阅读说明：本技术 一种检测癌细胞的表面增强拉曼散射磁性复合纳米材料 (Surface-enhanced Raman scattering magnetic composite nano material for detecting cancer cells ) 是由 薛婷 沈折玉 吴爱国 李勇 于 2019-02-27 设计创作，主要内容包括：本发明涉及一种检测癌细胞的表面增强拉曼散射磁性复合纳米材料。所述复合纳米材料包括：作为核心的磁性纳米粒子,所述磁性纳米粒子表面组装有贵金属纳米粒子；贵金属纳米粒子,所述贵金属纳米粒子组装于磁性纳米粒子表面；拉曼信号分子,所述拉曼信号分子固定于所述贵金属纳米粒子表面；亲水性高分子或生物大分子,所述亲水性高分子或生物大分子包裹在所述贵金属纳米粒子的表面；和位于表层的靶分子,所述靶分子偶联于所述亲水性高分子或生物大分子表面,且与待测物具有特异性相互作用。所述复合纳米材料可有效实现癌细胞的检测和分离。(The invention relates to a surface-enhanced Raman scattering magnetic composite nano material for detecting cancer cells. The composite nanomaterial comprises: the magnetic nano-particles are used as cores, and the surfaces of the magnetic nano-particles are assembled with noble metal nano-particles; the noble metal nano particles are assembled on the surfaces of the magnetic nano particles; a Raman signal molecule fixed on the surface of the noble metal nanoparticle; the hydrophilic polymer or the biological macromolecule is coated on the surface of the noble metal nano particle; and the target molecules are positioned on the surface layer, coupled to the surfaces of the hydrophilic macromolecules or the biological macromolecules and have specific interaction with the object to be detected. The composite nano material can effectively realize the detection and separation of cancer cells.)

1. A composite nanomaterial, the composite nanomaterial comprising:

1) magnetic nanoparticles as an inner core;

2) noble metal nanoparticles assembled on the surfaces of the magnetic nanoparticles;

3) raman signal molecules, wherein the Raman signal molecules are fixed and/or adsorbed on the surfaces of the noble metal nanoparticles;

4) a hydrophilic polymer and/or a biomacromolecule, wherein the hydrophilic polymer and/or the biomacromolecule is provided with a plurality of binding functional groups for binding with the noble metal nano-particles, and the hydrophilic polymer and/or the biomacromolecule is bound on the surface of the noble metal nano-particles through the plurality of binding functional groups;

5) the target molecule is coupled to the surface of the hydrophilic macromolecule and/or the biological macromolecule, and has specific interaction with an object to be detected.

2. The composite nanomaterial of claim 1, wherein the magnetic nanoparticle is located in the center of the composite nanomaterial, the noble metal nanoparticle is wrapped on the entire outer surface of the magnetic nanoparticle, and the noble metal nanoparticle is co-surface modified by a hydrophilic macromolecule and/or biomacromolecule and a Raman signal molecule, and the hydrophilic macromolecule and/or biomacromolecule is surface modified by a target molecule.

3. The composite nanomaterial of claim 2, wherein the magnetic nanoparticles have one or more characteristics selected from the group consisting of:

1) the magnetic nano particles are spherical or spheroidal;

2) the particle size of the magnetic nanoparticles is 80-500 nm;

3) the magnetic nanoparticles are composed of metal oxides formed by metal elements selected from the following group: fe. Co, Ni, Ce, La, Nd, Gd, or combinations thereof.

4. The composite nanomaterial of claim 2, wherein the noble metal nanoparticles have one or more characteristics selected from the group consisting of:

1) the noble metal nanoparticles have a shape selected from the group consisting of: spherical, spheroidal, rod-like, cubic, triangular flake, triangular pyramid, star-like;

2) the average particle size of the noble metal nano particles is 10-50 nm;

3) the average particle size of the noble metal nanoparticles is 5-20% of the average particle size of the magnetic nanoparticles;

4) the composition of the noble metal nanoparticles is selected from the group consisting of: au, Ag, Pt, or a combination thereof.

5. The composite nanomaterial of claim 2, wherein the composite nanomaterial has one or more characteristics selected from the group consisting of:

1) the loading density of the noble metal nanoparticles on the surfaces of the magnetic nanoparticles is 0.3 × 10⁶－1000×10⁶Per mm²；

2) In the composite nano material, the molar ratio of the magnetic nanoparticles, the noble metal nanoparticles, the Raman signal molecules, the hydrophilic macromolecules and/or the biological macromolecules to the target molecules is 1:1-10³:10-10⁷:1-10⁵:10-10⁷。

6. The composite nanomaterial of claim 2, wherein the composite nanomaterial has one or more characteristics selected from the group consisting of:

1) the raman signaling molecule is selected from the group consisting of: 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorosulphenol, rhodamine, crystal violet, navan, or a combination thereof;

2) the hydrophilic polymer is selected from the group consisting of: a polyether-based modified compound, a polyester-based modified compound, or a combination thereof, the modified compound having a group selected from the group consisting of: amino, carboxyl, hydroxyl, or combinations thereof;

3) the biomacromolecule is selected from the group consisting of: an oligopeptide, polypeptide, protein, polysaccharide, or combination thereof;

4) the target molecule is selected from the group consisting of: anti-epithelial cell adhesion molecule (EpCAM), folate, monoclonal antibody, galactosamine, RGD peptide, epidermal growth factor EGF, aptamer, or a combination thereof.

7. A method for preparing the composite nanomaterial of claim 1, comprising the steps of:

1) providing solutions respectively containing magnetic nanoparticles, noble metal nanoparticles, Raman signal molecules, hydrophilic macromolecules or biomacromolecules and target molecules;

2) dropwise adding the solution containing the magnetic nanoparticles into the solution containing the noble metal nanoparticles, and carrying out oscillation reaction to obtain a solution A containing the magnetic nanoparticles with the surface assembled noble metal nanoparticles;

3) after target molecules are activated, adding a solution containing the target molecules into a solution containing hydrophilic macromolecules or biomacromolecules, and stirring for reaction to obtain a solution B containing target molecule modified hydrophilic macromolecules or biomacromolecules;

4) and adding a solution containing Raman signal molecules into the solution A, after the solution A is subjected to oscillation reaction for the first time, adding the solution B into the solution after oscillation reaction, and continuing to perform oscillation reaction for the second time to obtain the composite nano material.

8. The method of claim 7, further comprising, after step 2), the steps of: magnetically washing the product obtained in the step 2), and dispersing the washed product in water.

9. A method of detecting cancer cells, comprising the steps of:

1) contacting the composite nanomaterial of claim 1 with a solution to be tested;

2) after culturing for a third time, centrifuging to remove the free composite nano material;

3) and detecting the SERS signal of the mixed solution obtained after centrifugation, and determining the content of the cancer cells in the solution to be detected based on the measured SERS signal.

10. A method of isolating a cancer cell, comprising the steps of:

1) contacting the composite nanomaterial of claim 1 with a solution containing cancer cells;

2) after culturing for a third time, centrifuging to remove the free composite nano material;

3) and (3) applying a magnetic field effect to the mixed liquid obtained by centrifugation to realize the enrichment and separation of cancer cells.

Technical Field

The invention relates to the field of materials, in particular to a surface-enhanced Raman scattering magnetic composite nano material for detecting cancer cells.

Background

When the tumor progresses to a particular stage, cancer cells are shed and enter the blood, forming Circulating Tumor Cells (CTCs). Cancer metastasis occurs when CTCs are transported to distant tissues and adapt to new environments [ Cell 2006,127, 679-. This is the leading cause of death in most cancer patients. The concentration of CTC in blood may reflect the degree of tumor progression [ Nature 2007,450,1235-1239 ]. Clinical studies have demonstrated that conventional methods (e.g., imaging diagnosis) fail to detect tumors when they are only 1-2 mm, but detect CTCs in blood [ chi J Cancer Res 2012,24, 403-. The definitive diagnosis of cancer is generally performed by invasive biopsy, which presents problems of sampling bias, difficulty in deep tumor sampling, and harm to the patient [ BJU int2011,108, 171-178; theranostics 2016,6, 1425-. The detection of CTC has no damage to patients, simple sampling, and important significance for early diagnosis in vitro, judgment after recovery and survival time, rapid judgment of treatment effect, detection of drug resistance in vivo, individualized treatment, tumor recurrence detection and the like [ Theransosics 2017,7,2606 and 2019; j Clin Oncol 2018,36, 2353-2354; cancer metastasis Rev 2012,31,663-71 ].

At present, CTC detection technologies include density gradient precipitation, size exclusion filtration, self-driven micromachines, magnetic beads, microfluidic chips, etc. [ Chem Soc Rev 2017,46,2038-]CTCs have a very low number in blood (5.0 × 10)⁹Each red blood cell and 1.0 × 10⁷Only a few CTCs in an individual leukocyte) and therefore detection of CTCs requires ultra-sensitive methods. Surface Enhanced Raman Scattering (SERS) is one of the best means for detecting CTC by utilizing the unique properties of noble metal nanoparticles and can be used for characterization at molecular level [ Nat Mater2017,16, 918-]。

However, the conventional method for detecting CTCs by using the SERS technology has a problem that effective detection and separation of CTCs cannot be simultaneously achieved, that is, detection of the number of CTCs and separation of cancer cells from blood cannot be achieved by using one material for further molecular typing characterization, quantitative Polymerase Chain Reaction (PCR), whole genome sequencing, transplantation research, and the like.

Disclosure of Invention

The invention aims to provide a composite nano material capable of effectively realizing detection and separation of cancer cells, a preparation method thereof and application thereof in the aspect of detection and separation of cancer cells.

In a first aspect of the present invention, there is provided a composite nanomaterial comprising:

1) magnetic nanoparticles as an inner core;

2) noble metal nanoparticles assembled on the surfaces of the magnetic nanoparticles;

3) raman signal molecules, wherein the Raman signal molecules are fixed and/or adsorbed on the surfaces of the noble metal nanoparticles;

4) a hydrophilic polymer and/or a biomacromolecule, wherein the hydrophilic polymer and/or the biomacromolecule is provided with a plurality of binding functional groups for binding with the noble metal nano-particles, and the hydrophilic polymer and/or the biomacromolecule is bound on the surface of the noble metal nano-particles through the plurality of binding functional groups;

5) the target molecule is coupled to the surface of the hydrophilic macromolecule and/or the biological macromolecule, and has specific interaction with an object to be detected.

In another preferred example, in the composite nanomaterial, the magnetic nanoparticle is located in the center of the composite nanomaterial, the precious metal nanoparticle is wrapped on the whole outer surface of the magnetic nanoparticle, the precious metal nanoparticle is modified by the co-surface of a hydrophilic macromolecule and/or a biological macromolecule and a raman signal molecule, and the hydrophilic macromolecule and/or the biological macromolecule is modified by the surface of a target molecule.

In another preferred embodiment, the composite nanomaterial has a spherical or spheroidal shape.

In another preferred embodiment, the diameter of the composite nanomaterial is 50-800nm, preferably 80-500nm, and more preferably 120-350 nm.

In another preferred embodiment, the test agent is a cancer cell selected from the group consisting of: circulating tumor cells, human cervical cancer cells, human esophageal cancer cells, human breast cancer cells, human ovarian cancer cells, human colorectal cancer cells, human liver cancer cells, human lung adenocarcinoma cells, human stomach cancer cells, human prostate cancer cells, human pancreatic cancer cells, or a combination thereof.

In another preferred embodiment, the magnetic nanoparticles have one or more characteristics selected from the group consisting of:

1) the magnetic nano particles are spherical or spheroidal;

2) the particle size of the magnetic nanoparticles is 80-500nm, preferably 100-280nm, more preferably 150-250 nm;

3) the magnetic nanoparticles are composed of metal oxides formed by metal elements selected from the following group: fe. Co, Ni, Ce, La, Nd, Gd, or combinations thereof.

In another preferred embodiment, the magnetic nanoparticles are selected from the group consisting of: fe₃O₄、LaFe₂O₄Or a combination thereof.

In another preferred embodiment, the noble metal nanoparticles have one or more characteristics selected from the group consisting of:

1) the noble metal nanoparticles have a shape selected from the group consisting of: spherical, spheroidal, rod-like, cubic, triangular flake, triangular pyramid, star-like;

2) the average particle diameter of the noble metal nano particles is 10-50nm, preferably 15-45nm, more preferably 20-40 nm;

3) the average particle diameter of the noble metal nanoparticles is 5-20%, preferably 8-18%, more preferably 10-15% of the average particle diameter of the magnetic nanoparticles;

4) the composition of the noble metal nanoparticles is selected from the group consisting of: au, Ag, Pt, or a combination thereof.

In another preferred embodiment, the composite nanomaterial has one or more characteristics selected from the group consisting of:

1) the loading density of the noble metal nanoparticles on the surfaces of the magnetic nanoparticles is 0.3 × 10⁶－1000×10⁶Per mm²Preferably 3.0 × 10⁶－100×10⁶Per mm²More preferably 20 × 10⁶－50×10⁶Per mm²；

2) In the composite nanomaterial, the magnetic nanoparticles, the noble metal nanoparticles, the Raman signal molecules, and the likeThe mole ratio of the hydrophilic polymer and/or biological macromolecule to the target molecule is 1:1-10³:10-10⁷:1-10⁵:10-10⁷Preferably 1:10-100:10³-10⁵:100-10⁴:10³-10⁵More preferably 1:40-60:10⁴-5×10⁴:10³-3×10³:10⁴-5×10⁴。

In another preferred embodiment, the composite nanomaterial has one or more characteristics selected from the group consisting of:

1) the raman signaling molecule is selected from the group consisting of: 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorosulphenol, rhodamine, crystal violet, navan, or a combination thereof;

2) the hydrophilic polymer is selected from the group consisting of: a polyether-based modified compound, a polyester-based modified compound, or a combination thereof, the modified compound having a group selected from the group consisting of: amino, carboxyl, hydroxyl, or combinations thereof;

3) the biomacromolecule is selected from the group consisting of: an oligopeptide, polypeptide, protein, polysaccharide, or combination thereof;

4) the target molecule is selected from the group consisting of: anti-epithelial cell adhesion molecule (EpCAM), folate, monoclonal antibody, galactosamine, RGD peptide, epidermal growth factor EGF, aptamer, or a combination thereof.

In another preferred embodiment, the hydrophilic polymer is selected from the group consisting of: polyacrylamide, polyacrylic acid, polyvinylpyrrolidone, polyvinyl alcohol, polymaleic anhydride and polyquaternary ammonium salt.

In another preferred example, the magnetic nanoparticles and/or the noble metal nanoparticles are modified with a polymer and then assembled with each other.

In another preferred embodiment, the polymer is selected from the group consisting of: PEI and PAA.

In another preferred example, the polymer modification makes the magnetic nanoparticles and/or the noble metal nanoparticles charged to facilitate assembly.

In another preferred embodiment, the polypeptide is selected from the group consisting of: polyglutamic acid, polyaspartic acid, glutamic acid-containing compounds, aspartic acid-containing compounds, polylysine, polyarginine, lysine-containing compounds, arginine-containing compounds, or combinations thereof.

In another preferred embodiment, the protein is a reduced protein, such as reduced bovine serum albumin (rBSA).

In another preferred embodiment, the polysaccharide is selected from the group consisting of: chitosan, dextran, chitin, cellulose, starch, agar, or combinations thereof.

In another preferred embodiment, the target molecule is coupled to the biomacromolecule or hydrophilic macromolecule by a group selected from the group consisting of: an amino group, a carboxyl group, a hydroxyl group, or a combination thereof, the target molecule being covalently bonded to the macromolecule through an amide linkage.

In a second aspect of the present invention, there is provided a method for preparing the composite nanomaterial of the first aspect of the present invention, comprising the steps of:

1) providing solutions respectively containing magnetic nanoparticles, noble metal nanoparticles, Raman signal molecules, hydrophilic macromolecules or biomacromolecules and target molecules;

2) dropwise adding the solution containing the magnetic nanoparticles into the solution containing the noble metal nanoparticles, and carrying out oscillation reaction to obtain a solution A containing the magnetic nanoparticles with the surface assembled noble metal nanoparticles;

3) after target molecules are activated, adding a solution containing the target molecules into a solution containing hydrophilic macromolecules or biomacromolecules, and stirring for reaction to obtain a solution B containing target molecule modified hydrophilic macromolecules or biomacromolecules;

4) and adding a solution containing Raman signal molecules into the solution A, after the solution A is subjected to oscillation reaction for the first time, adding the solution B into the solution after oscillation reaction, and continuing to perform oscillation reaction for the second time to obtain the composite nano material.

In another preferred example, the following steps are further included after the step 2): magnetically washing the product obtained in the step 2), and dispersing the washed product in water.

In another preferred embodiment, the first time is 3-20min, preferably 3-10 min.

In another preferred embodiment, the second time period is 3-20min, preferably 3-10 min.

In another preferred embodiment, in step 2), the reaction molar ratio of the magnetic nanoparticles to the noble metal nanoparticles is 1: 10-1000, preferably 1: 30-500, and more preferably 1: 50-100.

In another preferred embodiment, in step 3), the reaction molar ratio of the hydrophilic polymer or biomacromolecule to the target molecule is 1:10-100, preferably 1: 30-70, and more preferably 1: 40-60.

In another preferred example, in step 4), the reaction molar ratio of the magnetic nanoparticles with the surface assembled noble metal nanoparticles, the hydrophilic polymer or biomacromolecule modified by the target molecules and the raman signal molecules is 1:1-10⁵:10-10⁷Preferably 1:100-³-10⁵More 1:300-700:10⁴-5×10⁴。

In a third aspect of the present invention, there is provided a method for detecting cancer cells, comprising the steps of:

1) contacting the composite nanomaterial of the first aspect of the invention with a solution to be tested;

2) after culturing for a third time, centrifuging to remove the free composite nano material;

3) and detecting the SERS signal of the mixed solution obtained after centrifugation, and determining the content of the cancer cells in the solution to be detected based on the measured SERS signal.

In a fourth aspect of the invention, there is provided a method of isolating cancer cells comprising the steps of:

1) contacting the composite nanomaterial of the first aspect of the present invention with a solution containing cancer cells;

2) after culturing for a third time, centrifuging to remove the free composite nano material;

3) and (3) applying a magnetic field effect to the mixed liquid obtained by centrifugation to realize the enrichment and separation of cancer cells.

In another preferred embodiment, the third time period is 10-60min, preferably 20-50 min.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 is an electron microscope image of the nanoparticles prepared in example 1, wherein (a), (B), (C), and (D) are magnetic nanoparticles, noble metal nanoparticles assembled on the surface of the magnetic nanoparticles, and noble metal nanoparticles assembled on the surface of the magnetic nanoparticles and connected to raman signal molecules, natural hydrophilic polymers, and target molecules, respectively.

FIG. 2 shows the results of preliminary verification of the sensitivity and specificity of the composite nanomaterial 1 for detecting cancer cells in example 1, wherein (A) and (B) are the results of detection of the sensitivity of the composite nanomaterial 1 on cancer cells (e.g., HeLa, MCF-7), and (C) and (D) are the results of detection of the specificity of the composite nanomaterial 1 on cancer cells (e.g., HeLa, MCF-7).

FIG. 3 shows the results of the sensitivity and specificity of the composite nanomaterial 1 in example 1 for detecting cancer cells in blood, wherein (A) shows the results of the sensitivity detection of the composite nanomaterial 1 for cancer cells, and (B) shows the number of cancer cells and 1076cm^-1(C) is the specific detection result of the magnetic composite nanometer material 1 on cancer cells in blood.

FIG. 4 is an immunofluorescence plot of cancer cells in blood before and after separation by the nanocomposite material 1.

Fig. 5 is a graph comparing activities of cancer cells with or without release of the nanocomposite material 1, in which (a) is an inverted microscope photograph of cancer cells without any treatment, (B) and (C) are inverted microscope photographs of cancer cells without release and release of the nanocomposite material 1, and (D) is a result of a cell activity test without release and release of the nanocomposite material 1.

FIG. 6 is SERS signal spectra of silver triangular plate-MBA and magnetic nano particle @ noble metal nano particle-MBA at different time periods.

Detailed Description

Through long-term and intensive research, the inventor prepares a composite nano material which can simultaneously realize effective detection and separation of cancer cells by taking magnetic nanoparticles as a matrix and compositely assembling precious metal nanoparticles, Raman signal molecules, hydrophilic macromolecules/biological macromolecules and target molecules on the surface of the magnetic nanoparticles. On this basis, the inventors have completed the present invention.

Composite nanomaterial (composite or magnetic composite nanomaterial)

The present invention provides a compound comprising:

the magnetic nano-particles are assembled with precious metal nano-particles on the surfaces, and the precious metal nano-particles mainly comprise gold or silver nano-particles with different shapes (spheres, rods, cubes, triangular plates, stars and the like);

the Raman signal molecules are connected to the surface of the noble metal nano particle, and the surface Raman signal can be enhanced through an electromagnetic enhancement mechanism and a chemical enhancement mechanism;

the surface of the artificially synthesized or natural hydrophilic polymer or biological macromolecule is coated on the surface of the magnetic nanoparticle with the noble metal nanoparticle assembled on the surface, and the surface of the artificially synthesized or natural hydrophilic polymer or biological macromolecule is connected with a target molecule which is an antibody or a ligand capable of specifically interacting with a cancer cell surface antigen or receptor.

The magnetic nanoparticles can realize magnetic aggregation under the action of an external magnetic field.

The noble metal nanoparticles mainly comprise gold or silver nanoparticles with different shapes (spheres, rods, cubes, triangular plates, stars and the like), and can be connected to the surfaces of the magnetic nanoparticles through chemical bonds, coordination bonds, hydrogen bonds and electrostatic interaction force.

The synthetic or natural hydrophilic polymer or biological macromolecule has a plurality of binding functional groups (such as metal-S and metal-N) for binding with the noble metal nanoparticles, and can be bound with the noble metal nanoparticles through a plurality of the binding functional groups.

In the present invention, the "complex" means to be linked in a manner selected from the group consisting of (but not limited to): chemical bonds, coordination bonds, hydrogen bonds, electrostatic interaction forces.

In another preferred example, the noble metal nanoparticles are attached and assembled on the surfaces of the magnetic nanoparticles by a method selected from the group consisting of: chemical bonds, coordination bonds, hydrogen bonds, electrostatic interaction forces.

Specifically, precious metal nanoparticles are assembled on the surfaces of the magnetic nanoparticles through chemical bonds, coordination bonds, hydrogen bonds and electrostatic interaction force, and electromagnetic fields are generated between adjacent precious metal nanoparticles through the assembly of the precious metal nanoparticles to be superposed to form hot spots; subsequently, modifying Raman signal molecules on the surface of the composite nano particle to enable the composite ball to have an SERS signal; the hydrophilic macromolecule or biological macromolecule-target molecule is modified, the stability of the magnetic composite nanosphere is improved, the nonspecific interaction with normal cells is reduced, and simultaneously, cancer cells are specifically identified, so that the magnetic composite nanoparticles and tumor cells generate specific interaction. The magnetic composite nano material is used for detecting cancer cells, the detection limit is 1 cell/ml, and the linear relation between the cancer cell concentration and the SERS intensity is that Y is 4.6391X +70.178(R is²0.9953), cancer cells can be quantified based on SERS intensity. The excessive target molecule solution and the captured cells are co-cultured to realize the release of the magnetic composite nano material, the state of the cells releasing the magnetic composite nano material is superior to the cells not releasing the magnetic composite nano material, and the culture amplification, identification, molecular phenotype analysis and the like of cancer cells are further realized.

More specifically, the invention provides a SERS magnetic composite nano material for detecting cancer cells, which can specifically target various cancer cells and effectively realize the detection and separation of the cancer cells by means of a Raman spectrometer and an external magnetic field.

The SERS magnetic composite nano material for detecting cancer cells is composed of magnetic nanoparticles serving as cores, precious metal nanoparticles, Raman signal molecules, artificially synthesized or natural hydrophilic macromolecules or biological macromolecules and target molecules.

The magnetic nanoparticles can realize magnetic aggregation under the action of an external magnetic field, so that the enrichment and separation of CTC are realized; the noble metal nanoparticles mainly comprise gold or silver nanoparticles with different shapes (spheres, rods, cubes, triangular plates, stars and the like), and can be connected to the surfaces of the magnetic nanoparticles through chemical bonds, coordination bonds, hydrogen bonds and electrostatic interaction force; the Raman signal molecule is 2-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorosulphenol, rhodamine, crystal violet, NalLan, or a combination thereof; the synthetic or natural hydrophilic macromolecule or biological macromolecule has a plurality of binding functional groups for binding with the noble metal nanoparticles, and can be bound with the noble metal nanoparticles through a plurality of binding functional groups; the target molecule is a compound which has specific interaction with tumor cells and can target cancer cells, and is coupled to the artificially synthesized or natural hydrophilic polymer or biological macromolecule, preferably anti-epithelial cell adhesion molecule (EpCAM), folic acid, monoclonal antibody or galactosamine and the like.

More specifically, the composite material comprises:

a) the magnetic nano-particles as the inner cores have better magnetic responsiveness;

b) the noble metal nano particles are assembled on the surfaces of the magnetic nano particles;

c) raman signal molecules, wherein the Raman signal molecules are fixed and/or adsorbed on the surfaces of the noble metal nanoparticles;

d) a hydrophilic polymer or a biomacromolecule having a plurality of binding functional groups (-SH and/or-NH) that bind to the noble metal particles₂) And the hydrophilic macromolecule or the biological macromolecule is combined on the surface of the noble metal nanoparticle through a plurality of the combination functional groups (metal-S bond and/or metal-N bond).

In another preferred example, in the solution containing the analyte, the analyte and the composite nano material are combined through the target molecule to form a complex which is easy to be separated magnetically.

In another preferred embodiment, the test substance is selected from the group consisting of: circulating tumor cells, human cervical cancer cells, human esophageal cancer cells, human breast cancer cells, human ovarian cancer cells, human colorectal cancer cells, human liver cancer cells, human lung adenocarcinoma cells, human stomach cancer cells, human prostate cancer cells, human pancreatic cancer cells, or a combination thereof.

In another preferred embodiment, the composite nanomaterial has a spherical or spheroidal shape.

In another preferred embodiment, the diameter of the composite nanomaterial is 50-800nm, preferably 80-500nm, and more preferably 120-350 nm.

In another preferred embodiment, the magnetic nanoparticles are spherical or spheroidal in shape.

In another preferred embodiment, the particle size of the magnetic nanoparticles is 80-500nm, preferably 100-280nm, and more preferably 150-250 nm.

In another preferred embodiment, the magnetic nanoparticles are composed of a metal oxide formed from a metal element selected from the group consisting of: fe. Co, Ni, Ce, La, Nd, Gd, or combinations thereof.

In another preferred example, the noble metal nanoparticles have a shape selected from the group consisting of: spherical, spheroidal, rod-like, cubic, triangular flake, triangular pyramid, star-like.

In another preferred embodiment, the average particle size of the noble metal nanoparticles is 10 to 50nm, preferably 15 to 45nm, and more preferably 20 to 40 nm.

In another preferred embodiment, the average particle diameter of the noble metal nanoparticles is 5 to 20%, preferably 8 to 18%, more preferably 10 to 15% of the average particle diameter of the magnetic nanoparticles.

In another preferred embodiment, the composition of the noble metal nanoparticles is selected from the group consisting of: au, Ag, Pt, or a combination thereof.

In another preferred example, the loading density of the noble metal nanoparticles on the surfaces of the magnetic nanoparticles is 0.3 × 10⁶－1000×10⁶Per mm²Preferably 3.0 × 10⁶－100×10⁶Per mm²More preferably 20 × 10⁶－50×10⁶Per mm²。

In another preferred example, in the composite nanomaterial, the molar ratio of the magnetic nanoparticles, the noble metal nanoparticles, the raman signal molecules, the hydrophilic polymer or biomacromolecule and the target molecule is 1:1-10³:10-10⁷:1-10⁵:10-10⁷Preferably 1:10-100:10³-10⁵:100-10⁴:10³-10⁵More preferably 1:40-60:10⁴-5×10⁴:10³-3×10³:10⁴-5×10⁴。

In another preferred example, the raman signal molecule is an organic molecule having a distinct conjugate vibration in the raman spectrum.

In another preferred embodiment, the "immobilization" and/or the "adsorption" is chemical bonding and/or physical adsorption.

In another preferred embodiment, the raman signaling molecule is selected from the group consisting of: 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorosulphenol, rhodamine, crystal violet, nai's blue, or a combination thereof.

In another preferred example, each molecular chain of the hydrophilic macromolecule or the biological macromolecule is chemically bonded to the noble metal nanoparticle through a plurality of "metal-S bonds" and/or "metal-N bonds".

In another preferred embodiment, the hydrophilic polymer is selected from the group consisting of: a polyether-based modified compound, a polyester-based modified compound, or a combination thereof, the modified compound having a group selected from the group consisting of: amino, carboxyl, hydroxyl, or combinations thereof.

In another preferred embodiment, the biomacromolecule is selected from the group consisting of: an oligopeptide, a polypeptide, a protein, a polysaccharide, or a combination thereof.

In another preferred embodiment, the polypeptide is selected from the group consisting of: polyglutamic acid, polyaspartic acid, glutamic acid-containing compounds, aspartic acid-containing compounds, polylysine, polyarginine, lysine-containing compounds, arginine-containing compounds, or combinations thereof.

In another preferred embodiment, the protein is a reduced protein, such as reduced bovine serum albumin (rBSA).

In another preferred embodiment, the polysaccharide is selected from the group consisting of: chitosan, dextran, chitin, cellulose, starch, agar, or combinations thereof.

In another preferred embodiment, the target molecule is coupled to the biomacromolecule or hydrophilic macromolecule by a group selected from the group consisting of: amino, carboxyl, hydroxyl, or combinations thereof.

In another preferred embodiment, the target molecule is selected from the group consisting of: anti-epithelial cell adhesion molecule (EpCAM), folate, monoclonal antibody, galactosamine, RGD peptide, epidermal growth factor EGF, aptamer, or a combination thereof.

In another preferred embodiment, the target molecule is covalently bonded to the macromolecule through an amide bond.

Method for producing

Typically, the composite nanomaterial is prepared as follows:

(1) preparation of magnetic nanoparticles (in Fe)₃O₄Example of the design reside in

0.68g FeCl₃·6H₂O, 1.8g NaAc, and 0.75g PEI (i.e., polyetherimide) were dissolved in 20mL ethylene glycol, and the mixed solution was transferred to a reaction vessel, stored in an oven at 220 ℃ for 2 hours, and then taken out. After cooling to room temperature, ethanol and pure water were added, magnetic washed several times, and finally dispersed in 100mL of deionized water. After centrifugation (1000rpm) for 5 minutes, the supernatant was transferred to a new tube and stored in a refrigerator at 4 ℃.

(2) Preparation of noble metal nanoparticles

Heating the noble metal compound solution to boiling, quickly adding a reducing agent, heating for a period of time, cooling to room temperature, and storing in a refrigerator at 4 ℃. The noble metal compound is selected from the group consisting of: chloroauric acid, gold chloride, gold chlorite, potassium chloroaurate, sodium chloroaurate, silver nitrate, silver acetate, silver trifluoroacetate, silver trifluoromethanesulfonate, silver hexafluoroantimonate, silver tetrafluoroborate, or combinations thereof. The reducing agent is selected from the group consisting of: trisodium citrate, sodium borohydride, citric acid, trisodium citrate, sodium oxalate, gallic acid, ascorbic acid, hydrazine hydrate, sodium gluconate, dextran, hydrogen peroxide, or combinations thereof.

(3) Assembly of noble metal nanoparticles on the surface of magnetic nanoparticles

The magnetic nanoparticle solution was added dropwise to the noble metal nanoparticle solution, and the mixture was transferred to a shaker and shaken (200rpm) at room temperature for 20 min. Magnetic washing was performed several times and finally dispersed in 4mL of deionized water to give solution a.

It is understood that, in the above preparation method, the magnetic nanoparticle solution is added dropwise to the noble metal nanoparticle solution, if the order is reversed, the noble metal nanoparticles are immediately aggregated during the process of adding dropwise and dispersing the magnetic nanoparticle solution, and cannot be uniformly connected to the surfaces of the magnetic nanoparticles. The reaction molar ratio of the magnetic nanoparticles to the noble metal nanoparticles is 5-6: 10-45, preferably 5-6: 10-30, more preferably 5-6: 10-20.

(4) preparation of hydrophilic macromolecule or biomacromolecule-target molecule solution

Activating a target molecule by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), adding the target molecule into a hydrophilic macromolecule or biomacromolecule solution, and stirring overnight to obtain the hydrophilic macromolecule or biomacromolecule-target molecule solution. Through an ultrafiltration centrifuge tube (M)_W3kDa) to purify the hydrophilic macromolecule or biomacromolecule-target molecule solution to 1.1-6.0 mg/mL to obtain a solution B.

(5) Preparation of Raman signal molecule solution

And weighing Raman signal molecules, and dispersing the Raman signal molecules into an ethanol solution to prepare a Raman signal molecule solution C (0.25-2.5 mM).

(6) Preparation of magnetic composite nano material

Adding the Raman signal molecule solution C (0.25-2.5 mM) into 4mL of the solution A, then transferring the sample to a shaking table, shaking at room temperature (200rpm) for 5min, and taking the concentration of the added signal molecule solution with high signal as the optimal concentration through SERS detection. And then adding the solution B (1.1-6.0 mg/mL), oscillating at room temperature (200rpm) for 5min, performing SERS detection, taking the concentration of the added solution B as the optimal concentration, performing magnetic washing for a plurality of times, finally dispersing in 4mL of deionized water, centrifuging (1000rpm) for 5min, transferring the supernatant into a new tube, and finally obtaining the magnetic composite nano material.

Compared with the prior art, the invention has the following main advantages:

(1) the composite nano material can realize effective detection of cancer cells and can realize effective separation of the cancer cells under the action of an external magnetic field;

(2) the composite nano material has an excellent detection effect on cancer cells, the detection sensitivity can reach 1 cell/ml, the SERS intensity and the cancer cell concentration have an excellent linear relation during detection, and the cancer cell concentration can be conveniently judged according to the SERS intensity;

(3) the composite nano material has excellent separation effect on cancer cells, the density of the cancer cells of a simulated blood sample is 300 cells/ml, and after enrichment and magnetic separation operations, the immunofluorescence assay hardly detects leucocytes but detects the existence of the cancer cells;

(4) the preparation method of the composite nano material has the characteristics of simple process, low cost and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.