydrolyzes, and a SiO2 layer is formed on the surface of the Fe3O4 nanospheres to obtain the core-shell structure Fe3O4@SiO2;

[0019] 2) The core-shell structured Fe3O4@SiO2 was carboxyl modified with 3-(triethoxysilyl) propyl succinic anhydride, and then the coupling chemical reaction reagents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to activate the carboxyl groups on the silica surface. After the activation was completed, antibody Abs was added for incubation. Antibody Abs underwent a coupling reaction with the carboxyl-modified Fe3O4@SiO2. Finally, bovine serum albumin solution was added to block the sites, and the solid product was collected and washed to obtain the coupling structure of the antibody and Fe3O4@SiO2, which was the core-shell structured Fe3O4@SiO2-Abs.

[0020] Preferably, the composite magnetic nanospheres are core-shell structured Fe3O4@SiO2-Abs, and the preparation method is as follows:

[0021] 1) The Fe3O4 nanospheres were dispersed in 100 ml of ethanol at a dispersion concentration of 0.5 - 0.75 mg / ml. 100 - 200 μL of ammonia water was added dropwise and mixed well. Subsequently, tetraethyl orthosilicate in ethanol was added drop by drop, and the concentration of tetraethyl orthosilicate was 0.05 - 0.07 M. It was heated and stirred for 2 - 4 h, and the black product was collected for washing and vacuum drying to obtain the core-shell structured Fe3O4@SiO2.

[0022] 2) The core-shell structured Fe3O4@SiO2 was dispersed in ethanol to prepare a Fe3O4@SiO2 solution with a concentration of 0.5 - 1 mg / ml. 10 μL of 3-(triethoxysilyl) propyl succinic anhydride (TEPSA) was added to 1 ml of the Fe3O4@SiO2 solution, and the mixture was stirred and reacted for 4 - 8 h. The reaction product was washed to obtain the carboxyl-modified Fe3O4@SiO2.

[0023] 3) 10 μL of the coupling chemical reaction reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with a concentration of 10 mg / ml and 20 μL of N-hydroxysuccinimide (NHS) with a concentration of 10 mg / ml were added to the above carboxyl-modified Fe3O4@SiO2 to activate the carboxyl groups on the silica surface. After the activation was completed, 100 - 400 μL of the antibody diluent with an antibody concentration of 20 μg / ml was added. After thorough mixing, it was incubated at 4°C for 12 - 24 h. Then, 100 - 200 μL of a 5% bovine serum albumin solution was added to block the sites, and the solid product was washed with phosphate buffer solution PBS to obtain the coupling structure of the antibody and Fe3O4@SiO2, which was the core-shell structured Fe3O4@SiO2-Abs.

[0024] Preferably, the preparation method of the Fe3O4 nanospheres is as follows: FeCl3·6H2O, trisodium citrate, sodium acetate, and ethylene glycol were mixed and subjected to a hydrothermal reaction to prepare Fe3O4 nanospheres.

[0025] Preferably, the preparation method of the Fe3O4 nanospheres is as follows: in an ethylene glycol solvent, FeCl3·6H2O and trisodium citrate are sequentially added and stirred until dissolved, and then sodium acetate NaAc is added and stirred until dissolved to obtain a mixture; wherein, the mass ratio of FeCl3·6H2O, trisodium citrate and sodium acetate NaAc is 3:1:6, and the addition concentration of FeCl3·6H2O in the ethylene glycol solvent is 0.1-0.2 M; the mixture is transferred to an autoclave and kept at 180-200 °C for 8-12 h, and the black product is collected, washed and dried under vacuum to obtain Fe3O4 nanospheres with a particle size of 300-500 nm.

[0026] Preferably, the Raman probe in step S2 is an Ag / Au-tag@SiO2-Abs with a core-shell structure. The core of the Raman probe is an Ag or Au nanoparticle with a particle size of 20-50 nm. A Raman signal reporter molecule (tag) is modified on the surface of the Ag or Au nanoparticle, and a SiO2 shell layer with a thickness of 1-5 nm is coated outside, and an antibody Abs capable of specifically adsorbing tumor markers is modified on the surface of the SiO2 shell layer.

[0027] Preferably, the preparation method of the Raman probe is as follows:

[0028] 1) Sodium citrate and ascorbic acid are used to reduce silver nitrate to prepare a sodium citrate ligand-stabilized and monodisperse spherical Ag nanoparticle solution;

[0029] Alternatively, sodium citrate is used to reduce chloroauric acid to prepare a sodium citrate ligand-stabilized and monodisperse spherical Au nanoparticle solution;

[0030] 2) 4-Mercaptobenzoic acid is used to modify the Ag / Au nanoparticles to obtain Ag / Au nanoparticles labeled with the reporter molecule tag, namely Ag / Au-tag;

[0031] 3) 3-Aminopropyltrimethoxysilane solution and sodium silicate solution are added to Ag / Au-tag to coat a SiO2 layer on the surface of Ag / Au-tag to obtain Ag / Au-tag@SiO2;

[0032] 4) 3-(Triethoxysilyl)propyl succinic anhydride (TEPSA) was used to modify the carboxyl group of Ag / Au-tag@SiO2. Then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to activate the carboxyl groups on the silica surface. After the activation, antibody Abs was added for incubation. Antibody Abs underwent a coupling reaction with the activated Ag / Au-tag@SiO2. Finally, a bovine serum albumin solution was added to block the sites, and the solid product was collected and washed to obtain the coupling structure of the antibody and Ag / Au-tag@SiO2, which was the Ag / Au-tag@SiO2-Abs Raman probe.

[0033] Preferably, the Raman probe is Ag-tag@SiO2-Abs, and the preparation method is as follows:

[0034] 1) Take 1 ml of a 34.3 mM sodium citrate solution, 0.25 - 0.5 ml of a 59.5 mM silver nitrate solution, and 0.2 ml of a 20 mM sodium chloride solution, mix them at room temperature for 5 - 10 minutes to obtain a mixed solution. The above mixed solution and 70 - 80 μL of a 100 mM ascorbic acid solution were quickly added to 50 ml of boiling water, heated and stirred under reflux for 60 - 90 minutes, and then cooled to room temperature to obtain a monodisperse spherical Ag nanoparticle solution stabilized by sodium citrate ligands with a particle size of 25 - 30 nm.

[0035] 2) Take 10 ml of the above Ag nanoparticle solution, add 100 - 200 μL of a 1 mM 4-mercaptobenzoic acid solution, and stir for 8 - 12 h to obtain a reporter molecule-labeled Ag nanoparticle solution.

[0036] 3) Take 10 ml of the above reporter molecule-labeled Ag nanoparticle solution, add 100 - 200 μL of a 2 mM 3-aminopropyltrimethoxysilane solution to it. After sufficient stirring, add 0.5 ml of freshly prepared sodium silicate solution with a concentration of 0.54 wt%, stir, and then place it at 90 - 95 °C for heating for 40 - 90 minutes. After the solution cools, add 10 - 20 ml of absolute ethanol, let it stand for 18 - 24 h, and wash the resulting yellow solution to obtain the Ag-tag@SiO2 solution.

[0037] 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 mL of the above Ag-tag@SiO2 solution, stir and react for 4 - 8 h. After washing the reaction product, carboxyl-modified Ag-tag@SiO2 is obtained. Subsequently, add 5 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide (NHS) with a concentration of 10 mg / mL in sequence to activate the carboxyl groups on the silica surface. After activation, add 100 - 400 μL of an antibody dilution with an antibody concentration of 20 μg / mL, mix well, incubate at 4 °C for 12 - 24 h, then add 100 - 200 μL of a bovine serum albumin solution with a concentration of 5 wt% to block the sites. Collect the solid product and wash it with PBS multiple times to obtain an antibody-Ag-tag@SiO2 conjugate structure, which is the Ag-tag@SiO2-Abs Raman probe.

[0038] Preferably, the Raman probe is Au-tag@SiO2-Abs, and the preparation method is as follows:

[0039] 1) Take 0.75 mL of a 1 wt% sodium citrate solution and add it to 50 mL of a boiling 0.4 mM HAuCl4·3H2O solution. After continuously boiling for 30 min, cool to room temperature to obtain a monodisperse spherical Au nanoparticle solution stabilized by sodium citrate ligands with a particle size of 20 nm, and store it at 4 °C for later use;

[0040] 2) Take 10 mL of the above Au nanoparticle solution, add 20 - 50 μL of a 1 mM 4-mercaptobenzoic acid solution, and stir for 8 - 16 h to obtain a reporter molecule-labeled Au nanoparticle solution;

[0041] 3) Take 10 mL of the above reporter molecule-labeled Au nanoparticle solution, add 100 - 200 μL of freshly prepared 3-aminopropyltrimethoxysilane solution with a concentration of 2.0 mM, stir well, then add 0.5 mL of freshly prepared sodium silicate solution with a concentration of 0.54 wt%, stir, let it stand at room temperature for 24 h, then add 10 - 20 mL of ethanol, and let it stand at room temperature for 24 h again. Wash the resulting solution to obtain the Au-tag@SiO2 solution;

[0042] 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 mL of the above Au-tag@SiO2 solution, stir and react for 4 - 8 h. After washing the reaction product, carboxyl-modified Ag-tag@SiO2 is obtained; then, 5 μL of coupling chemical reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with a concentration of 10 mg / ml and 10 μL of N-hydroxysuccinimide (NHS) with a concentration of 10 mg / ml are added successively to activate the carboxyl groups on the silica surface. After activation, 100 - 400 μL of antibody diluent with an antibody concentration of 20 μg / ml is added, and after thorough mixing, it is incubated at 4 °C for 12 - 24 h. Then, 100 - 200 μL of bovine serum albumin solution with a concentration of 5 wt% is added to block the sites, and finally, it is washed multiple times with PBS to obtain the antibody-Au-tag@SiO2 conjugate structure, which is the Au-tag@SiO2-Abs Raman probe.

[0043] Preferably, the desorption process used in step S3 is a chemical desorption, mechanical desorption, or a desorption process combining chemical desorption and mechanical desorption.

[0044] Preferably, for the chemical desorption, 10 - 20 wt% dilute ammonia water is added to the "capture agent - tumor marker - Raman probe" composite structure as the desorbent. For the mechanical desorption, deionized water is added to the "capture agent - tumor marker - Raman probe" composite structure, and then desorption is carried out by ultrasonic oscillation; for the combination of chemical desorption and mechanical desorption, 10 - 20 wt% dilute ammonia water is added to the "capture agent - tumor marker - Raman probe" composite structure as the desorbent, and desorption is carried out by ultrasonic oscillation.

[0045] Preferably, in step S3, an Ag nanoparticle solution with a concentration of 0.05 mg / ml and a size of 30 - 70 nm is added.

[0046] Preferably, the superhydrophobic SERS substrate is a thin film material with a surface hydrophobic angle greater than 150 degrees or a gold-silver nanostructure SERS substrate.

[0047] Preferably, the superhydrophobic SERS substrate is a commercial superhydrophobic thin film material, or a thin film material after hydrophobic treatment, or a gold-silver nanostructure SERS substrate after superhydrophobic treatment.

[0048] The beneficial effects of the present invention compared with the prior art are as follows:

[0049] 1. The present invention provides a method for SERS detection of tumor markers based on magnetic separation and superhydrophobic aggregation strategies. It adopts a detection strategy of "magnetic separation capture → probe separation → probe aggregation", and detects tumor markers based on SERS spectra. The specific steps are as follows:

[0050] 1) The present invention uses composite nanomagnetic spheres as tumor marker capture agents. The composite nanomagnetic spheres can be selected as core-shell structure Fe3O4@TiO2 or Fe3O4@SiO2-Abs. When the tumor marker is exosome, the shell layer TiO2 in the core-shell structure Fe3O4@TiO2 can form a chemical bond with the phospholipid bilayer of exosomes to achieve magnetic separation and purification of exosomes from complex samples such as serum. The Fe3O4 magnetic spheres in the core can achieve purification of tumor markers in complex samples. When the tumor marker is other proteins except exosomes, Fe3O4@SiO2-Abs is used as the capture agent, and the specific antibodies modified on the surface of the SiO2 shell layer can respectively recognize and capture exosomes and tumor protein markers in the sample. The composite nanomagnetic spheres can quickly extract tumor markers from complex samples such as serum, with rapid and simple operation, avoiding the complex and time-consuming pretreatment processes such as purification of tumor markers in blood samples.

[0051] The present invention provides a preparation method for a tumor marker capture agent Fe3O4@TiO2 or Fe3O4@SiO2-Abs. By dispersing Fe3O4 nanospheres in ethanol and adjusting the amount of ammonia water added to control the hydrolysis reaction rate of tetrabutyl titanate / tetraethyl orthosilicate, a TiO2 / SiO2 layer is coated on the surface of the iron tetroxide to form monodisperse composite core-shell structure Fe3O4@TiO2 and Fe3O4@SiO2 particles.

[0052] On the basis of the above synthesis of Fe3O4@SiO2 particles, by introducing carboxyl groups on the surface of the shell layer SiO2 and using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to activate the carboxyl groups, the antibody can be covalently coupled to the surface of the carboxylated nanoparticles. The reactive intermediate formed by this reaction can react quickly with the antibody to form a stable amide bond. The reactive intermediate formed by the EDC reaction is unstable and is easily hydrolyzed by water. This competitive reaction with water can cleave the reactive intermediate to regenerate the carboxyl group. To prevent the rapid hydrolysis of the reactive intermediate, N-hydroxysuccinimide (NHS) can be added to the reaction to activate the carboxyl groups on the surface of SiO2 to form a more stable NHS ester intermediate, which reacts slowly with the antibody to form a stable amide bond, improving the covalent coupling binding efficiency of the antibody. Add a phosphate buffer solution containing antibody Abs for incubation, and a coupling reaction occurs. Finally, add a bovine serum albumin solution to block the sites to obtain the core-shell structure Fe3O4@SiO2-Abs.

[0053] 2) The present invention uses a Raman probe that can specifically bind to tumor markers and adds it to the "capture agent-tumor marker" enrichment solution for incubation. The Raman probe specifically binds to the tumor marker to form an immune sandwich structure; then, through magnetic separation, the unspecifically bound Raman probe is removed, and a composite structure of "capture agent-tumor marker-Raman probe" can be obtained.

[0054] The Raman probe adopted by the present invention can be Ag / Au-tag@SiO2-Abs with a core-shell structure. The core of the Raman probe is Ag / Au nanoparticles. After modifying Raman signal reporter molecule tag on the surface of the Ag / Au nanoparticles, it is wrapped with a SiO2 shell layer, and antibodies Abs capable of specifically adsorbing cancer tumor markers are modified on the surface of the SiO2 shell layer, which has a stronger SERS enhancement effect than the traditional gold nanosphere Raman probe.

[0055] The present invention provides a preparation method of the Raman probe Ag / Au-tag@SiO2-Abs. This method prepares monodisperse and uniform-sized Ag / Au nanoparticles by a reduction method. The uniform-sized SERS probe guarantees the reproducibility of subsequent SERS detection signals. 4-Mercaptobenzoic acid is used to modify the Ag / Au nanoparticles, and then a SiO2 layer is coated to obtain Ag / Au-tag@SiO2. The SiO2 shell layer prevents the loss of the reporter molecule modified on the surface of the Ag nanoparticles during storage and rinsing, which affects the SERS signal, improves the stability of the SERS probe, and provides a binding site for subsequent antibody modification;

[0056] On the basis of the above synthesis of Ag / Au-tag@SiO2 particles, by introducing carboxyl groups on the surface of the SiO2 shell layer and activating the carboxyl groups with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), the antibody can be covalently coupled to the surface of the carboxylated nanoparticles. The reactive intermediate formed by this reaction can react quickly with the antibody to form stable amide bonds. The reactive intermediate formed by the EDC reaction is unstable and is easily hydrolyzed by water. This competitive reaction with water can cleave the reactive intermediate to regenerate the carboxyl group. To prevent the rapid hydrolysis of the reactive intermediate, N-hydroxysuccinimide (NHS) can be added to the reaction to activate the carboxyl groups on the surface of the SiO2 to form a more stable NHS ester intermediate, which reacts slowly with the antibody to form stable amide bonds, improving the covalent coupling binding efficiency of the antibody. Add a phosphate buffer solution containing antibody Abs for incubation, and a coupling reaction occurs. Finally, add a bovine serum albumin solution to block the sites to obtain the Ag / Au-tag@SiO2-Abs Raman probe.

[0057] 3) In the present invention, through chemical desorption and / or mechanical desorption, the immune binding between the antigen and the antibody in the "capture agent-tumor marker-Raman probe" composite structure is invalidated, and the immune sandwich structure is disrupted, so that the Raman probe specifically bound to the tumor marker is separated. The number of separated Raman probes is positively correlated with the concentration of the tumor marker. The Raman probes are free in the solution. Then, add Ag nanoparticles to the solution and mix well, and magnetically separate to obtain a mixture containing only Ag nanoparticles and desorbed Raman probes.

[0058] 4) In the present invention, a mixed solution containing only Ag nanoparticles and desorbed Raman probes is dropped onto a superhydrophobic SERS substrate, and after drying, the signals of the Raman probes are collected. The superhydrophobic SERS substrate used has universality. It is converged in a small area on the micron scale (equivalent to the incident laser spot during detection) for detection, ensuring that all SERS probe signals can be collected each time, without interference from other impurities, and ensuring signal reproducibility. At the same time, a large number of SERS hot spots will be generated between the aggregated probes, enhancing the signals of the reporter molecules located between the probes, thereby improving the detection sensitivity. The surface of the Raman probe is coated with 4-mercaptobenzoic acid molecules that are prone to generate Raman spectral signals, and the content of tumor markers can be quantitatively detected according to the signal intensity thereof. Preferably, the content of tumor markers can be quantified according to the intensity of the Raman signal peak of 4-mercaptobenzoic acid molecules at 1590 cm -1 -1.

[0059] 2. The detection method of the present invention has the advantages of strong specificity, high sensitivity, and good reproducibility, solving the bottleneck problems in the practical application of SERS early screening of tumor markers, such as low detection sensitivity and poor signal reproducibility of existing detection methods, and has great clinical application potential. The samples used in the present invention include blood, serum, etc., samples containing exosomes secreted by tumor cells, or samples containing specific proteins secreted by tumor cells. By changing the types of specific antibodies modified on the Raman probes in the embodiments of the present invention, the detection method of the present invention can be applied to the detection of other tumor markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 are the TEM photograph and the elemental surface distribution diagram of the Fe3O4@TiO2 core-shell structure in Example 2 of the present invention.

[0061] Figure 2 are the TEM photograph and the elemental surface distribution diagram of the Fe3O4@SiO2 core-shell structure in Example 3 of the present invention.

[0062] Figure 3 are the TEM photograph and the UV-Vis absorption spectrum of the Raman probe Ag-tag@SiO2-Abs in Example 4 of the present invention.

[0063] Figure 4 are the TEM photograph and the UV-Vis absorption spectrum of the Raman probe Au-tag@SiO2-Abs in Example 5 of the present invention.

[0064] Figure 5 are the SEM photographs of the superhydrophobic mercury nanocolumn array prepared in Example 6 of the present invention, the superhydrophobic aluminum sheet prepared in Example 7, and the commercial superhydrophobic glass fiber SERS substrate.

[0065] Figure 6 It is a schematic flow chart of SERS for detecting tumor markers based on the magnetic separation and superhydrophobic convergence strategies proposed by the present invention.

[0066] Figure 7 It is a result diagram of SERS for detecting exosomes of lung cancer cells based on the magnetic separation and superhydrophobic convergence strategies in Example 8 of the present invention.

[0067] Figure 8 It is a relationship diagram between the logarithm of the exosome concentration and the average intensity of the SERS spectrum at 1590 cm -1 in the SERS detection of exosomes of lung cancer cells based on the magnetic separation and superhydrophobic convergence strategies in Example 8 of the present invention. Detailed implementation manners

[0068] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0069] Example 1

[0070] This example provides a preparation method of Fe3O4 nanospheres, which specifically includes the following steps:

[0071] 1) In 50 ml of ethylene glycol solvent, 1.5 g of FeCl3·6H2O and 0.5 g of trisodium citrate are added in sequence and stirred until dissolved, and then 3 g of NaAc (sodium acetate) is added and stirred until dissolved to obtain a mixture; wherein, the mass ratio of FeCl3·6H2O, trisodium citrate and sodium acetate NaAc is 3:1:6, and the addition concentration of FeCl3·6H2O in the ethylene glycol solvent is 0.15 M;

[0072] 2) Transfer the above mixture to an autoclave, heat and keep it warm at 200°C for 10 h, collect the black product, wash it and dry it under vacuum to obtain Fe3O4 nanospheres.

[0073] After testing, the particle size of the Fe3O4 nanospheres is 300 - 500 nm.

[0074] Example 2

[0075] This example provides a preparation method for a tumor marker capture agent (Fe3O4@TiO2), which specifically includes the following steps: Take 0.06 g of the Fe3O4 nanospheres in Example 1 and disperse them in 100 mL of ethanol at a dispersion concentration of 0.6 mg / ml. Add 350 μL of ammonia water and mix well by ultrasonic treatment. Then, gradually add tetrabutyl titanate (TBOT) to the ethanol at a concentration of 20 mM in the ethanol. Heat and mechanically stir for 24 h. Wash the black product three times with ethanol and water respectively, and dry it to obtain the core-shell structure Fe3O4@TiO2.

[0076] Figure 2 Figure 4 is the TEM photograph of the core-shell structure Fe3O4@TiO2 prepared in Example 2 and the elemental surface distribution diagram of the Fe3O4@TiO2 core-shell structure; Figure 2 It can be seen that the core-shell structure consists of an Fe3O4 nanosphere with a diameter of 300 nm as the inner core and a TiO2 layer with a thickness of 90 nm as the shell layer. The successful preparation of the Fe3O4@TiO2 core-shell structure is further confirmed by the elemental surface distribution diagram.

[0077] Example 3

[0078] This example provides a preparation method for a tumor marker capture agent (Fe3O4@SiO2-Abs), which specifically includes the following steps:

[0079] 1) Take 0.045 g of the Fe3O4 nanospheres in Example 1 and disperse them in 100 mL of ethanol at a dispersion concentration of 0.5 mg / ml. Add 500 μL of ammonia water and mix well by ultrasonic treatment. Then, gradually add tetraethyl orthosilicate to the ethanol at a concentration of 0.06 M in the ethanol. Heat and mechanically stir for 4 h. Wash the black product three times with ethanol and water respectively, and dry it to obtain the core-shell structure Fe3O4@SiO 2；

[0080] 2) Disperse the core-shell structure Fe3O4@SiO2 in ethanol to prepare an Fe3O4@SiO2 solution with a concentration of 0.6 mg / ml. Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 ml of the Fe3O4@SiO2 solution and stir and react for 6 h. The reaction product is washed to obtain carboxyl-modified Fe3O4@SiO2;

[0081] 3) Add 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), a coupling chemical reaction reagent with a concentration of 10 mg / ml, and 20 μL of N-hydroxysuccinimide (NHS) with a concentration of 10 mg / ml to the above carboxyl-modified Fe3O4@SiO2 to activate the carboxyl groups on the silica surface. After the activation is completed, add 200 μL of an antibody diluent with an antibody concentration of 20 μg / ml. After thorough mixing, incubate at 4 °C for 20 h, then add 150 μL of a 5% bovine serum albumin solution to block the sites, and wash the solid product with phosphate buffer solution PBS to obtain the conjugate structure of the antibody and Fe3O4@SiO2, which is the core-shell structure Fe3O4@SiO2-Abs.

[0082] When the antibody in the selected antibody diluent is alpha-fetoprotein antibody, the finally prepared tumor marker capture agent is Fe3O4@SiO2-AFP;

[0083] When the antibody in the selected antibody diluent is carcinoembryonic antigen antibody, the finally prepared tumor marker capture agent is Fe3O4@SiO2-CEA.

[0084] Figure 3 It is the TEM photograph of the core-shell structure Fe3O4@SiO2 prepared in Example 3 and the elemental surface distribution map of the Fe3O4@SiO2 core-shell structure; Figure 3 It can be seen that the core-shell structure consists of a Fe3O4 nanosphere with a diameter of 300 nm as the inner core and a SiO2 layer with a thickness of 20 nm as the shell. The successful preparation of the Fe3O4@SiO2 core-shell structure is further confirmed by the elemental surface distribution map.

[0085] Dynamic light scattering (DLS) was used to measure the changes in the particle sizes of the Fe3O4@SiO2 core-shell structure and the Fe3O4@SiO2-Abs core-shell structure. The results show that the hydrodynamic diameter of the Fe3O4@SiO2-Abs core-shell structure nanoparticles after antibody modification increases, confirming that the antibody Abs is coupled to the surface of the SiO2 shell layer.

[0086] Example 4

[0087] This example provides a preparation method of a Raman probe (Ag-tag@SiO2-Abs), which specifically includes the following steps:

[0088] 1) 1 mL of 34.3 mM trisodium citrate solution, 0.5 mL of 59.5 mM silver nitrate solution and 0.2 mL of 20 mM sodium chloride solution were mixed at room temperature for 5 minutes to obtain a mixed solution; the mixed solution and 80 μL of 100 mM ascorbic acid solution were quickly added to 50 ml of boiling water, heated and stirred under reflux for 60 minutes, and then cooled to room temperature to obtain a monodisperse spherical Ag nanoparticle solution with a stable trisodium citrate ligand and a particle size of 25-30 nm;

[0089] 2) Take 10 ml of the above Ag nanoparticle solution, add 100 μL of freshly prepared 4-mercaptobenzoic acid solution with a concentration of 1 mM, stir and react for 12 hours to obtain a Ag nanoparticle solution labeled with a reporter molecule;

[0090] 3) Take 10 ml of the above reporter molecule-labeled Ag nanoparticle solution, add 100 μL of 2 mM 3-aminopropyltrimethoxysilane solution, stir thoroughly, add 0.5 ml of freshly prepared 0.54 wt% sodium silicate solution, stir and heat at 90 ° C for 60 minutes, after the solution is cooled, add 10 ml of anhydrous ethanol, let it stand for 24 hours, wash the obtained yellow solution, and obtain Ag-tag@SiO2 solution;

[0091] 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 ml of the above Ag-tag@SiO2 solution, stir and react for 4 h, and wash the reaction product to obtain carboxyl-modified Ag-tag@SiO2; then add 5 μL of 10 mg / ml coupling chemical reaction reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 10 μL of 10 mg / ml N-hydroxysuccinimide (NHS) to activate the carboxyl groups on the silica surface, add 200 μL of antibody diluent with an antibody concentration of 20 μg / ml after activation, mix thoroughly, incubate at 4 °C for 20 h, then add 150 μL of 5 wt% bovine serum albumin solution to block the site, collect the solid product and wash it with PBS several times to obtain the antibody and Ag-tag@SiO2 coupling structure, which is the Ag-tag@SiO2-Abs Raman probe.

[0092] When the antibody in the selected antibody diluent is PDL-1 antibody, the Raman probe Ag-tag@SiO2-PDL-1 is finally prepared;

[0093] When the antibody in the selected antibody diluent is HER2 antibody, the Raman probe Ag-tag@SiO2-HER2 is finally prepared;

[0094] When the antibody in the selected antibody diluent is an alpha-fetoprotein antibody, the Raman probe Ag-tag@SiO2-AFP is finally prepared.

[0095] Figure 4 are the TEM photograph and Uv-vis absorption spectrum of the Raman probe Ag-tag@SiO2-Abs in Example 4 of the present invention. As can be Figure 4 seen, the core of the Raman probe is Ag nanoparticles with a size of 25-30 nm, and the shell layer is SiO2 with a thickness of 3 nm. From the Uv-vis absorption spectrum, it can be seen that as SiO2 is coated on the surface of Ag nanoparticles and the antibody is modified, the LSPR peak shows a red shift in turn, further confirming the successful preparation of the Raman probe Ag-tag@SiO2-Abs.

[0096] The dynamic light scattering (DLS) is used to measure the change in the particle size of the Ag-tag@SiO2 core-shell structure and the Ag-tag@SiO2-Abs core-shell structure. The results show that the hydrodynamic diameter of the Ag-tag@SiO2-Abs core-shell structure nanoparticles after antibody modification increases, proving that the antibody Abs is coupled to the surface of the shell layer SiO2.

[0097] Example 5

[0098] This example provides a preparation method of a Raman probe (Au-tag@SiO2-Abs), which specifically includes the following steps:

[0099] 1) Take 0.75 ml of a 1 wt% trisodium citrate solution, add it to 50 ml of a boiling 0.4 mM HAuCl4·3H2O solution, continuously boil for 30 min and then cool to room temperature to obtain a monodisperse spherical Au nanoparticle solution with a size of 20 nm stabilized by trisodium citrate ligand, and store it at 4°C for later use;

[0100] 2) Take 10 ml of the above Au nanoparticle solution, add 30 μL of a 1 mM 4-mercaptobenzoic acid solution, and stir for 8 h to obtain a Au nanoparticle solution labeled with a reporter molecule;

[0101] 3) Add 100 μl of a freshly prepared 2.0 mM 3-aminopropyltrimethoxysilane solution to the above Au nanoparticle solution labeled with a reporter molecule, stir well, then add 0.5 ml of a freshly prepared 0.54 wt% sodium silicate solution, stir and let it stand at room temperature for 24 h, then add 10 ml of ethanol, and let it stand at room temperature for another 24 h, and wash the obtained solution to obtain a Au-tag@SiO2 solution;

[0102] 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 mL of the above Au-tag@SiO2 solution, stir and react for 5 h. After washing the reaction product, carboxyl-modified Ag-tag@SiO2 is obtained. Subsequently, 5 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with a concentration of 10 mg / mL and 10 μL of N-hydroxysuccinimide (NHS) with a concentration of 10 mg / mL are added successively to activate the carboxyl groups on the silica surface. After activation, 200 μL of antibody diluent with a concentration of 20 μg / mL is added, and after thorough mixing, it is incubated at 4 °C for 20 h. Then, 150 μL of bovine serum albumin solution with a concentration of 5 wt% is added to block the sites, and finally, it is washed multiple times with PBS to obtain the antibody-Au-tag@SiO2 conjugate structure, which is the Au-tag@SiO2-Abs Raman probe.

[0103] When the antibody in the selected antibody diluent is the anti-carcinoembryonic antigen antibody, the Raman probe Au-tag@SiO2-CEA is finally prepared.

[0104] Figure 5 is the TEM photograph and Uv-vis absorption spectrum of the Raman probe Au-tag@SiO2-Abs in Example 5 of the present invention. From Figure 5 It can be seen that the core of the Raman probe is 20-nm Au nanoparticles, and the shell layer is 3-nm SiO2. From the Uv-vis absorption spectrum, it can be seen that after coating SiO2 on the surface of Au nanoparticles, the LSPR peak undergoes a red shift, further confirming the successful preparation of the Raman probe Au-tag@SiO2.

[0105] Dynamic light scattering (DLS) is used to measure the particle size changes of the Au-tag@SiO2 core-shell structure and the Au-tag@SiO2-Abs core-shell structure. The results show that for the Au-tag@SiO2-Abs core-shell structure after antibody modification, the hydrodynamic diameter of the nanoparticles increases, confirming that the antibody Abs is coupled to the surface of the shell layer SiO2.

[0106] Example 6

[0107] This example provides a preparation method for a superhydrophobic SERS substrate (superhydrophobic mercury nanocolumn array structure), which specifically includes the following steps:

[0108] 1) Use photolithography technology to etch cylindrical pits arranged in a square array on a 2 cm × 2 cm silicon wafer. The depth of the cylindrical pits is 200 nm, the diameter is 200 nm, and the interval between adjacent pits is 500 nm to obtain a silicon wafer template.

[0109] 2) Uniformly coat the polyvinylidene fluoride (PVDF) solution on the silicon wafer template, then heat it at 60 °C for 2 h. After the silicon wafer cools to room temperature, separate the PVDF film attached to the silicon wafer template to obtain the PVDF substrate with a nano-cylindrical array;

[0110] 3) Place the PVDF substrate with a nano-cylindrical array in an ion sputtering machine. Under a current of 40 mA, sputter a silver film on the surface where the nano-cylindrical array is located for 8 minutes to obtain a silver nano-column array PVDF substrate film with surface-enhanced Raman effect;

[0111] 4) Immerse the above silver nano-column array PVDF substrate film with surface-enhanced Raman effect in an ethanol solution of perfluorodecanethiol for 24 h. The concentration of perfluorodecanethiol is 0.05 M. Then take it out and rinse it three times with ethanol. After drying, a superhydrophobic silver nano-column array structure is obtained. After testing, the surface hydrophobic angle is greater than 150 degrees.

[0112] Example 7

[0113] This example provides a method for preparing a superhydrophobic SERS substrate (superhydrophobic aluminum sheet), which specifically includes the following steps:

[0114] 1) Immerse the polished aluminum sheet in a 0.1 M HCl solution for etching for 20 minutes to obtain an aluminum sheet with a micro-nano structure;

[0115] 2) Immerse the above aluminum sheet with a micro-nano structure in a 0.05 M 1H,1H,2H,2H-perfluorodecyltrimethoxysilane solution for 24 h. Then take it out, wash it three times with ethanol and dry it to obtain a superhydrophobic aluminum sheet. After testing, the surface hydrophobic angle is greater than 150 degrees.

[0116] Figure 6 are the SEM photos of the superhydrophobic silver nano-column array structure prepared in Example 6 of the present invention, the superhydrophobic aluminum sheet prepared in Example 7, and the commercial superhydrophobic glass fiber SERS substrate (produced by Delv Technology, model DL-MCE0.22μm). As can be Figure 6 seen, the three substrates have complex micro-nano structures, providing excellent superhydrophobic properties for the substrates.

[0117] Example 8

[0118] This example provides a method for highly sensitive detection of tumor marker - lung cancer exosomes by SERS based on magnetic separation and superhydrophobic convergence strategy. The detection process refers to Figure 6 , and specifically includes the following steps:

[0119] Step S1: Take 0.5 mg of the tumor marker capture agent (Fe3O4@TiO2) prepared in Example 2 and add it to 500 μL of solutions with concentrations of 1*10 8cells / ml, 1*10 7 cells / ml, 1*10 6 cells / ml, 1*10 5 cells / ml, 1*10 4 cells / ml, 1*10 3 cells / ml and 1*10 2 cells / ml of lung cancer exosome samples (A549 lung cancer cell culture supernatant) were co-incubated for 8 minutes to achieve the capture of exosomes in the samples; then the composite magnetic nanospheres after capturing tumor markers were separated from the sample solution to obtain the "capturing agent - tumor marker" enrichment solution;

[0120] Step S2: Add 30 μL of the Raman probe Ag-tag@SiO2-PDL-1 prepared in Example 4 to the "capturing agent - tumor marker" enrichment solution. The PDL-1 antibody modified on the probe surface can specifically bind to lung cancer exosomes; co-incubate for 30 minutes to form an immune sandwich structure, and wash 3 times by magnetic separation to remove the Raman probes that did not specifically bind to tumor markers. The precipitate obtained is the "capturing agent - tumor marker - Raman probe" composite structure;

[0121] Step S3: Add 100 μL of 10 wt% dilute ammonia water as a desorbing agent to the "capturing agent - tumor marker - Raman probe" composite structure, so that the Raman probe specifically bound to the tumor marker is stripped off and free in the solution; then add 30 μL of Ag nanoparticles with a concentration of 0.05 mg / ml and a diameter of 30 nm to the solution and mix well, and then obtain a mixed solution containing only the stripped Raman probe and Ag nanoparticles by magnetic separation;

[0122] Step S4: Use the superhydrophobic aluminum sheet prepared in Example 7 as the superhydrophobic SERS substrate. Take 10 μL of the above mixed solution and drop it on the superhydrophobic SERS substrate. After drying, Raman signal collection is carried out to achieve the measurement of the content of tumor markers.

[0123] Figure 6 is the schematic diagram of the SERS detection process of lung cancer exosomes based on magnetic separation and superhydrophobic convergence strategy in Example 8 of the present invention.

[0124] Figure 7 is the SERS detection result diagram of lung cancer exosomes based on magnetic separation and superhydrophobic convergence strategy in Example 8 of the present invention. Figure 8 is the relationship diagram of the logarithm of exosome concentration and the average intensity of the SERS spectrum at 1590 cm -1 in the SERS detection of lung cancer cell exosomes based on magnetic separation and superhydrophobic convergence strategy in Example 8 of the present invention.

[0125] From Figure 7It can be seen that as the concentration of lung cancer exosomes continues to decrease (from 1×10 8 per ml to 1×10 2 per ml), the intensity of the SERS spectrum continues to decrease. When the exosome concentration is 1×10 2 per ml, the technical solution of the present invention can still clearly detect the presence of exosomes. From the Figure 8 relationship diagram of the logarithm of exosome concentration and the average intensity of the SERS spectrum at 1590 cm -1 , it can be seen that a good linear relationship is established between the signal intensity of the Raman probe and the concentration of exosomes, and the detection limit is 1×10 2 per ml. The technical solution of the present invention has strong sensitivity and repeatability.

[0126] Example 9

[0127] This example provides a method for highly sensitive SERS detection of tumor markers - breast cancer exosomes based on magnetic separation and superhydrophobic aggregation strategy. The detection process refers to Figure 6 , and specifically includes the following steps:

[0128] Step S1: Take 0.5 mg of the tumor marker capture agent (Fe3O4@TiO2) prepared in Example 2, and add it to 500 μL of samples of breast cancer exosomes with concentrations of 1×10 8 per ml, 1×10 7 per ml, 1×10 6 per ml, 1×10 5 per ml, 1×10 4 per ml, 1×10 3 per ml, and 1×10 2 per ml (SKBR-3 breast cancer cell culture supernatant), and incubate for 8 minutes to achieve the capture of exosomes in the sample; then separate the composite magnetic nanospheres captured with the tumor marker from the sample solution to obtain an enriched solution of "capture agent - tumor marker";

[0129] Step S2: Add 30 μL of the Raman probe Ag-tag@SiO2-HER2 prepared in Example 4 to the enriched solution of "capture agent - tumor marker". The HER2 monoclonal antibody modified on the probe surface can specifically bind to breast cancer exosomes; incubate for 30 minutes to form an immune sandwich structure, and wash 3 times by magnetic separation to remove the Raman probes that do not specifically bind to the tumor marker. The obtained precipitate is a "capture agent - tumor marker - Raman probe" composite structure;

[0130] Step S3: Add 100 μL of 10 wt% dilute ammonia water as a desorbent to the "capture agent - tumor marker - Raman probe" composite structure, so that the Raman probe specifically bound to the tumor marker is stripped off and free in the solution; then add 30 μL of Ag nanoparticles with a diameter of 30 nm to the solution and mix well, and then obtain a mixture containing only the stripped Raman probe and Ag nanoparticles through magnetic separation;

[0131] Step S4: Use the superhydrophobic mercury nanocolumn array prepared in Example 6 as the superhydrophobic SERS substrate, take 10 μL of the above mixture and drop it on the superhydrophobic SERS substrate, and perform Raman signal acquisition after drying to realize the measurement of the content of the tumor marker.

[0132] According to the detection results, as the concentration of breast cancer exosomes continuously decreases (from 1*10 8 per ml to 1*10 2 per ml), the intensity of the SERS spectrum continuously decreases, and a good linear relationship is established between the signal intensity of the Raman probe and the concentration of breast cancer exosomes. The technical solution of the present invention has strong sensitivity and repeatability.

[0133] Example 10

[0134] This example provides a method for highly sensitive detection of tumor marker - alpha - fetoprotein by SERS based on magnetic separation and superhydrophobic aggregation strategy. The detection process refers to Figure 6 , and specifically includes the following steps:

[0135] Step S1: Take 0.9 mg of the tumor marker capture agent (Fe3O4@SiO2 - AFP) prepared in Example 3 and add it to 500 μL of alpha - fetoprotein samples with concentrations of 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 50 ng / ml, and 100 ng / ml respectively. The AFP monoclonal antibody modified on the surface of the capture agent can specifically bind to alpha - fetoprotein, and incubate together for 45 minutes to achieve the capture of alpha - fetoprotein in the sample; then separate the composite magnetic nanospheres after capturing the tumor marker from the sample solution to obtain the "capture agent - tumor marker" enrichment solution;

[0136] Step S2: Add 30 μL of the Raman probe Ag - tag@SiO2 - AFP prepared in Example 4 to the "capture agent - tumor marker" enrichment solution. The AFP monoclonal antibody modified on the surface of the probe can specifically bind to alpha - fetoprotein; incubate together for 30 minutes to form an immune sandwich structure, and wash it 3 times through magnetic separation to remove the Raman probe that has not specifically bound to the tumor marker. The obtained precipitate is the "capture agent - tumor marker - Raman probe" composite structure;

[0137] Step S3: Add 100 μL of dilute ammonia water with a concentration of 10 wt% as a desorbent to the "capture agent - tumor marker - Raman probe" composite structure, so that the Raman probe specifically bound to the tumor marker is stripped off and free in the solution; then add 30 μL of Ag nanoparticles with a diameter of 30 nm to the solution and mix well, and then obtain a mixture containing only the stripped Raman probe and Ag nanoparticles by magnetic separation;

[0138] Step S4: Use the superhydrophobic aluminum sheet prepared in Example 7 as the superhydrophobic SERS substrate, take 10 μL of the above mixture and drop it on the superhydrophobic SERS substrate, and perform Raman signal acquisition after drying to realize the measurement of the content of the tumor marker.

[0139] According to the detection results, as the concentration of alpha-fetoprotein continuously decreases (from 100 ng / ml to 0.1 ng / ml), the intensity of the SERS spectrum continuously decreases, and a good linear relationship is established between the signal intensity of the Raman probe and the concentration of alpha-fetoprotein. The technical solution of the present invention has strong sensitivity and repeatability.

[0140] Example 11

[0141] This example provides a method for highly sensitive detection of tumor marker carcinoembryonic antigen based on magnetic separation and superhydrophobic aggregation strategy, and the detection process refers to Figure 6 , and specifically includes the following steps:

[0142] Step S1: Add 0.6 mg of the tumor marker capture agent (Fe3O4@SiO2-CEA) prepared in Example 3 to a carcinoembryonic antigen sample containing 500 μL with concentrations of 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 50 ng / ml, and 100 ng / ml respectively. The CEA monoclonal antibody modified on the surface of the capture agent can specifically bind to carcinoembryonic antigen; co-incubate for 30 minutes to achieve the capture of carcinoembryonic antigen in the sample; then separate the composite nanomagnetic beads after capturing the tumor marker from the sample solution to obtain a "capture agent - tumor marker" enrichment solution;

[0143] Step S2: Add 30 μL of the Raman probe Au-tag@SiO2-CEA prepared in Example 5 to the "capture agent - tumor marker" enrichment solution. The CEA monoclonal antibody modified on the surface of the probe can specifically bind to carcinoembryonic antigen; co-incubate for 30 minutes to form an immunological sandwich structure, and wash it 3 times by magnetic separation to remove the Raman probe that has not specifically bound to the tumor marker. The obtained precipitate is a "capture agent - tumor marker - Raman probe" composite structure;

[0144] Step S3: Add 100 μL of dilute ammonia water with a concentration of 10 wt% as a desorbent to the "capture agent - tumor marker - Raman probe" composite structure, so that the Raman probe specifically bound to the tumor marker is stripped off and free in the solution; then add 30 μL of Ag nanoparticles with a diameter of 30 nm to the solution and mix well, and then obtain a mixture containing only the stripped Raman probe and Ag nanoparticles by magnetic separation;

[0145] Step S4: Use the superhydrophobic aluminum sheet prepared in Example 7 as the superhydrophobic SERS substrate, take 10 μL of the above mixture and drop it on the superhydrophobic SERS substrate, and collect Raman signals after drying to realize the measurement of the content of tumor markers.

[0146] According to the detection results, as the concentration of carcinoembryonic antigen continuously decreases (from 100 ng / ml to 0.1 ng / ml), the intensity of the SERS spectrum continuously decreases, and a good linear relationship is established between the signal intensity of the Raman probe and the concentration of carcinoembryonic antigen. The technical solution of the present invention has strong sensitivity and repeatability.

[0147] Those skilled in the art should understand that the above are only several specific embodiments of the present invention, rather than all embodiments. It should be pointed out that many modifications and improvements can be made for those of ordinary skill in the art. All modifications or improvements that do not exceed the scope of the claims should be regarded as the protection scope of the present invention.

Claims

1. A method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy, characterized in that, It includes the following steps: Step S1: Use a composite nano-magnetic sphere as a capture agent, add it to a sample containing tumor markers, and incubate them together to capture the tumor markers in the sample; then separate the composite nano-magnetic sphere after capturing the tumor markers from the solution to obtain an "enrichment solution of capture agent - tumor marker". When the tumor marker is exosome, the composite nano-magnetic sphere is a core-shell structure Fe3O4@TiO2. The core of the core-shell structure Fe3O4@TiO2 is a Fe3O4 nano-sphere with a diameter of 200 - 400 nm, and the outside is coated with a TiO2 shell layer with a thickness of 20 - 100 nm. When the tumor marker is other proteins except exosome, the composite nano-magnetic sphere is a core-shell structure Fe3O4@SiO2-Abs. The core of the core-shell structure Fe3O4@SiO2-Abs is a Fe3O4 nano-sphere with a diameter of 200 - 400 nm, and the outside is coated with a SiO2 shell layer with a thickness of 20 - 100 nm, and the surface of the SiO2 shell layer is modified with an antibody Abs that can specifically bind to the tumor marker to be detected. Step S2: Add a Raman probe that can specifically bind to the tumor marker to the "enrichment solution of capture agent - tumor marker" for incubation, and magnetically separate to remove the Raman probe that has not specifically bound to obtain a "composite structure of capture agent - tumor marker - Raman probe". The Raman probe is a core-shell structure Ag / Au-tag@SiO2-Abs. The core of this Raman probe is an Ag or Au nano-particle with a particle size of 20 - 50 nm. The surface of the Ag or Au nano-particle is modified with a Raman signal reporter molecule tag, and the outside is coated with a SiO2 shell layer with a thickness of 1 - 5 nm. The surface of the SiO2 shell layer is modified with an antibody Abs that can specifically adsorb the tumor marker. Step S3: Adopt a desorption process to strip the Raman probe from the "composite structure of capture agent - tumor marker - Raman probe" and make it free in the solution to obtain a desorbed Raman probe. Magnetically separate to remove the desorbed "capture agent - tumor marker", and then add an Ag nano-particle solution to the solution and mix evenly to obtain a mixed solution containing Ag nano-particles and the desorbed Raman probe. Step S4: Drop the mixed solution in Step S3 onto a super-hydrophobic SERS substrate with a surface hydrophobic angle greater than 150 degrees, dry it, and collect Raman signals to realize the rapid measurement of the content of tumor markers.

2. The method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy according to claim 1, characterized in that, The composite nano-magnetic sphere is a core-shell structure Fe3O4@TiO2, and its preparation method is as follows: Disperse Fe3O4 nano-spheres in ethanol, slowly add ammonia water and tetrabutyl titanate, and tetrabutyl titanate hydrolyzes to form a TiO2 layer on the surface of the Fe3O4 nano-spheres to obtain the core-shell structure Fe3O4@TiO2.

3. The method for SERS detection of tumor markers based on magnetic separation and superhydrophobic aggregation strategy according to claim 1, wherein The composite nano-magnetic sphere is a core-shell structure Fe3O4@SiO2-Abs, and its preparation method is as follows: 1) Disperse Fe3O4 nano-spheres in ethanol, slowly add ammonia water and tetraethyl orthosilicate, and tetraethyl orthosilicate hydrolyzes to form a SiO2 layer on the surface of the Fe3O4 nano-spheres to obtain a core-shell structure Fe3O4@SiO2; 2) The core-shell structure Fe3O4@SiO2 was carboxyl modified with 3-(triethoxysilyl)propyl succinic anhydride, and then the coupling chemical reaction reagents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to activate the carboxyl groups on the surface of silica. After the activation was completed, the antibody Abs was added for incubation. The antibody Abs underwent a coupling reaction with the carboxyl-modified Fe3O4@SiO2 nanoparticles. Finally, a bovine serum albumin solution was added to block the sites, and the solid product was collected and washed to obtain the coupling structure of the antibody and Fe3O4@SiO2, which was the core-shell structure Fe3O4@SiO2-Abs.

4. The method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy according to claim 1, characterized in that The preparation method of the Raman probe is as follows: 1) Sodium citrate and ascorbic acid were used to reduce silver nitrate to prepare a sodium citrate ligand-stabilized, monodisperse spherical Ag nanoparticle solution; Alternatively, sodium citrate was used to reduce chloroauric acid to prepare a sodium citrate ligand-stabilized, monodisperse spherical Au nanoparticle solution; 2) 4-Mercaptobenzoic acid was used to modify the Ag / Au nanoparticles to obtain Ag / Au nanoparticles labeled with the reporter molecule tag, which was Ag / Au-tag; 3) A 3-aminopropyltrimethoxysilane solution and a sodium silicate solution were added to the Ag / Au-tag to coat a SiO2 layer on the surface of the Ag / Au-tag to obtain Ag / Au-tag@SiO2; 4) 3-(Triethoxysilyl)propyl succinic anhydride was used to carboxyl modify the Ag / Au-tag@SiO2, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to activate the carboxyl groups on the surface of silica. After the activation was completed, the antibody Abs was added for incubation. The antibody Abs underwent a coupling reaction with the activated Ag / Au-tag@SiO2. Finally, a bovine serum albumin solution was added to block the sites, and the solid product was collected and washed to obtain the coupling structure of the antibody and Ag / Au-tag@SiO2, which was the Ag / Au-tag@SiO2-Abs Raman probe.

5. The method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy according to claim 1, characterized in that, The desorption process used in step S3 is a chemical desorption, mechanical desorption, or a desorption process combining chemical desorption and mechanical desorption.

6. The method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy according to claim 1, characterized in that, In step S3, an Ag nanoparticle solution with a concentration of 0.05 mg / ml and a particle size of 30-70 nm was added.

7. The method for SERS detection of tumor markers based on magnetic separation and superhydrophobic convergence strategy according to claim 1, wherein The superhydrophobic SERS substrate is a thin film material or a SERS substrate of gold and silver nanostructures.

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