Method for detecting tumor marker based on magnetic separation and super-hydrophobic convergence strategy SERS (Surface Enhanced Raman Scattering)

Abstract

The invention belongs to the technical field of tumor marker detection, and particularly relates to a method for detecting a tumor marker based on magnetic separation and super-hydrophobic convergence strategy SERS (Surface Enhanced Raman Scattering). A strategy of magnetic separation capture-probe separation-probe convergence is adopted, the magnetic balls capable of capturing the tumor markers in the sample are added, the tumor markers are rapidly extracted from the sample through magnetic separation, and the operation is simple and convenient; adding an SERS probe capable of being specifically combined with the marker, and separating the Raman probe on the surface of the tumor marker from the magnetically separated sample by adopting a desorption process; finally, the desorbed Raman probes are converged through the super-hydrophobic SERS substrate, Raman signals are collected, the method has the advantages of being high in specificity and sensitivity, good in reproducibility and the like, the sensitivity and reproducibility of SERS detection of tumor markers are improved, and the bottleneck problem that an existing detection method restricts actual application of SERS in early screening of the tumor markers is solved.

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 main diseases plaguing human survival and 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 cancer cell metastasis and provide more treatment opportunities and higher cure rates for patients. Therefore, developing highly sensitive and rapid cancer early 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, and metabolites, etc. by non-tumor cells. Substances that can reflect the occurrence and development of tumors are called tumor markers, which are widely present in body fluids such as blood, urine, and saliva. Currently, the detection of tumor markers mainly has three major targeted objectives, namely 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. 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 been gradually introduced into the field of cancer diagnosis due to its advantages such as rapidity, accuracy, reliability, and sensitivity. 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 involve modifying the substrate with antibodies that can specifically capture tumor markers. After capture, a Raman probe that can specifically recognize the tumor marker is added to form a typical sandwich structure, and then Raman testing is directly performed. However, the Raman probe is 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 disadvantages such as 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 detection strategy of "magnetic separation capture → probe separation → probe convergence" 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, a SERS probe is added to label the cancerous tumor markers. Then, a desorbing agent is added to separate the Raman probe 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 is used in the detection to quickly extract tumor markers from complex matrix samples such as serum or even whole blood, with rapid 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: Step S1: Use a composite nano-magnetic bead as a capture agent, add it to a sample containing tumor markers and incubate together to capture the tumor markers in the sample; then separate the composite nano-magnetic bead after capturing the tumor markers from the solution to obtain a "capture agent - tumor marker" enrichment solution; Step S2: Add a Raman probe that can specifically bind to the tumor marker to the "capture agent - tumor marker" enrichment solution and incubate, and magnetically separate to remove the Raman probe that is not specifically bound to obtain a "capture agent - tumor marker - Raman probe" composite structure; Step S3: Adopt a desorption process to strip the Raman probe in the "capture agent - tumor marker - Raman probe" composite structure and make it free in the solution to obtain desorbed Raman probes, magnetically separate to remove 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; Step S4: Drop the mixed solution of Step S3 onto a superhydrophobic SERS substrate, dry it and collect Raman signals to achieve rapid measurement of the content of tumor markers.

[0008] As a further improvement of the SERS detection method for tumor markers based on magnetic separation and superhydrophobic aggregation strategy: Preferably, when the tumor marker is exosome, the composite magnetic nanosphere in step S1 is a core-shell structure Fe 3 O 4 @TiO 2 , the inner core of the core-shell structure Fe 3 O 4 @TiO 2 is an Fe 3 O 4 nanosphere with a diameter of 200 - 400 nm, and is coated with a TiO 2 shell layer with a thickness of 20 - 100 nm.

[0009] Preferably, when the tumor marker is other proteins except exosome, the composite magnetic nanosphere in step S1 is a core-shell structure Fe 3 O 4 @SiO 2 -Abs, the inner core of the core-shell structure Fe 3 O 4 @SiO 2 -Abs is an Fe 3 O 4 nanosphere with a diameter of 200 - 400 nm, and is coated with a SiO 2 shell layer with a thickness of 20 - 100 nm, and the surface of the SiO 2 shell layer is modified with an antibody Abs that can specifically bind to the tumor marker to be detected.

[0010] Preferably, the composite magnetic nanosphere is a core-shell structure Fe 3 O 4 @TiO 2 , and the preparation method is as follows: Disperse the Fe 3 O 4 nanospheres in ethanol, slowly add ammonia water and tetrabutyl titanate, and tetrabutyl titanate hydrolyzes to form a TiO 3 O 4 layer on the surface of the Fe 2 nanospheres to obtain the core-shell structure Fe 3 O 4 @TiO 2 ; Preferably, the composite magnetic nanosphere is a core-shell structure Fe 3 O 4 @TiO 2 , and the preparation method is as follows: Fe 3 O 4The nanospheres are dispersed in 100 ml of ethanol at a dispersion concentration of 0.5 - 0.75 mg / ml. 200 - 400 μL of ammonia water is added dropwise and mixed thoroughly. Subsequently, tetrabutyl titanate is added dropwise to the ethanol, and the concentration of tetrabutyl titanate in ethanol is 10 - 30 mM. It is heated and stirred for 16 - 24 h, and the black product is collected, washed, and dried in vacuum to obtain the core - shell structure Fe 3 O 4 @TiO 2。

[0011] Preferably, the composite nanomagnetic sphere is a core - shell structure Fe 3 O 4 @SiO 2 -Abs, and the preparation method is as follows: 1) Disperse the Fe 3 O 4 nanospheres in ethanol, slowly add ammonia water and tetraethyl orthosilicate. Tetraethyl orthosilicate hydrolyzes to form a SiO 3 O 4 layer on the surface of the Fe 2 nanospheres to obtain the core - shell structure Fe 3 O 4 @SiO 2 ; 2) Use 3 - (triethoxysilyl) propyl succinic anhydride to modify the carboxyl group of the core - shell structure Fe 3 O 4 @SiO 2 . Then add the coupling chemical reaction reagents 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide and N - hydroxysuccinimide to activate the carboxyl group on the surface of silica. After the activation is completed, add the antibody Abs for incubation. The antibody Abs reacts with the carboxyl - modified Fe 3 O 4 @SiO 2 to occur a coupling reaction. Finally, add bovine serum albumin solution to block the sites, collect the solid product and wash it to obtain the coupling structure of the antibody and Fe 3 O 4 @SiO 2 , which is the core - shell structure Fe 3 O 4 @SiO 2 -Abs.

[0012] Preferably, the composite nanomagnetic sphere is a core - shell structure Fe 3 O 4 @SiO 2 -Abs, and the preparation method is as follows: 1) Disperse the Fe 3 O 4The nanospheres are dispersed in 100 ml of ethanol at a dispersion concentration of 0.5 - 0.75 mg / ml. 100 - 200 μL of ammonia water is added dropwise and mixed well. Subsequently, tetraethyl orthosilicate is added dropwise to the ethanol, and the concentration of tetraethyl orthosilicate is 0.05 - 0.07 M. It is heated and stirred for 2 - 4 h, and the black product is collected, washed, and dried in vacuum to obtain the core - shell structure Fe 3 O 4 @SiO 2 ; 2) The core - shell structure Fe 3 O 4 @SiO 2 is dispersed in ethanol to prepare a Fe 3 O 4 @SiO 2 solution with a concentration of 0.5 - 1 mg / ml. 10 μL of 3 - (triethoxysilyl) propyl succinic anhydride (TEPSA) is added to 1 ml of the Fe 3 O 4 @SiO 2 solution, and the mixture is stirred and reacted for 4 - 8 h. The reaction product is washed to obtain carboxyl - modified Fe 3 O 4 @SiO 2 ; 3) 10 μL of 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, which are coupling chemical reaction reagents, are added to the above - mentioned carboxyl - modified Fe 3 O 4 @SiO 2 to activate the carboxyl groups on the silica surface. After the activation is completed, 100 - 400 μL of an antibody dilution solution with an antibody concentration of 20 μg / ml is added. After thorough mixing, it is incubated at 4 °C for 12 - 24 h. Then, 100 - 200 μL of a 5% bovine serum albumin solution is added to block the sites, and the solid product is washed with phosphate - buffered saline PBS to obtain the coupling structure of the antibody and Fe 3 O 4 @SiO 2 , which is the core - shell structure Fe 3 O 4 @SiO 2 -Abs.

[0013] Preferably, the preparation method of the Fe 3 O 4 nanospheres is as follows: FeCl 3 ·6H 2 O, trisodium citrate, sodium acetate, and ethylene glycol are mixed, and a hydrothermal reaction is carried out to prepare Fe 3 O 4Nanospheres.

[0014] Preferably, the Fe 3 O 4 The preparation method of nanospheres is as follows: In an ethylene glycol solvent, FeCl 3 ·6H 2 O and trisodium citrate are added in sequence and stirred until dissolved, then sodium acetate NaAc is added and stirred until dissolved to obtain a mixture; wherein, the mass ratio of FeCl 3 ·6H 2 O, trisodium citrate and sodium acetate NaAc is 3:1:6, and the addition concentration of FeCl 3 ·6H 2 O 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 Fe 3 O 4 nanospheres with a particle size of 300 - 500 nm.

[0015] Preferably, the Raman probe in step S2 is a core - shell structured Ag / Au - tag@SiO 2 -Abs. The core of this Raman probe is Ag or Au nanoparticles with a particle size of 20 - 50 nm. A Raman signal reporting molecule (tag) is modified on the surface of the Ag or Au nanoparticles, and a SiO 2 shell layer with a thickness of 1 - 5 nm is coated outside. The surface of the SiO 2 shell layer is modified with an antibody Abs that can specifically adsorb tumor markers.

[0016] Preferably, the preparation method of the Raman probe is as follows: 1) Use trisodium citrate and ascorbic acid to reduce silver nitrate to prepare a trisodium citrate ligand - stabilized and monodisperse spherical Ag nanoparticle solution; Or, use trisodium citrate to reduce chloroauric acid to prepare a trisodium citrate ligand - stabilized and monodisperse spherical Au nanoparticle solution; 2) Modify the Ag / Au nanoparticles with 4 - mercaptobenzoic acid to obtain Ag / Au nanoparticles labeled with the reporting molecule tag, namely Ag / Au - tag; 3) Add 3 - aminopropyltrimethoxysilane solution and sodium silicate solution to Ag / Au - tag to coat a SiO 2 layer on the surface of Ag / Au - tag to obtain Ag / Au - tag@SiO 2 ; 4) Use 3 - (triethoxysilyl) propyl succinic anhydride (TEPSA) to modify Ag / Au - tag@SiO 2Carboxyl modification was carried out, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to activate the carboxyl groups on the surface of silica. After the activation was completed, antibody Abs was added for incubation. Antibody Abs was coupled with the activated Ag / Au-tag@SiO 2 to occur a coupling reaction. 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 Ag / Au-tag@SiO 2 , which was the Ag / Au-tag@SiO 2 -Abs Raman probe.

[0017] Preferably, the Raman probe is Ag-tag@SiO 2 -Abs, and the preparation method is as follows: 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; quickly add the above mixed solution and 70 - 80 μL of a 100 mM ascorbic acid solution to 50 ml of boiling water, heat and stir under reflux for 60 - 90 minutes, and then cool to room temperature to obtain a monodisperse spherical Ag nanoparticle solution with a sodium citrate ligand stabilization and a particle size of 25 - 30 nm; 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 report molecule-labeled Ag nanoparticle solution; 3) Take 10 ml of the above report molecule-labeled Ag nanoparticle solution, add 100 - 200 μL of a 2 mM 3-aminopropyltrimethoxysilane solution to it, stir well, then add 0.5 ml of freshly prepared sodium silicate solution with a concentration of 0.54 wt%, stir, and place it at 90 - 95 °C for heating for 40 - 90 minutes. After the solution is cooled, add 10 - 20 ml of absolute ethanol, let it stand for 18 - 24 h, and wash the obtained yellow solution to obtain the Ag-tag@SiO 2 solution; 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 ml of the above Ag-tag@SiO 2 solution, stir and react for 4 - 8 h, and after washing the reaction product, obtain the carboxyl-modified Ag-tag@SiO 2; Subsequently, 5 μL of coupling chemical reaction 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 were 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 was added. After thorough mixing, it was incubated at 4 °C for 12 - 24 h. Subsequently, 100 - 200 μL of bovine serum albumin solution with a concentration of 5 wt% was added to block the sites. The solid product was collected and washed multiple times with PBS to obtain the antibody and Ag-tag@SiO 2 coupling structure, which is Ag-tag@SiO 2 -Abs Raman probe.

[0018] Preferably, the Raman probe is Au-tag@SiO 2 -Abs, and the preparation method is as follows: 1) Take 0.75 ml of trisodium citrate solution with a concentration of 1 wt%, and add it to 50 ml of boiling HAuCl 4 ·3H 2 O solution. After continuous boiling for 30 min, it was cooled to room temperature to obtain a monodisperse spherical Au nanoparticle solution with a particle size of 20 nm stabilized by trisodium citrate ligand, and it was stored at 4 °C for later use; 2) Take 10 ml of the above Au nanoparticle solution, add 20 - 50 μL of 4-mercaptobenzoic acid solution with a concentration of 1 mM, and stir for 8 - 16 h to obtain a report molecule-labeled Au nanoparticle solution; 3) Take 10 ml of the above report molecule-labeled Au nanoparticle solution, add 100 - 200 μL of freshly prepared 3-aminopropyltrimethoxysilane solution with a concentration of 2.0 mM. After thorough stirring, add 0.5 ml of freshly prepared sodium silicate solution with a concentration of 0.54 wt%. After stirring, it was left standing at room temperature for 24 h, then 10 - 20 ml of ethanol was added, and it was left standing at room temperature for another 24 h. The resulting solution was washed to obtain Au-tag@SiO 2 solution; 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 ml of the above Au-tag@SiO 2 solution, stir and react for 4 - 8 h. After washing the reaction product, carboxyl group-modified Ag-tag@SiO 2; Subsequently, 5 μL of coupling chemical reaction 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 were added in sequence 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 was added. After thorough mixing, it was incubated at 4°C for 12 - 24 h. Subsequently, 100 - 200 μL of bovine serum albumin solution with a concentration of 5 wt% was added to block the sites, and finally, it was washed multiple times with PBS to obtain the antibody-Au-tag@SiO 2 coupling structure, namely Au-tag@SiO 2 -Abs Raman probe.

[0019] Preferably, the desorption process adopted in step S3 is a chemical desorption, mechanical desorption, or a desorption process combining chemical desorption and mechanical desorption.

[0020] Preferably, the chemical desorption is to add 10 - 20 wt% dilute ammonia water as a desorbent to the "capture agent-tumor marker-Raman probe" composite structure, and the mechanical desorption is to add deionized water to the "capture agent-tumor marker-Raman probe" composite structure and then desorb it by ultrasonic oscillation; the combination of chemical desorption and mechanical desorption is to add 10 - 20 wt% dilute ammonia water as a desorbent to the "capture agent-tumor marker-Raman probe" composite structure and desorb it by ultrasonic oscillation.

[0021] Preferably, a Ag nanoparticle solution with a concentration of 0.05 mg / ml and a size of 30 - 70 nm is added in step S3.

[0022] Preferably, the superhydrophobic SERS substrate is a thin film material with a hydrophobic angle greater than 150 degrees or a Au-Ag nanostructure SERS substrate.

[0023] Preferably, the superhydrophobic SERS substrate is a commercial superhydrophobic thin film material, or a thin film material after hydrophobic treatment, or a Au-Ag nanostructure SERS substrate after superhydrophobic treatment.

[0024] The beneficial effects of the present invention compared with the prior art are as follows: 1. The present invention provides a method for detecting tumor markers by SERS based on magnetic separation and superhydrophobic aggregation strategies, adopting a detection strategy of "magnetic separation capture → probe separation → probe aggregation", and detecting tumor markers based on SERS spectra. The specific steps are as follows: 1) The present invention uses composite magnetic nanospheres as tumor marker capture agents. The composite magnetic nanospheres can be selected as core-shell structure Fe 3 O 4 @TiO 2 or Fe3 O 4 @SiO 2 -Abs; When the tumor marker is exosome, the shell layer TiO in the core-shell structured Fe 3 O 4 @TiO 2 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 Fe 2 magnetic spheres in the core can achieve purification of tumor markers in complex samples. When the tumor marker is other proteins except exosomes, Fe 3 O 4 @SiO 3 O 4 @SiO 2 -Abs is used as a capture agent, and the specific antibody modified on the surface of the SiO 2 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.

[0025] The present invention provides a preparation method of a tumor marker capture agent Fe 3 O 4 @TiO 2 or Fe 3 O 4 @SiO 2 -Abs. By dispersing Fe 3 O 4 nanospheres in ethanol and adjusting the amount of ammonia water added to control the hydrolysis reaction rate of tetrabutyl titanate / tetraethyl orthosilicate, a TiO 2 / SiO 2 layer is coated on the surface of magnetite to form monodisperse composite core-shell structured Fe 3 O 4 @TiO 2 and Fe 3 O 4 @SiO 2 particles.

[0026] Based on the above synthesis of Fe 3 O 4 @SiO 2 particles, by coating the shell layer SiO 2The surface is introduced with carboxyl groups. Using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to activate the carboxyl groups enables the covalent coupling of antibodies to the surface of carboxylated nanoparticles. The reactive intermediates formed in this reaction can react rapidly with antibodies to form stable amide bonds. The reactive intermediates formed by the EDC reaction are unstable and are easily hydrolyzed by water. This competitive reaction with water can cleave the reactive intermediates to regenerate the carboxyl groups. To prevent the rapid hydrolysis of the reactive intermediates, N-hydroxysuccinimide (NHS) can be added to the reaction to activate the carboxyl groups on the SiO 2 surface to form more stable NHS ester intermediates, which react slowly with antibodies to form stable amide bonds, improving the covalent coupling binding efficiency of antibodies. Add a phosphate buffer solution containing antibody Abs for incubation to carry out the coupling reaction. Finally, add a bovine serum albumin solution to block the sites to obtain the core-shell structure Fe 3 O 4 @SiO 2 -Abs.

[0027] 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. This Raman probe specifically binds to the tumor marker to form an immune sandwich structure; then, through magnetic separation, the Raman probes that have not specifically bound are removed, and a composite structure of "capture agent - tumor marker - Raman probe" can be obtained.

[0028] The Raman probe used in the present invention can be the core-shell structure Ag / Au-tag@SiO 2 -Abs. The inner core of this Raman probe is Ag / Au nanoparticles. After modifying Raman signal reporter molecule tag on the surface of the Ag / Au nanoparticles, it is encapsulated with a SiO 2 shell layer. On the surface of the SiO 2 shell layer, antibodies Abs that can specifically adsorb cancerous tumor markers are modified, and it has a stronger SERS enhancement effect compared to traditional gold nanosphere Raman probes.

[0029] The present invention provides a preparation method for the Raman probe Ag / Au-tag@SiO 2 -Abs. This method prepares monodisperse and uniformly sized Ag / Au nanoparticles by a reduction method. The uniformly sized SERS probes provide a guarantee for the reproducibility of subsequent SERS detection signals. Modify the Ag / Au nanoparticles with 4-mercaptobenzoic acid, and then coat a SiO 2 layer to obtain Ag / Au-tag@SiO 2 , SiO 2The shell prevents the loss of the reporter molecule modified on the surface of Ag nanoparticles during storage and rinsing, which affects the SERS signal, improves the stability of the SERS probe, and provides a binding site for the subsequent modification of antibodies; On the basis of the above-synthesized Ag / Au-tag@SiO 2 particles, by introducing carboxyl groups on the surface of the shell SiO 2 and using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to activate the carboxyl groups, antibodies can be covalently coupled to the surface of the carboxylated nanoparticles. The reactive intermediate formed by this reaction can react quickly with antibodies 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 carboxyl groups. 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 SiO 2 to form a more stable NHS ester intermediate, which reacts slowly with antibodies to form stable amide bonds, improving the binding efficiency of antibody covalent coupling. Add a phosphate buffer solution containing antibody Abs for incubation, and the coupling reaction occurs. Finally, add a bovine serum albumin solution to block the sites to prepare the Ag / Au-tag@SiO 2 -Abs Raman probe.

[0030] 3) In the present invention, through chemical desorption and / or mechanical desorption, the immune binding between antigens and antibodies in the "capture agent-tumor marker-Raman probe" composite structure is invalidated, breaking the immune sandwich structure, separating the Raman probe specifically binding to the tumor marker, and 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.

[0031] 4) The present invention drops the mixture containing only Ag nanoparticles and desorbed Raman probes onto a superhydrophobic SERS substrate, and after drying, collects the signals of the Raman probes. The used superhydrophobic SERS substrate has universality. It is converged in a small area on the micron scale (equivalent to the incident laser spot during detection) for detection to ensure that all SERS probe signals can be collected each time, and there is no interference from other impurities, ensuring the reproducibility of the signals. 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 the tumor marker can be quantitatively detected according to the signal intensity. Preferably, the intensity of the Raman signal peak of 4-mercaptobenzoic acid molecules at 1590 cm -1 can be used to quantify the content of the tumor marker.

[0032] 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 for tumor markers, such as low detection sensitivity and poor signal reproducibility of existing detection methods, and has great potential for clinical application. 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 by Raman probes in the embodiments of the present invention, the detection method of the present invention can be applied to detect other tumor markers. Description of the Drawings

[0033] Figure 1 is the TEM photograph of the core-shell structure of Fe 3 O 4 @TiO 2 and its elemental surface distribution map in Example 2 of the present invention.

[0034] Figure 2 is the TEM photograph of the core-shell structure of Fe 3 O 4 @SiO 2 and its elemental surface distribution map in Example 3 of the present invention.

[0035] Figure 3 is the TEM photograph and UV-Vis absorption spectrum of the Raman probe Ag-tag@SiO 2 -Abs in Example 4 of the present invention.

[0036] Figure 4 is the TEM photograph and UV-Vis absorption spectrum of the Raman probe Au-tag@SiO 2 -Abs in Example 5 of the present invention.

[0037] Figure 5 is the SEM photograph 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.

[0038] Figure 6 is the schematic diagram of the SERS detection process of tumor markers based on magnetic separation and superhydrophobic convergence strategy proposed by the present invention.

[0039] Figure 7 is the result diagram of SERS detection of lung cancer cell exosomes based on magnetic separation and superhydrophobic convergence strategy in Example 8 of the present invention.

[0040] Figure 8 is the logarithm of the exosome concentration and the SERS spectrum at 1590 cm in the SERS detection of lung cancer cell exosomes based on magnetic separation and superhydrophobic convergence strategy in Example 8 of the present invention-1 Graph of average intensity relationship at a certain position Specific implementation manners

[0041] 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 in conjunction with embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0042] Embodiment 1 This embodiment provides a preparation method of Fe 3 O 4 nanospheres, which specifically includes the following steps: 1) In 50 ml of ethylene glycol solvent, 1.5 g of FeCl 3 ·6H 2 O 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 FeCl 3 ·6H 2 O, trisodium citrate and sodium acetate NaAc is 3:1:6, and the addition concentration of FeCl 3 ·6H 2 O in the ethylene glycol solvent is 0.15 M; 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 Fe 3 O 4 nanospheres.

[0043] After testing, the particle size of the Fe 3 O 4 nanospheres is 300 - 500 nm.

[0044] Embodiment 2 This embodiment provides a preparation method of a tumor marker capture agent (Fe 3 O 4 @TiO 2 ), which specifically includes the following steps: Take 0.06 g of the Fe 3 O 4 nanospheres in Embodiment 1 and disperse them in 100 mL of ethanol, with a dispersion concentration of 0.6 mg / ml. Dropwise add 350 μL of ammonia water and mix well by ultrasonic waves, and then gradually add tetrabutyl titanate (TBOT) to the ethanol. The concentration of tetrabutyl titanate in the ethanol is 20 mM. Heat and mechanically stir for 24 h. The black product is washed three times with ethanol and water respectively, and after drying, a core-shell structure Fe 3 O 4 @TiO 2 is obtained.

[0045] Figure 2 is the core-shell structured Fe prepared in Example 2 3 O 4 @TiO 2 TEM photograph and the elemental mapping of the Fe 3 O 4 @TiO 2 core-shell structure; It can be seen from Figure 2 that the core-shell structure consists of an inner core of Fe nanospheres with a diameter of 300 nm 3 O 4 and a shell layer of TiO with a thickness of 90 nm 2 which further confirms the successful preparation of the Fe 3 O 4 @TiO 2 core-shell structure through the elemental mapping.

[0046] Example 3 This example provides a preparation method of a tumor marker capturer (Fe 3 O 4 @SiO 2 -Abs), which specifically includes the following steps: 1) Take 0.045 g of the Fe nanospheres from Example 1 3 O 4 and disperse them in 100 mL of ethanol at a dispersion concentration of 0.5 mg / ml. Dropwise add 500 μL of ammonia water and mix thoroughly by ultrasonic treatment. Then gradually add tetraethyl orthosilicate to the ethanol, with the concentration of tetraethyl orthosilicate in ethanol being 0.06 M. Heat and mechanically stir for 4 h. Wash the black product three times with ethanol and water respectively, and dry to obtain the core-shell structured Fe 3 O 4 @SiO 2； 2) Disperse the core-shell structured Fe 3 O 4 @SiO 2 in ethanol to prepare a solution with a concentration of 0.6 mg / ml of Fe 3 O 4 @SiO 2 . Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 ml of the Fe 3 O 4 @SiO 2 solution and stir for 6 h. The reaction product is washed to obtain the carboxyl group-modified Fe 3 O 4 @SiO 2 ; 3) Add to the above carboxyl group-modified Fe 3 O4 @SiO 2 Add 10 μL of coupling chemical 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 to activate the carboxyl groups on the surface of silica. After the activation is completed, add 200 μL of 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 5% bovine serum albumin solution to block the sites, and wash the solid product with phosphate buffer solution PBS to obtain the coupling structure of antibody and Fe 3 O 4 @SiO 2 which is the core-shell structure Fe 3 O 4 @SiO 2 -Abs.

[0047] When the antibody in the selected antibody diluent is alpha-fetoprotein antibody, the finally prepared tumor marker capture agent Fe 3 O 4 @SiO 2 -AFP; When the antibody in the selected antibody diluent is carcinoembryonic antigen antibody, the finally prepared tumor marker capture agent Fe 3 O 4 @SiO 2 -CEA.

[0048] Figure 3 is the TEM photo of the core-shell structure Fe 3 O 4 @SiO 2 prepared in Example 3 and the elemental surface distribution map of the Fe 3 O 4 @SiO 2 core-shell structure; It can be seen from Figure 3 that this core-shell structure consists of an inner core of Fe 3 O 4 nano-spheres with a diameter of 300 nm and a shell layer of SiO 2 with a thickness of 20 nm. The successful preparation of the Fe 3 O 4 @SiO 2 core-shell structure is further confirmed by the elemental surface distribution map.

[0049] Dynamic light scattering (DLS) is used to measure the Fe 3 O 4 @SiO 2 core-shell structure and Fe 3 O 4 @SiO 2-Variation in the particle size of the Fe@SiO₂-Abs core-shell structure. The results showed that after antibody modification, the hydrodynamic diameter of the Fe@SiO₂-Abs core-shell structure nanoparticles increased, confirming that the antibody Abs was coupled to the surface of the SiO₂ shell. 3 O 4 @SiO 2 -Abs core-shell structure nanoparticles increased, confirming that the antibody Abs was coupled to the surface of the SiO₂ shell. 2

[0050] Example 4 This example provides a method for preparing a Raman probe (Ag-tag@SiO₂-Abs), which specifically includes the following steps: 2 1) Mix 1 mL of a 34.3 mM sodium citrate solution, 0.5 mL of a 59.5 mM silver nitrate solution, and 0.2 mL of a 20 mM sodium chloride solution at room temperature for 5 minutes to obtain a mixed solution; quickly add the above mixed solution and 80 μL of a 100 mM ascorbic acid solution to 50 ml of boiling water, heat and stir under reflux for 60 minutes, and then cool to room temperature to obtain a monodisperse spherical Ag nanoparticle solution stabilized by sodium citrate ligands with a particle size of 25 - 30 nm; 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, and stir and react for 12 h to obtain a reporter molecule-labeled Ag nanoparticle solution; 3) Take 10 ml of the above reporter molecule-labeled Ag nanoparticle solution, add 100 μL of a 3-aminopropyltrimethoxysilane solution with a concentration of 2 mM thereto, stir well, then add 0.5 ml of freshly prepared sodium silicate solution with a concentration of 0.54 wt%, stir, and heat at 90 °C for 60 minutes. After the solution cools, add 10 ml of absolute ethanol thereto, let it stand for 24 h, wash the resulting yellow solution to obtain an Ag-tag@SiO₂ solution; 2 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 after washing the reaction product, obtain a carboxyl-modified Ag-tag@SiO₂ 2 2; Subsequently, 5 μL of coupling chemical reaction 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 were added in sequence to activate the carboxyl groups on the silica surface. After activation, 200 μL of antibody dilution with an antibody concentration of 20 μg / ml was added. After thorough mixing, it was incubated at 4 °C for 20 h. Subsequently, 150 μL of bovine serum albumin solution with a concentration of 5 wt% was added to block the sites. The solid product was collected and washed multiple times with PBS to obtain the antibody and Ag-tag@SiO 2 coupling structure, namely Ag-tag@SiO 2 -Abs Raman probe.

[0051] When the antibody in the selected antibody dilution is PDL-1 antibody, the finally prepared Raman probe is Ag-tag@SiO 2 -PDL-1; When the antibody in the selected antibody dilution is HER2 antibody, the finally prepared Raman probe is Ag-tag@SiO 2 -HER2; When the antibody in the selected antibody dilution is alpha-fetoprotein antibody, the finally prepared Raman probe is Ag-tag@SiO 2 -AFP.

[0052] Figure 4 is the TEM photograph and Uv-vis absorption spectrum of the Raman probe Ag-tag@SiO 2 -Abs in Example 4 of the present invention. From Figure 4 it can be seen that the core of the Raman probe is Ag nanoparticles with a size of 25-30 nm, and the shell layer is 3 nm of SiO 2 . It can be seen from the Uv-vis absorption spectrum that with the coating of SiO 2 on the surface of Ag nanoparticles and the modification of antibodies, its LSPR peak shows a red shift in sequence, further confirming the successful preparation of the Raman probe Ag-tag@SiO 2 -Abs.

[0053] Dynamic light scattering (DLS) was used to measure the change in the particle size of the core-shell structure of Ag-tag@SiO 2 and the core-shell structure of Ag-tag@SiO 2 -Abs. The results show that the hydrodynamic diameter of the core-shell structure nanoparticles of Ag-tag@SiO 2 -Abs increases after antibody modification, confirming that antibodies Abs are coupled to the surface of the shell layer SiO 2 .

[0054] Example 5 This embodiment provides a preparation method of a Raman probe (Au-tag@SiO 2 -Abs), which specifically includes the following steps: 1) Take 0.75 ml of a 1 wt% trisodium citrate solution and add it to 50 ml of a 0.4 mM boiling HAuCl 4 ·3H 2 O solution. Keep boiling for 30 min and then cool to room temperature to obtain a monodisperse spherical Au nanoparticle solution stabilized by trisodium citrate ligand with a particle size of 20 nm, and store it at 4 °C for later use; 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 reporter molecule-labeled Au nanoparticle solution; 3) Add 100 μl of freshly prepared 2.0 mM 3-aminopropyltrimethoxysilane solution to the above reporter molecule-labeled Au nanoparticle solution. After sufficient stirring, add 0.5 ml of freshly prepared 0.54 wt% sodium silicate solution, stir and let stand at room temperature for 24 h, then add 10 ml of ethanol, and let stand at room temperature for another 24 h. Wash the resulting solution to obtain an Au-tag@SiO 2 solution; 4) Add 10 μL of 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) to 1 ml of the above Au-tag@SiO 2 solution, stir and react for 5 h. After washing the reaction product, a carboxyl-modified Ag-tag@SiO 2 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 200 μL of an antibody diluent with a concentration of 20 μg / ml, mix well, incubate at 4 °C for 20 h, then add 150 μL of a 5 wt% bovine serum albumin solution to block the sites, and finally wash with PBS multiple times to obtain an antibody-Au-tag@SiO 2 coupling structure, which is the Au-tag@SiO 2 -Abs Raman probe.

[0055] When the antibody in the selected antibody diluent is an anti-carcinoembryonic antigen antibody, the finally prepared Raman probe is Au-tag@SiO 2 -CEA; Figure 5 is the TEM photo and Uv-vis absorption spectrum of the Raman probe Au-tag@SiO 2 -Abs in Example 5 of the present invention. FromFigure 5 It is known that the core of the Raman probe is 20 nm Au nanoparticles, and the shell is 3 nm SiO 2 . It can be seen from the Uv-vis absorption spectrum that with the coating of SiO on the surface of Au nanoparticles 2 , the LSPR peak shows a red shift, further confirming the successful preparation of the Raman probe Au-tag@SiO 2 .

[0056] Dynamic light scattering (DLS) was used to measure the particle size changes of the Au-tag@SiO 2 core-shell structure and the Au-tag@SiO 2 -Abs core-shell structure. The results show that after antibody modification of the Au-tag@SiO 2 -Abs core-shell structure, the hydrodynamic diameter of the nanoparticles increases, confirming that the antibody Abs is coupled to the surface of the SiO 2 shell.

[0057] Example 6 This example provides a method for preparing a superhydrophobic SERS substrate (superhydrophobic silver nanorod array structure), which specifically includes the following steps: 1) Use lithography 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; 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 is cooled to room temperature, separate the PVDF film attached to the silicon wafer template to obtain a PVDF substrate with a nanocylindrical array; 3) Place the PVDF substrate with a nanocylindrical array in an ion sputtering machine. Under a current of 40 mA, sputter a silver film on the surface where the nanocylindrical array is located for 8 minutes to obtain a silver nanorod array PVDF substrate film with surface-enhanced Raman effect; 4) Immerse the above silver nanorod 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, and dry it to obtain a superhydrophobic silver nanorod array structure. After testing, the surface hydrophobic angle is greater than 150 degrees.

[0058] Example 7 This example provides a method for preparing a superhydrophobic SERS substrate (superhydrophobic aluminum sheet), which specifically includes the following steps: 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; 2) Immerse the above aluminum sheet with micro-nano structure in a 0.05 M solution of 1H,1H,2H,2H-perfluorodecyltrimethoxysilane 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.

[0059] Figure 6 SEM photographs of the superhydrophobic mercury nanocolumn 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). From Figure 6 it can be seen that the three substrates have complex micro-nano structures, providing excellent superhydrophobic properties for the substrates.

[0060] Example 8 This example provides a method for highly sensitive SERS detection of tumor markers - lung cancer exosomes based on magnetic separation and superhydrophobic aggregation strategies. The detection process refers to Figure 6 and specifically includes the following steps: Step S1: Take 0.5 mg of the tumor marker capture agent (Fe 3 O 4 @TiO 2 ) prepared in Example 2 and add it to 500 μL of samples (A549 lung cancer cell culture supernatant) of lung cancer exosomes with concentrations of 1×10 8 cells / 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, and co-incubate for 8 minutes to achieve the capture of exosomes in the sample; then separate the composite magnetic nanospheres after capturing the tumor marker from the sample solution to obtain a "capture agent - tumor marker" enrichment solution; Step S2: Add 30 μL of the Raman probe Ag-tag@SiO 2 -PDL-1 prepared in Example 4 to the "capture 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 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; 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 concentration of 0.05 mg / ml and 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; 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 tumor marker content.

[0061] Figure 6 It is a schematic diagram of the SERS detection process of lung cancer exosomes based on magnetic separation and superhydrophobic aggregation strategy in Example 8 of the present invention.

[0062] Figure 7 It is a graph of the SERS detection results of lung cancer exosomes based on magnetic separation and superhydrophobic aggregation strategy in Example 8 of the present invention. Figure 8 For the SERS detection of lung cancer cell exosomes based on magnetic separation and superhydrophobic aggregation strategy in Example 8 of the present invention, it is a graph of the relationship between the logarithm of exosome concentration and the average intensity of the SERS spectrum at 1590 cm -1 -1

[0063] As can be seen from Figure 7 that as the concentration of lung cancer exosomes continues to decrease (from 1×10 8 9 2 to 1×10 2 6 Figure 8 / ml), the intensity of the SERS spectrum is continuously decreasing. When the exosome concentration is 1×10 -1 6 2 / ml, the technical solution of the present invention can still clearly detect the presence of exosomes. From the graph of the relationship between the logarithm of exosome concentration and the average intensity of the SERS spectrum at 1590 cm

[0064] Example 9 This example provides a method for highly sensitive SERS detection of tumor marker - breast cancer exosomes based on magnetic separation and superhydrophobic aggregation strategy. The detection process refers to Figure 6 , and specifically includes the following steps: Step S1: Take 0.5 mg of the tumor marker capture agent (Fe 3 3 4 @TiO2 ), and added to 500 μL of samples of breast cancer exosomes with concentrations of 1×10 8 cells / 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 (SKBR-3 breast cancer cell culture supernatant) and co-incubated for 8 minutes to achieve the capture of exosomes in the samples; then the composite magnetic nanospheres after capturing the tumor markers were separated from the sample solution to obtain an enriched solution of "capture agent - tumor marker"; Step S2, add 30 μL of the Raman probe Ag-tag@SiO 2 -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; co-incubate for 30 minutes to form an immune sandwich structure, and wash it 3 times by magnetic separation to remove the Raman probes that did not specifically bind to the tumor markers. The obtained precipitate is a "capture agent - tumor marker - Raman probe" composite structure; Step S3, add 100 μL of 10 wt% dilute ammonia water as a desorbing agent 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 mixed solution containing only the stripped Raman probe and Ag nanoparticles by magnetic separation; Step S4, use the superhydrophobic mercury nanocolumn array prepared in Example 6 as the superhydrophobic SERS substrate, take 10 μL of the above mixed solution and drop it on the superhydrophobic SERS substrate, dry it and then collect Raman signals to realize the measurement of the content of the tumor marker.

[0065] According to the detection results, as the concentration of breast cancer exosomes continuously decreases (from 1×10 8 cells / ml to 1×10 2 cells / 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.

[0066] Example 10 This example provides a method for highly sensitive SERS detection of tumor marker - alpha-fetoprotein based on magnetic separation and superhydrophobic aggregation strategy. The detection process refers to Figure 6 , and specifically includes the following steps: Step S1: Take 0.9 mg of the tumor marker capture agent (Fe 3 O 4 @SiO 2 -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. Incubate them together for 45 minutes to achieve the capture of alpha-fetoprotein in the sample; then separate the composite nanomagnetic beads after capturing the tumor marker from the sample solution to obtain the "capture agent-tumor marker" enrichment solution; Step S2: Add 30 μL of the Raman probe Ag-tag@SiO 2 -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 them together for 30 minutes to form an immunological sandwich structure, and wash it 3 times by magnetic separation to remove the Raman probes that did not specifically bind to the tumor marker. The precipitate obtained is the "capture agent-tumor marker-Raman probe" composite structure; Step S3: Add 100 μL of 10 wt% dilute ammonia water as a desorbing agent to the "capture agent-tumor marker-Raman probe" composite structure to strip the Raman probe specifically bound to the tumor marker and make it free in the solution; then add 30 μL of Ag nanoparticles with a diameter of 30 nanometers to the solution and mix well, and then obtain a mixture containing only the stripped Raman probe and Ag nanoparticles by magnetic separation; 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 onto the superhydrophobic SERS substrate. After drying, collect the Raman signal to achieve the measurement of the content of the tumor marker.

[0067] 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.

[0068] Example 11 This example provides a method for highly sensitive SERS detection of tumor marker - carcinoembryonic antigen based on magnetic separation and superhydrophobic aggregation strategy. The detection process refers to Figure 6 , and specifically includes the following steps: Step S1: Add 0.6 mg of the tumor marker capture agent (Fe 3 O 4 @SiO 2 -CEA) prepared in Example 3 to cancer embryo antigen samples with a volume of 500 μL and 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 cancer embryo antigen. Incubate them together for 30 minutes to achieve the capture of cancer embryo antigen in the samples. Then separate the composite nanomagnetic beads after capturing the tumor marker from the sample solution to obtain the "capture agent - tumor marker" enrichment solution. Step S2: Add 30 μL of the Raman probe Au-tag@SiO 2 -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 cancer embryo antigen. Incubate them together for 30 minutes to form an immune sandwich structure. Wash it 3 times by magnetic separation to remove the Raman probes that did not specifically bind to the tumor marker. The precipitate obtained is the "capture agent - tumor marker - Raman probe" composite structure. Step S3: Add 100 μL of 10 wt% dilute ammonia water as a desorbing agent 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. Then obtain a mixed solution containing only the stripped Raman probe and Ag nanoparticles by magnetic separation. 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, collect Raman signals to achieve the measurement of the content of the tumor marker.

[0069] According to the detection results, as the concentration of cancer embryo 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 cancer embryo antigen. The technical solution of the present invention has strong sensitivity and repeatability.

[0070] 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 for those of ordinary skill in the art, many modifications and improvements can be made. 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 detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS, characterized in that: The following steps are involved: Step S1, using composite nano-magnetic spheres as a capture agent, adding them to a sample containing tumor markers for co-incubation, and capturing the tumor markers in the sample; then separating the composite nano-magnetic spheres after capturing the tumor markers from the solution to obtain a "capture agent-tumor marker" enrichment solution; Step S2, adding a Raman probe that can specifically bind to the tumor marker to the "capture agent-tumor marker" enrichment solution for incubation, and removing the Raman probe that is not specifically bound by magnetic separation to obtain a "capture agent-tumor marker-Raman probe" composite structure; Step S3, using a desorption process to peel off the Raman probe in the composite structure of "capture agent-tumor marker-Raman probe" and release it in the solution to obtain a desorbed Raman probe, removing the desorbed "capture agent-tumor marker" by magnetic separation, and then adding the Ag nanoparticle solution to the solution and mixing it to obtain a mixed solution containing Ag nanoparticles and the desorbed Raman probe; Step S4, dropping the mixed solution of step S3 onto the super-hydrophobic SERS substrate, collecting Raman signals after drying, thereby realizing rapid measurement of tumor marker content.

2. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 1, characterized in that: When the tumor marker is exosome, the composite nanomagnetic sphere in step S1 is a core-shell structure Fe3O4@TiO2, wherein the core of the core-shell structure Fe3O4@TiO2 is a Fe3O4 nanosphere with a diameter of 200-400nm, and the outside is coated with a TiO2 shell layer with a thickness of 20-100nm.

3. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 1, characterized in that: When the tumor marker is a protein other than exosomes, the composite nanomagnetic sphere in step S1 is a core-shell structure Fe3O4@SiO2-Abs, wherein the core of the core-shell structure Fe3O4@SiO2-Abs is a Fe3O4 nanosphere 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 antibody Abs that can specifically bind to the tumor marker to be tested.

4. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 2, characterized in that: The composite nano magnetic sphere is a core-shell structure Fe3O4@TiO2, and the preparation method is as follows: disperse Fe3O4 nano spheres in ethanol, slowly add ammonia water and tetrabutyl titanate, hydrolyze tetrabutyl titanate, form a TiO2 layer on the surface of the Fe3O4 nano spheres, and prepare the core-shell structure Fe3O4@TiO2.

5. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 3, characterized in that: The composite nano-magnetic sphere is a core-shell structure Fe3O4@SiO2-Abs, and the preparation method is as follows: 1) Disperse Fe3O4 nanospheres in ethanol, slowly add ammonia water and tetraethyl orthosilicate, and tetraethyl orthosilicate is hydrolyzed to form a SiO2 layer on the surface of the Fe3O4 nanospheres to obtain a core-shell structure Fe3O4@SiO2; 2) The core-shell structure Fe3O4@SiO2 was carboxyl-modified using 3-(triethoxysilyl)propylsuccinic 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, antibody Abs were added for incubation. The antibody Abs and the carboxyl-modified Fe3O4@SiO2 nanoparticles underwent coupling reaction. Finally, bovine serum albumin solution was added to seal the sites, and the solid product was collected and washed to obtain the coupling structure of the antibody and Fe3O4@SiO2, which is the core-shell structure Fe3O4@SiO2-Abs.

6. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 1, characterized in that: The Raman probe described in step S2 is a core-shell structured Ag / Au-tag@SiO2-Abs, wherein the core of the Raman probe is an Ag or Au nanoparticle with a particle size of 20-50 nm, the surface of the Ag or Au nanoparticle 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, and the surface of the SiO2 shell layer is modified with antibody Abs that can specifically adsorb tumor markers.

7. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 1 or 6, characterized in that: The preparation method of the Raman probe is as follows: 1) Using trisodium citrate and ascorbic acid to reduce silver nitrate, a trisodium citrate ligand-stabilized, monodispersed, spherical Ag nanoparticle solution was prepared; Alternatively, trisodium citrate is used to reduce chloroauric acid to prepare a trisodium citrate ligand-stabilized, monodispersed, spherical Au nanoparticle solution; 2) Modify Ag / Au nanoparticles with 4-mercaptobenzoic acid to obtain Ag / Au nanoparticles labeled with reporter molecules, namely Ag / Au-tag; 3) Add 3-aminopropyltrimethoxysilane solution and sodium silicate solution to the Ag / Au-tag, and coat the surface of the Ag / Au-tag with a SiO2 layer to obtain Ag / Au-tag@SiO2; 4) 3-(triethoxysilyl)propyl succinic anhydride was used to modify the carboxyl group of Ag / Au-tag@SiO2, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide were added to activate the carboxyl group on the surface of silica. After the activation, antibody Abs were added for incubation. The antibody Abs and the activated Ag / Au-tag@SiO2 underwent a coupling reaction. Finally, bovine serum albumin solution was added to block the site, and the solid product was collected and washed to obtain the coupling structure of the antibody and Ag / Au-tag@SiO2, which is the Ag / Au-tag@SiO2-Abs Raman probe.

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

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

10. The method for detecting tumor markers based on magnetic separation and superhydrophobic aggregation strategy SERS according to claim 1, characterized in that: The super-hydrophobic SERS substrate is a thin film material with a surface hydrophobic angle greater than 150 degrees or a SERS substrate with a gold-silver nanostructure.

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