Functional multimodal sers nanoprobe, preparation method and application thereof

Abstract

The application belongs to the technical field of life science, and relates to a functional multi-modal SERS nanoprobe as well as a preparation method and application thereof. The functional multi-modal SERS nanoprobe comprises, from inside to outside, a magnetic particle, a Raman / fluorescence signal molecule layer, a polymer layer and a targeting antibody layer; and the magnetic particle is a transition metal doped iron-based nanoparticle. The SERS nanoprobe constructed by using the transition metal doped iron-based nanoparticle as the magnetic particle has a better SERS enhancement effect.

Description

Technical Field

[0001] This invention belongs to the field of life science technology and relates to a functional multimodal SERS nanoprobe, its preparation method and application. Background Technology

[0002] Malignant tumors are a serious threat to human health worldwide. Circulating tumor cells (CTCs) are tumor cells that are spontaneously released from solid tumors or metastatic lesions into the peripheral blood circulation system, either through diagnostic or treatment procedures. They are a necessary condition for tumor metastasis. CTC detection, as an important in vitro liquid biopsy technique, has the advantages of being rapid, simple, dynamic, and real-time. It can greatly shorten the time from examination to treatment and provide a highly feasible and informative new diagnostic method for early diagnosis, early treatment, and personalized treatment of patients with solid tumors such as breast cancer, lung cancer, esophageal cancer, cervical cancer, and liver cancer.

[0003] Surface-enhanced Raman scattering (SERS) is a phenomenon based on localized plasmon resonance in noble metal or metal compound nanostructures, which significantly enhances the Raman signal, up to 10⁻⁶ times. 6 -10 10 Surface-enhanced Raman scattering (SERS) spectroscopy boasts advantages such as high selectivity, high sensitivity, no photobleaching, interference resistance, and rapid, non-destructive operation, leading to its wide application in environmental analysis and biomedicine. However, nanomaterials with surface-enhanced Raman scattering capabilities used for detecting circulating tumor cells (CTCs) generally suffer from low detection sensitivity and require a high concentration of cancer cells in the test solution. This undoubtedly limits the efficient detection of circulating tumor cells to some extent. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a functional multimodal SERS nanoprobe, its preparation method, and its applications.

[0005] One objective of this invention is achieved through the following technical solution:

[0006] A functional multimodal SERS nanoprobe, comprising, from the inside out: magnetic particles, a Raman / fluorescent signal molecule layer, a polymer layer, and a targeting antibody layer;

[0007] The magnetic particles are iron-based nanoparticles doped with transition metals.

[0008] Preferably, the transition metal is one or more of zinc, titanium, cobalt, nickel, manganese, vanadium, and chromium; the iron-based nanoparticles are one or more of iron nanoparticles, iron oxide nanoparticles, and iron(II,III) oxide nanoparticles.

[0009] Preferably, the molar ratio of the transition metal to iron is 1:(0.01 to 1000). More preferably, it is 1:(0.1 to 100), and even more preferably, it is 1:(1 to 10).

[0010] Preferably, the transition metals are zinc and cobalt, with a molar ratio of zinc to cobalt of 1:(0.1-10).

[0011] Preferably, the transition metals are zinc and manganese, with a molar ratio of zinc to manganese of 1:(0.1-10).

[0012] When zinc and cobalt, or zinc and manganese co-doped iron-based nanoparticles are used, the resulting SERS nanoprobes exhibit better performance.

[0013] Preferably, the magnetic particles have a particle size of 1–1000 nm.

[0014] Preferably, the particle size of the magnetic particles is any one value or a range between any two values ​​from 1, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, to 1000 nm.

[0015] The Raman / fluorescence signal molecule layer is a layered structure formed by Raman / fluorescence signal molecules connected to the surface of magnetic particles. Preferably, the Raman / fluorescence signal molecules are substances that have both fluorescence and Raman properties, including but not limited to one or more of IR783, IR780, 3,3'-diethylthiotricarbazocyanine iodide (DTTC), rhodamine, crystal violet, alizarin red, and Nell blue.

[0016] The polymer layer is a layered structure formed by polymers coating the surface of the Raman / fluorescence signal molecular layer; preferably, the polymer is one or more of polydopamine, bovine serum albumin, and polyethylene glycol.

[0017] The targeted antibody layer is a layered structure formed by antibodies coupled to the surface of the polymer layer. Preferably, the antibody is an antibody against a tumor marker, such as tumor cells, protein markers, exosomes, CtDNA, etc.

[0018] Preferably, the mass ratio of the magnetic particles, Raman / fluorescence signal molecule layer, polymer layer, and targeting antibody layer is 1-1200:1-2000:1-1000:0.01-500.

[0019] The functional multimodal SERS nanoprobe of this invention has excellent binding to tumor cells. After binding, the functional multimodal SERS nanoprobe can be rapidly enriched with tumor cells through a magnetic field, which is beneficial to improving the detection sensitivity and accuracy of tumor cells.

[0020] Another objective of this invention is achieved through the following technical solution:

[0021] A method for preparing a functional multimodal SERS nanoprobe includes the following steps:

[0022] S1. Obtain magnetic particles;

[0023] S2. Raman / fluorescent signal molecules are attached to the surface of magnetic particles to form a Raman / fluorescent signal molecule layer, thus obtaining magnetic particle II;

[0024] S3. A polymer layer is formed by coating the surface of the Raman / fluorescent signal molecular layer with polymer to obtain magnetic particles III.

[0025] S4. Antibodies are coupled to the surface of a polymer layer to form a targeting antibody layer, resulting in a functional multimodal SERS nanoprobe.

[0026] Preferably, in step S1, a solution of iron salt and transition metal salt is mixed with a solution formed by an alkaline inorganic substance, a long alkyl chain organic acid, and a polar organic solvent to react and obtain initial magnetic particles, followed by phase inversion to obtain magnetic particles. The magnetic particles prepared in this way have SERS properties.

[0027] Iron salts include, but are not limited to, one or more of the following: ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, ferrous ammonium sulfate, ferric nitrate, and ferrous nitrate.

[0028] Transition metal salts include, but are not limited to, one or more of the following: chloride salts, sulfate salts, and nitrate salts of transition metals.

[0029] Alkaline inorganic substances include, but are not limited to, barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, and ammonia; long alkyl chain organic acids include, but are not limited to, oleic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid; polar organic solvents include, but are not limited to, highly polar organic solvents such as methanol, ethanol, and isopropanol.

[0030] Solutions of ferric salts and transition metal salts are solutions formed by dissolving ferric salts and transition metal salts in water. The concentration of ferric salts in these solutions ranges from 2 to 200 mmol / L.

[0031] The solution formed by the basic inorganic substance, the long alkyl chain organic acid and the polar organic solvent is formed by adding the basic inorganic substance to the long alkyl chain organic acid and the polar organic solvent, with each gram of basic inorganic substance added to 1 to 100 ml of the long alkyl chain organic acid and 1 to 100 ml of the polar organic solvent.

[0032] Preferably, the volume ratio of the solution of iron salt and transition metal salt to the solution formed by the basic inorganic substance, the long alkyl chain organic acid and the polar organic solvent is 1:10 to 10:1.

[0033] Preferably, a solution of iron salt and transition metal salt is mixed with a solution formed by an alkaline inorganic substance, a long alkyl chain organic acid and a polar organic solvent, and the mixture is reacted at a temperature of 100–500°C for a time of 1–50 h.

[0034] Preferably, the reaction time is any one of 1, 2, 3, 5, 8, or 10 hours, or a range between any two values.

[0035] Optionally, the reaction temperature can be any one of 100, 200, 250, 300, 350, 400, 450, 500°C or a range between any two of these values.

[0036] Preferably, the phase inversion includes the following steps: adding the initial magnetic particles and citric acid to an organic solvent and stirring at room temperature for 1 to 50 hours.

[0037] In subsequent steps, the magnetic particles need to be dispersed in water for reaction. In the preparation of magnetic particles, this invention uses a phase inversion reaction to convert the nanoparticles from the oil phase to the water phase, thereby improving the dispersibility of the nanoparticles in water and enhancing the SERS performance of the magnetic particles.

[0038] Optionally, the stirring time is any one of 5, 8, 10, 15, or 20 hours, or a range between any two values.

[0039] Organic solvents include chloroform, N,N-dimethylformamide, chloroform, carbon tetrachloride, formamide, DMSO, tetrahydrofuran, pyridine, etc.

[0040] Preferably, in step S2, the Raman / fluorescent signal molecule solution and the magnetic particle solution are mixed and stirred to obtain magnetic particle II.

[0041] Raman / fluorescent signal molecule solution is formed by dissolving Raman / fluorescent signal molecules in an organic solvent; any organic solvent capable of dissolving Raman / fluorescent signal molecules is acceptable. Magnetic particle solution is formed by dispersing magnetic particles in water.

[0042] Preferably, the concentration of the Raman / fluorescent signal molecule solution is 0.001–10,000,000 mmol; and the concentration of the magnetic particle solution is 0.0001–800,000 mg / mL.

[0043] Preferably, the concentration of the Raman / fluorescence signal molecule solution is any one of 0.01, 0.1, 1, 5, 10, 30, 50, 80, 100, 1000, or 100000 mmol, or a range between any two values. The concentration of the magnetic particle solution is any one of 0.001, 0.01, 0.1, 1, 5, 10, 30, 50, 80, 800, or 8000 mg / mL, or a range between any two values.

[0044] Preferably, the volume ratio of the Raman / fluorescent signal molecule solution to the magnetic particle solution is 1:1000 to 10:1. More preferably, the volume ratio is 1:500 to 1:1, and even more preferably, the volume ratio is 1:500 to 1:100.

[0045] Preferably, the stirring time for obtaining magnetic particles II is 0.1 to 10 hours. Preferably, the stirring time is any one of 0.5, 1, 3, 5, and 8 hours, or a range between any two values.

[0046] Preferably, in step S3, the magnetic particle II solution, ethanol, and ammonia are mixed and stirred, and then a polymer solution is added to react and obtain magnetic particle III.

[0047] The magnetic particle II solution is formed by dispersing magnetic particles II in water. Preferably, the concentration of the magnetic particle II solution is 0.01 to 10 mg / ml. More preferably, the concentration of the magnetic particle II solution is any one of 0.01, 0.1, 0.3, 0.5, 1, 3, and 5 mg / ml or a range between any two values.

[0048] The polymer solution is formed by dissolving a polymer in water. Preferably, the concentration of the polymer solution is 1–100 mg / ml. Optionally, the concentration of the polymer solution is any one of 1, 20, 30, 40, 50, 60, and 100 mg / ml, or a range between any two values.

[0049] Preferably, the volume ratio of the magnetic particle II solution, ethanol, ammonia, and polymer solution is 5–55: 2–16: 0.3–1.5: 0.4–10.

[0050] Preferably, the magnetic particle II solution, ethanol, and ammonia are mixed and stirred for 1 to 100 minutes. Optionally, the stirring time is any one of 1, 10, 20, 30, 50, 80, and 100 minutes, or a range between any two values.

[0051] Preferably, the reaction time to obtain magnetic particles III is 1 to 10 hours. Optionally, the reaction time is any one of 1, 3, 5, 8, and 10 hours, or a range between any two values.

[0052] Preferably, in step S4, the solution containing magnetic particles III, buffer solution, and antibody is mixed and stirred to obtain the functional multimodal SERS nanoprobe.

[0053] Preferably, the buffer solution includes, but is not limited to, at least one of Tris-HCl solution and ammonia alkaline solution.

[0054] Preferably, the ratio of the mass of magnetic particle III (mg): the volume of buffer solution (ml): the mass of antibody (μg) is 0.0005–1000: 0.001–10000: 0.0001–5000. More preferably, the ratio of the mass of magnetic particle III (mg): the volume of buffer solution (ml): the mass of antibody (μg) is 0.05–100: 0.01–1000: 0.001–500; even more preferably, the ratio of the mass of magnetic particle III (mg): the volume of buffer solution (ml): the mass of antibody (μg) is 0.5–10: 0.1–100: 0.01–50.

[0055] Preferably, the solution containing magnetic particles III, buffer solution, and antibody is mixed and stirred for 0.1–72 h at a temperature of 1–80 °C. More preferably, the stirring time is any one or a range between any two of the following values: 1, 2, 10, 12, 15, 20, 30, 50, and 72 h. More preferably, the stirring temperature is any one or a range between any two of the following values: 15, 20, 25, 30, 35, and 40 °C.

[0056] Another objective of this invention is achieved through the following technical solution:

[0057] The above-mentioned functional multimodal SERS nanoprobes are applied in one or more of SERS detection, fluorescence detection, and nuclear magnetic resonance detection. This enables the application of nanoprobes in single-modal, dual-modal, or multimodal modes.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] 1. The functional multimodal SERS nanoprobe provided by this invention can be used in any one or more of SERS detection, fluorescence detection, and nuclear magnetic resonance detection, realizing the multimodal application of nanoprobes;

[0060] 2. The functional multimodal SERS nanoprobes provided by this invention can effectively bind to tumor cells;

[0061] 3. The functional multimodal SERS nanoprobes provided by this invention can be rapidly enriched with tumor cells by a magnetic field after binding with tumor cells, which is beneficial to improving the detection sensitivity and accuracy of tumor cells.

[0062] 4. The present invention uses iron-based nanoparticles doped with transition metals as magnetic particles, and the constructed SERS nanoprobe has a better SERS enhancement effect compared with iron-based nanoparticles.

[0063] 5. The present invention can further employ two or more transition metal-doped iron-based nanoparticles, thereby constructing SERS nanoprobes with better performance than single transition metal doping.

[0064] 6. In the preparation of magnetic particles, the present invention uses a phase inversion reaction to convert the nanoparticles from the oil phase to the water phase, thereby improving the dispersibility of the nanoparticles in water and facilitating subsequent reactions. Attached Figure Description

[0065] Figure 1 Zn in Example 1 0.2 Fe 2.8 TEM images of O4 magnetic particles;

[0066] Figure 2 Zn in Example 1 0.2 Fe 2.8 SERS spectra of O4-IR780-PDA nanoparticles with different concentrations of IR780 molecules;

[0067] Figure 3 Zn in Example 1 0.2 Fe 2.8 Fluorescence spectrum of O4-IR780-PDA nanoparticles;

[0068] Figure 4 Zn in Example 1 0.2 Fe 2.8 O4-IR780-PDA nanoparticle MRI magnetic resonance imaging;

[0069] Figure 5 This is a diagram showing the enrichment and capture of tumor cells by the SERS nanoprobe in Example 1.

[0070] Figure 6 a is a SERS imaging image of the SERS nanoprobe used in Example 2 against HCC1806 triple-negative breast cancer. Figure 6 b is a SERS imaging image of the SERS nanoprobe of Example 2 on MCF7 estrogen-type breast cancer;

[0071] Figure 7 Zn in Example 1 0.2 Fe 2.8 SERS spectra of methylene blue molecules from O4 magnetic particles and Fe3O4 magnetic particles of Comparative Example 1. Detailed Implementation

[0072] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0073] Example 1

[0074] (1)Zn 0.2 Fe 2.8 Preparation of O4 magnetic particles

[0075] 1.73 mmol of ferrous ammonium sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate were added to 20 mL of ultrapure water to prepare a solution. 1 g of sodium hydroxide was added to a mixture of 10 mL of oleic acid and 10 mL of ethanol, stirred until completely dissolved, and then 20 mL of the ferrous ammonium sulfate and zinc sulfate solution was added. Once the mixture turned brownish-red, it was transferred to a 50 mL reactor and heated at 230 °C for 16 h. After the reactor cooled, it was removed, washed three times by centrifugation with ethanol, and dispersed in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Then, 2 g of citric acid and 20 mL of the oil-soluble magnetic nanoparticles were added to a 30 mL mixture of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed three times by centrifugation with ethanol, and dispersed in 200 mL of water to obtain the desired Zn. 0.2 Fe 2.8 O4 magnetic particles, such as Figure 1 As shown.

[0076] (2)Zn 0.2 Fe 2.8 Preparation of O4-IR780 nanoparticles

[0077] Add 160 μl of 1 mmol / L IR780 ethanol solution to a final volume of 16 mL Zn. 0.2 Fe 2.8 In an O4 magnetic particle solution (solvent: water, concentration: 0.21 mg / ml), the mixture was stirred with a polytetrafluoroethylene rod for 2 hours, thoroughly washed with deionized water, and finally dispersed in 18 ml of deionized water to obtain Zn. 0.2 Fe 2.8 O4-IR780 solution.

[0078] (3)Zn 0.2 Fe 2.8 Preparation of O4-IR780-PDA nanoparticles

[0079] 18ml Zn 0.2 Fe 2.8 A solution of O4-IR780 (solvent: water, concentration: 0.19 mg / ml) was mixed with 8 ml of CH3CH2OH and 600 μl of NH3·H2O. The mixture was stirred with a polytetrafluoroethylene rod for 20 min, and then 2 ml of polydopamine solution (40 mg / ml) was slowly added. After 5 hours, the mixture was thoroughly washed with deionized water and dispersed in 8 ml of deionized water to prepare Zn. 0.2 Fe 2.8 O4-IR780-PDA nanoparticle solution.

[0080] (4)Zn 0.2 Fe 2.8 Preparation of O4-IR780-PDA-anti-trop2 nanoprobe

[0081] Take 4ml of Zn 0.2 Fe 2.8 O4-IR780-PDA nanoparticle solution, 5.84 mg of Zn was obtained by magnetic adsorption. 0.2 Fe 2.8 O4-IR780-PDA nanoparticles were mixed, the supernatant was discarded, and then 4 ml of Tris-HCl solution (10 mM, pH = 8.5) was added. Following this, 40 μg of anti-trop2 antibody was added, and the mixture was stirred at room temperature for 12 h. After washing three times with PBS, the nanoparticles were finally dispersed in 4 ml of PBS to obtain Zn. 0.2 Fe 2.8 The O4-IR780-PDA-anti-trop2 nanoprobe has a particle size of approximately 50 nm.

[0082] To demonstrate that the magnetic particles of Example 1 possess the ability to detect and image tumor cells using SERS, SERS spectra of different concentrations of IR780 molecules were performed using them as a SERS substrate under an excitation wavelength of 633 nm, generating SERS spectra as follows: Figure 2 As shown, Zn 0.2 Fe 2.8 O4 magnetic particles exhibit excellent SERS detection and imaging performance for low concentrations of IR780 molecules.

[0083] To demonstrate that the magnetic particles of Example 1 possess the ability to detect and image tumor cells using fluorescence, the surface of the magnetic particle material was modified with the luminescent indicator CCK-8 for characterization, and the fluorescence spectrum was obtained, as shown in the figure. Figure 3 As shown.

[0084] To demonstrate that the magnetic particles of Example 1 possess the ability to detect and image tumor cells using MRI, an MRI contrast-enhanced image was generated using a MesoMR23–060H-I Niumag instrument at 0.47T. Figure 4 As shown.

[0085] Zn 0.2 Fe 2.8 The O4-IR780-PDA-anti-trop2 nanoprobe was co-incubated with tumor cells, and combined with the magnetic enrichment module of the circulating tumor cell detection device, the enrichment effect was as follows: Figure 5 As shown, tumor cells and Zn 0.2 Fe 2.8 The O4-IR780-PDA-anti-trop2 magnetic particles exhibit excellent binding properties. After magnetic enrichment, tumor cells were enriched, and no tumor cells were found in the filtered waste liquid, indicating that Zn... 0.2 Fe 2.8 O4-IR780-PDA-anti-trop2 magnetic particles have the ability to enrich and capture tumor cells.

[0086] Example 2

[0087] The difference between Example 2 and Example 1 is that step (4) in Example 2 is as follows:

[0088] Take 4ml of Zn 0.2 Fe 2.8 O4-IR780-PDA nanoparticle solution, 5.84 mg of Zn was obtained by magnetic adsorption. 0.2 Fe 2.8 O4-IR780-PDA nanoparticles were discarded, and 5 ml of Tris-HCl solution (10 mM, pH 8.5) was added. Then, 50 μg of GE11 peptide was added, and the mixture was stirred at room temperature for 10 h. After washing three times with PBS, the nanoparticles were finally dispersed in 4 ml of PBS to obtain Zn. 0.2 Fe 2.8 The O4-IR780-PDA-GE11 peptide nanoprobe has a particle size of approximately 50 nm.

[0089] SERS nanoprobe (Zn) as described in Example 2 0.2 Fe 2.8 The O4-IR780-PDA-GE11 peptide nanoprobe was used to identify MCF7 (estrogen-positive breast cancer) and HCC1806 (triple-negative breast cancer) using SERS imaging. Figure 6 a is a SERS image of HCC1806 triple-negative breast cancer. Figure 6b is a SERS image of MCF7 estrogen-type breast cancer. The breast cancer subtype was identified by differentiating the uptake of SERS nanoprobes by MCF7 and HCC1806 through SERS imaging.

[0090] Example 3

[0091] The difference between Example 3 and Example 1 is that step (1) of Example 3 is as follows:

[0092] (1)Zn 0.1 Co 0.1 Fe 2.8 Preparation of O4 magnetic particles

[0093] 1.73 mmol of ferrous ammonium sulfate hexahydrate, 0.275 mmol of zinc sulfate heptahydrate, and 0.275 mmol of cobalt sulfate heptahydrate were added to 30 mL of ultrapure water to prepare a solution. 2 g of sodium hydroxide was added to a mixture of 20 mL of oleic acid and 20 mL of ethanol, stirred until completely dissolved, and then 30 mL of the ferrous ammonium sulfate, zinc sulfate, and cobalt sulfate solution was added. Once the mixture turned brownish-red, it was transferred to an 80 mL reactor and heated at 250 °C for 8 h. After the reactor cooled, it was removed, washed three times by centrifugation with ethanol, and dispersed in 30 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Then, 3 g of citric acid and 30 mL of the oil-soluble magnetic nanoparticles were added to a 50 mL mixture of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed three times by centrifugation with ethanol, and dispersed in 300 mL of water to obtain the desired Zn. 0.1 Co 0.1 Fe 2.8 O4 magnetic particles.

[0094] Everything else is the same as in Example 1.

[0095] Example 4

[0096] The difference between Example 4 and Example 1 is that step (1) of Example 4 is as follows:

[0097] (1)Zn 0.1 Mn 0.1 Fe 2.8 Preparation of O4 magnetic particles

[0098] 1.73 mmol of ferrous ammonium sulfate hexahydrate, 0.26 mmol of zinc sulfate heptahydrate, and 0.26 mmol of manganese sulfate pentahydrate were added to 50 mL of ultrapure water to prepare a solution. 3 g of potassium hydroxide was added to a mixture of 30 mL of octadecanoic acid and 30 mL of methanol, stirred until completely dissolved, and then 50 mL of the ferrous ammonium sulfate, zinc sulfate, and manganese sulfate solution was added. Once the mixture turned brownish-red, it was transferred to a 150 mL reactor and heated at 300 °C for 6 h. After cooling, the reactor was removed, washed three times by centrifugation with ethanol, and dispersed in 30 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Then, 3 g of citric acid and 30 mL of the oil-soluble magnetic nanoparticles were added to a 50 mL mixture of chloroform / DMF (v / v: 1 / 1), stirred for 10 h, washed three times by centrifugation with ethanol, and dispersed in 300 mL of water to obtain the desired Zn. 0.1 Mn 0.1 Fe 2.8 O4 magnetic particles.

[0099] Everything else is the same as in Example 1.

[0100] Under the same conditions, magnetic particles prepared by dual doping of Zn and Co or dual doping of Zn and Mn have better SERS imaging performance than nanoprobes prepared by single doping of Zn, Co, or Mn.

[0101] Comparative Example 1

[0102] Preparation of Fe3O4 magnetic particles

[0103] 2 mmol of ferrous ammonium sulfate hexahydrate was added to 20 mL of ultrapure water to prepare a solution. 1 g of sodium hydroxide was added to a mixture of 10 mL of oleic acid and 10 mL of ethanol, stirred until completely dissolved, and then 20 mL of ferrous ammonium sulfate solution was added. Once the mixture turned brownish-red, it was transferred to a 50 mL reactor and heated at 230 °C for 16 h. After cooling, the reactor was removed, washed three times by centrifugation with ethanol, and dispersed in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Then, 2 g of citric acid and 20 mL of the oil-soluble magnetic nanoparticles were added to a 30 mL mixture of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed three times by centrifugation with ethanol, and dispersed in 200 mL of water to obtain Fe3O4 magnetic particles.

[0104] Zn in Comparative Example 1 0.2 Fe 2.8 The SERS detection and imaging capabilities of O4 magnetic particles and Fe3O4 magnetic particles (Comparative Example 1) were demonstrated using them as SERS substrates under an excitation wavelength of 532 nm, respectively, for a concentration of 1×10⁻⁶. -5 SERS spectroscopy was performed on methylene blue molecules at a concentration of mol / L to generate SERS spectra, such as... Figure 7 As shown, Zn-doped nanoparticles exhibit a better SERS enhancement effect, possibly because Zn doping provides more orbital energy levels, which is beneficial for charge transfer between the material and molecules, thereby enhancing the SERS effect.

[0105] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0106] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0107] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A functional multimodal SERS nanoprobe, characterized in that, The functional multi-modal SERS nanoprobe comprises, from inside to outside, a magnetic particle, a Raman / fluorescent signal molecule layer, a polymer layer, and a targeting antibody layer. The magnetic particle is a transition metal-doped ferroferric oxide nanoparticle, which has SERS performance, and the transition metal is any one of zinc, zinc and cobalt, and zinc and manganese. The preparation method of the transition metal-doped ferroferric oxide nanoparticle comprises the following steps: The solution of the iron salt and the transition metal salt is mixed with a solution of the alkaline inorganic substance, the long-chain alkyl organic acid, and the polar organic solvent, and the initial magnetic particle is obtained by reacting at 100-500°C for 1-50 hours, and phase inversion is performed to obtain the transition metal-doped ferroferric oxide nanoparticle. The phase inversion comprises the following steps: the initial magnetic particle and citric acid are added to a mixed solution of chloroform and DMF, and stirring is performed at room temperature for 1-50 hours. The iron salt is one or more of ferrous chloride, ferrous sulfate, ferrous ammonium sulfate, and ferrous nitrate. The transition metal salt is one or more of a chloride salt of the transition metal, a sulfate salt of the transition metal, and a nitrate salt of the transition metal. The molar ratio of the transition metal to iron is 1:(1-10). The Raman / fluorescent signal molecule layer is a layered structure formed by Raman / fluorescent signal molecules connected to the surface of the magnetic particle, and the Raman / fluorescent signal molecules are substances that have both fluorescence and Raman properties, including one or more of IR783, IR780, 3,3'-diethylthiatri-carbocyanine iodide, rhodamine, crystal violet, alizarin red, and Nile blue. The polymer layer is a layered structure formed by polymers coated on the surface of the Raman / fluorescent signal molecule layer, and the polymers are one or more of polydopamine, bovine serum albumin, and polyethylene glycol.

2. The functional multimodal SERS nanoprobes of claim 1, wherein, The transition metal is zinc and cobalt, and the molar ratio of zinc to cobalt is 1:(0.1-10). The transition metal is zinc and manganese, and the molar ratio of zinc to manganese is 1:(0.1-10).

3. The functional multimodal SERS nanoprobes of claim 1, wherein, The particle size of the transition metal-doped ferroferric oxide nanoparticle is 1-100 nm.

4. The functional multimodal SERS nanoprobes of claim 1, wherein, The alkaline inorganic substance is barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, or ammonia water; the long-chain alkyl organic acid is oleic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, or dodecanoic acid; and the polar organic solvent is methanol, ethanol, or isopropanol.

5. The functional multimodal SERS nanoprobes of claim 1, wherein, The solution of the alkaline inorganic substance, the long-chain alkyl organic acid, and the polar organic solvent is formed by adding the alkaline inorganic substance to the long-chain alkyl organic acid and the polar organic solvent, and 1-100 ml of the long-chain alkyl organic acid and 1-100 ml of the polar organic solvent are added per gram of the alkaline inorganic substance.

6. The functional multimodal SERS nanoprobes of claim 1, wherein, The targeting antibody layer is a layered structure formed by antibodies coupled to the surface of the polymer layer, and the antibodies are antibodies of tumor markers.

7. A method of preparing the functional multimodal SERS nanoprobe of claim 1, characterized in that, The method comprises the following steps: S1, a solution of an iron salt and a transition metal salt is mixed with a solution of an alkaline inorganic substance, a long-chain alkyl organic acid, and a polar organic solvent, and an initial magnetic particle is obtained by reacting at 100-500°C for 1-50 hours, and phase inversion is performed to obtain a magnetic particle; S2, a Raman / fluorescent signal molecule is connected to the surface of the magnetic particle to form a Raman / fluorescent signal molecule layer, and a magnetic particle II is obtained; S3, coating the polymer on the surface of the Raman / fluorescent signal molecule layer to form a polymer layer, and obtaining magnetic particles III; S4, coupling the antibody on the surface of the polymer layer to form a targeting antibody layer, and obtaining the functional multi-modal SERS nanoprobe.

8. The production method according to claim 7, characterized by, In step S2, the Raman / fluorescent signal molecule solution and the magnetic particle solution are mixed and stirred to obtain magnetic particles II; In step S3, the magnetic particle II solution, ethanol, ammonia water are mixed and stirred, and then the polymer solution is added to react to obtain magnetic particles III; In step S4, the solution containing magnetic particles III, buffer and antibody is mixed and stirred to obtain the functional multi-modal SERS nanoprobe.

9. The preparation method according to claim 7, characterized in that, In step S2, the volume ratio of the Raman / fluorescent signal molecule solution to the magnetic particle solution is 1:1000-10:

1. In step S2, the volume ratio of the Raman / fluorescent signal molecule solution to the magnetic particle solution is 1:1000-10:

1.

10. The preparation method according to claim 7, characterized in that, In step S3, the volume ratio of the magnetic particle II solution, ethanol, ammonia water and polymer solution is 5-55:2-16:0.3-1.5:0.4-10. In step S3, the magnetic particle II solution, ethanol, ammonia water are mixed and stirred for 1-100 min. In step S3, the magnetic particle II solution, ethanol, ammonia water are mixed and stirred for 1-100 min. In step S3, the reaction time for obtaining magnetic particles III is 1-10 h.

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