Multifunctional composite bioprobe and its preparation method and application

Abstract

The present invention provides a multifunctional composite bioprobe and its preparation method and application. The multifunctional composite bioprobe includes one or more precious metal SERS bioprobes and one or more magnetic semiconductor SERS bioprobes. The present invention realizes the addition of the functions of the precious metal SERS bioprobes and the magnetic semiconductor SERS bioprobes in the most simple and effective manner, and improves the sensitivity, accuracy and magnetic concentration of the detection method.

Description

[Technical field]

[0001] The present invention belongs to the technical field of life science, and relates to a multifunctional composite bioprobe and its preparation method and application. [Background technology]

[0002] Surface-enhanced Raman scattering (SERS) is a phenomenon that significantly amplifies Raman signals based on the localized plasmon resonance of noble metal or metal compound nanostructures. 6 ~10 10 Due to the advantages of SERS spectrum technology such as 1× amplification, high selectivity, high sensitivity, no photobleaching effect, interference resistance, fast and damage-free, surface enhanced Raman scattering spectrum etc. shows great application prospects in the field of optical detection such as in vitro diagnosis and liquid biopsy. At present, ordinary SERS bioprobes are divided into noble metal probes with the advantage of high detection sensitivity and semiconductor bioprobes with the functional advantages of high signal stability, optical, electrical and magnetic response properties, etc. In order to integrate the advantages of both, noble metals and functional semiconductor materials are usually composited by chemical synthesis, but the method is relatively complicated and it is difficult to obtain composite materials with high uniformity, which severely limits the application and development of noble metals and semiconductor materials in the field of SERS. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the present invention provides a multifunctional composite bioprobe and its preparation method and application.

[0004] One object of the present invention is achieved by a technical solution.

[0005] The multifunctional composite bioprobe includes one or more types of noble metal SERS bioprobes and one or more types of magnetic semiconductor SERS bioprobes.

[0006] Preferably, the particle size of the noble metal SERS bioprobe is 0.1 nm to 10,000 nm, more preferably, the particle size is 0.1 to 1,000 nm, even more preferably, the particle size is 0.1 to 800 nm, and even more preferably, the particle size is 1 to 500 nm.

[0007] Preferably, the particle size of the magnetic semiconductor SERS bioprobe is 0.1 nm to 10,000 nm, more preferably, the particle size is 0.1 to 1,000 nm, more preferably, the particle size is 0.1 to 800 nm, and even more preferably, the particle size is 1 to 500 nm.

[0008] Preferably, the shape of the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe includes, but is not limited to, one of the following: single layer, tetrahedron, hexahedron, octahedron, dodecahedron, hollow cage, circular granule, and rod.

[0009] Preferably, the noble metal SERS bioprobe comprises a noble metal material having SERS performance, the noble metal material being one or more of a single material and a composite material, such as, but not limited to, gold, silver, palladium, copper, etc., and the composite material is a composite material containing gold, silver, palladium, or copper.

[0010] Preferably, the particle size of the precious metal material is 0.1 to 500 nm, and optionally, the particle size of the precious metal material is any one of 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or a range between any two of these values.

[0011] Preferably, the magnetic semiconductor SERS bioprobe comprises a magnetic metal oxide having SERS capabilities, including, but not limited to, oxide materials containing one or more of Fe, Zn, Co, Ni, Cr, Mn.

[0012] Preferably, the particle size of the magnetic metal oxide is 0.1 to 500 nm, and optionally, the particle size of the magnetic metal oxide is any one value or a range between any two values ​​of 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500. More preferably, the particle size is 1 to 50 nm.

[0013] Preferably, the magnetic metal oxide is an oxide material formed by one or more of Zn, Co, Ni, Cr, Mn and Fe, which has a higher SERS enhancing effect on the magnetic semiconductor SERS bioprobe constructed from the magnetic metal oxide formed by doping with Fe oxide, thereby helping to improve the detection sensitivity and accuracy.

[0014] Preferably, the magnetic metal oxide is Zn x Fe 3-x O4, where 0 <x<3である。

[0015] Preferably, the magnetic metal oxide preparation method includes the steps of mixing and reacting a solution of one or more of zinc salt, cobalt salt, nickel salt, chromium salt, manganese salt and iron salt with a solution formed from a basic inorganic substance, a long alkyl chain organic acid and a polar organic solvent to obtain initial magnetic granules, and then performing phase inversion to obtain magnetic metal oxide nanoparticles. There are many methods for preparing magnetic metal oxides, but the magnetic metal oxide nanoparticles prepared by many of the methods do not have SERS performance. Through the study of the present invention, it has been found that the magnetic metal oxide nanoparticles prepared by the method of the present invention have SERS performance.

[0016] Iron salts include, but are not limited to, one or more of ferric chloride, ferrous chloride, ferrous sulfate, ferrous sulfate, ammonium iron(II) sulfate, ferrous nitrate, and ferrous nitrate; zinc salts include, but are not limited to, one or more of zinc chloride, zinc sulfate, and zinc nitrate; cobalt salts include, but are not limited to, one or more of cobalt chloride, cobalt sulfate, and cobalt nitrate; nickel salts include, but are not limited to, one or more of nickel chloride, nickel sulfate, and nickel nitrate; chromium salts include, but are not limited to, one or more of chromium chloride, chromium sulfate, and chromium nitrate. Examples of manganese salts include, but are not limited to, one or more of manganese chloride, manganese sulfate, and manganese nitrate; examples of basic inorganic substances include, but are not limited to, barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, and aqueous ammonia; examples of long alkyl chain organic acids include, but are not limited to, oleic acid, stearic acid, hexadecanoic acid, tetradecanoic acid, dodecanoic acid, and the like; and examples of polar organic solvents include, but are not limited to, highly polar organic solvents such as methanol, ethanol, and isopropyl alcohol.

[0017] The solution of one or more of a zinc salt, a cobalt salt, a nickel salt, a chromium salt, and a manganese salt and an iron salt is a solution formed by dissolving one or more of a zinc salt, a cobalt salt, a nickel salt, a chromium salt, and a manganese salt and an iron salt in water, and in the solution of one or more of a zinc salt, a cobalt salt, a nickel salt, a chromium salt, and a manganese salt and an iron salt, the concentration of the iron salt is 2 to 200 mmol / L.

[0018] A solution formed from a basic inorganic substance, a long alkyl chain organic acid, and a polar organic solvent is formed by adding the basic inorganic substance to the long alkyl chain organic acid and the polar organic solvent, whereby per gram of the basic inorganic substance, 1-100 ml of the long alkyl chain organic acid and 1-100 ml of the polar organic solvent are added.

[0019] Preferably, the volume ratio of a solution of one or more of a zinc salt, a cobalt salt, a nickel salt, a chromium salt, and a manganese salt and an iron salt to a solution formed from a basic inorganic substance, a long alkyl chain organic acid, and a polar organic solvent is 1:10 to 10:1.

[0020] Preferably, a solution of at least one of a zinc salt, a cobalt salt, a nickel salt, a chromium salt, and a manganese salt and an iron salt is mixed with a solution formed from a basic inorganic substance, a long alkyl chain organic acid, and a polar organic solvent to react with each other, the reaction temperature being 100 to 500°C, and the reaction time being 1 to 50 hours.

[0021] Preferably, the reaction time is 1, 2, 3, 5, 8, 10 h or any two of the following values: 100, 200, 250, 300, 350, 400, 450, 500 °C or any two of the following values:

[0022] Preferably, the phase inversion includes adding the initial magnetic granules and citric acid to an organic solvent and stirring at room temperature for 1 to 50 h. Optionally, the stirring time is any one of 5, 8, 10, 15, and 20 h, or a range value between any two of these values. The organic solvent may be chloroform, N,N-dimethylformamide, trichloromethane, carbon tetrachloride, formamide, DMSO, tetrahydrofuran, or pyridine.

[0023] The phase inversion reaction converts the nanoparticles from the oil phase to the aqueous phase, thereby improving the dispersibility of the nanoparticles in water and improving or expressing the SERS performance of the nanoparticles.

[0024] Preferably, the noble metal SERS bioprobe comprises, from the inside out, a noble metal material, a Raman / fluorescence signal molecule, a biopolymer and a target antibody protein, and the magnetic semiconductor SERS bioprobe comprises, from the inside out, a magnetic metal oxide, a Raman / fluorescence signal molecule, a biopolymer and a target antibody protein.

[0025] Preferably, the Raman / fluorescence signal molecule is a substance that simultaneously has fluorescence and Raman properties, including, but not limited to, one or more of IR783, IR780, 3,3'-diethylthiacarbocyanine iodide (DTTC), rhodamine, crystal violet, alizarin red, Nile blue A, and methylene blue.

[0026] Preferably, the biopolymer includes one or more of polydopamine, dopamine hydrochloride, bovine serum albumin, reduced bovine serum protein, and polyethylene glycol, but is not limited thereto, and anything having the properties of a biopolymer may be a biopolymer of the present invention.

[0027] Preferably, the target antibody protein is a protein and polypeptide that targets a tumor marker, and examples of the tumor marker include tumor cells, protein markers, exosomes, CtDNA, etc., and examples of the tumor include breast cancer, liver cancer, lung cancer, esophageal cancer, etc., and examples of the above proteins and polypeptides include folate antibody protein, Trop2 antibody protein, GE11 polypeptide, etc., but are not limited to the above proteins and polypeptides. Any proteins and polypeptides that have the property of targeting a tumor may be the target antibody protein of the present invention.

[0028] The Raman / fluorescence signal molecules and biopolymers in the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe can be the same or different, and the target antibody proteins in the two types of probes are the same substances; thus, the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe may target the same tumor marker.

[0029] Another object of the present invention is achieved by the following technical solutions.

[0030] The method for preparing the multifunctional composite bioprobe includes the steps of mixing one or more kinds of noble metal SERS bioprobes and one or more kinds of magnetic semiconductor SERS bioprobes in liquid or solid form to obtain a multifunctional composite bioprobe.

[0031] Another object of the present invention is achieved by the following technical solutions.

[0032] The application of the multifunctional composite bioprobe to in vitro detection includes a step of mixing one or more of the noble metal SERS bioprobes and one or more of the magnetic semiconductor SERS bioprobes in liquid or solid form and then adding the resulting mixture to a test system, or a step of sequentially adding one or more of the noble metal SERS bioprobes and one or more of the magnetic semiconductor SERS bioprobes in liquid or solid form to a test system.

[0033] Preferably, the application further includes the steps of adding the multifunctional composite bioprobe to a test system, binding the multifunctional composite bioprobe to a target substance in the test system, and determining the concentration of the target substance in the test system through magnetic concentration and separation, and then measuring Raman spectrum and / or fluorescence spectrum.

[0034] The wavelength of the excitation light used in the Raman spectrum and / or fluorescence spectrum measurement is 266 to 1064 nm, and preferably the lower limit of the wavelength of the excitation light is 266 nm, and the upper limit is selected from any one of 325, 488, 514, 532, 633, 647, 785, and 1064 nm, and preferably the wavelength of the excitation light is selected from any one of 266 nm, 325 nm, 488 nm, 514 nm, 532 nm, 633 nm, 647 nm, 785 nm, and 1064 nm.

[0035] Another object of the present invention is achieved by the following technical solutions.

[0036] An in vitro detection device including the multifunctional composite bioprobe. The in vitro detection device material includes a sensor, a detector, a spectral responder, and the like.

[0037] Compared with the prior art, the present invention has the following beneficial effects: (1) In the multifunctional composite bioprobe of the present invention, the precious metal SERS bioprobe has the property of high detection sensitivity, and the magnetic semiconductor SERS bioprobe has the property of magnetic concentration. The precious metal SERS bioprobe and the magnetic semiconductor SERS bioprobe target tumor cells, and can realize rapid concentration of the nanoprobe and tumor cells through a magnetic field, which is helpful in improving the detection sensitivity and accuracy of tumor cells. (2) Both the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe can provide Raman spectrum signals and fluorescence spectrum signals, thereby improving the accuracy of the detection system through the dual signal molecular mode, and the magnetic semiconductor SERS bioprobe also has the ability to provide magnetic resonance contrast imaging. Therefore, the combination of the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe can realize the multi-modal application of Raman, fluorescence and nuclear magnetic resonance. (3) The precious metal SERS bioprobe and the magnetic semiconductor SERS bioprobe targeted and identified the same tumor cells, and Raman spectrum and / or fluorescence spectrum measurements were performed. As a result, the test cells showed two and more Raman and / or fluorescence signals simultaneously, which ensured the accuracy of tumor detection and eliminated the interference caused by false positives of blood cells in peripheral blood samples. (4) The noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe provided by the present invention can be added to a test system by a simple mixing method and reacted, thereby avoiding the need to obtain a noble metal-semiconductor composite material through complicated chemical synthesis. The process is simple, the equipment is simplified, the cost is low, and the bioprobe is safe and feasible. (5) In the present invention, the addition of functions of a noble metal SERS bioprobe and a magnetic semiconductor SERS bioprobe is realized in the most simple and effective manner, thereby improving the sensitivity, accuracy and property of imparting magnetic concentration to the detection method. (6) The magnetic metal oxide in the magnetic semiconductor SERS bioprobe of the present invention is preferably an oxide material of Fe and one or more of Zn, Co, Ni, Cr, and Mn, and the magnetic metal oxide has a higher SERS enhancing effect. [Brief description of the drawings]

[0038] [Figure 1] TEM chromatogram of the precious metal gold nanomaterial prepared in Example 1. [Diagram 2] TEM chromatogram of the precious metal gold nanomaterial prepared in Example 2. [Diagram 3] TEM chromatogram of the precious metal gold nanomaterial prepared in Example 3. [Figure 4] FIG. 1 is a TEM chromatogram of Zn0.2Fe2.8O4 magnetic nanoparticles prepared in Example 4. [Diagram 5] TEM chromatogram of Zn0.2Fe2.8O4 magnetic nanoparticles prepared in Example 5. [Figure 6] TEM chromatogram of Zn0.2Fe2.8O4 magnetic nanoparticles prepared in Example 6. [Figure 7] FIG. 7a is a surface-enhanced Raman spectrogram corresponding to 4MBA molecules on gold nanoparticles of Example 1, and FIG. 7b is a surface-enhanced Raman spectrogram corresponding to crystal violet molecules on Zn0.2Fe2.8O4 magnetic nanoparticles of Example 5. [Figure 8] FIG. 8(a) is a fluorescence spectrogram of the surface modification of the magnetic nanoparticles of Example 5 with the luminescent indicator CCK-8, and FIG. 8(b) is a fluorescence spectrogram of the surface modification of the precious metal gold nanomaterial of Example 1 with the luminescent indicator CCK-8. [Figure 9] 1 shows SERS spectrograms of the magnetic nanoparticles of Example 5 and the Fe3O4 magnetic nanoparticles of Comparative Example 1 against methylene blue molecules. [Figure 10]FIG. 13 shows the concentrated capture of tumor cells by the Zn0.2Fe2.8O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe of Example 8. [Figure 11] 13 is a SERS spectrogram after binding of the Au-IR783-rBSA-Trop2 antibody protein nanoprobe of Example 7 and the Zn0.2Fe2.8O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe of Example 8 to MCF7 breast cancer cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The technical solution of the present invention will be further described below with specific examples and drawings. It should be understood that the specific examples described herein are merely for the purpose of understanding the present invention, and do not specifically limit the present invention. In addition, the drawings used in this specification are merely for the purpose of better explaining the contents disclosed in the present invention, and do not limit the scope of protection. Unless otherwise stated, all raw materials used in the examples of the present invention are raw materials commonly used in this field, and all methods used in the examples are common methods in this field.

[0040] <Example 1> Preparation of 3nm noble metal gold nanomaterials 2mL 5mM HAuCl4·4H2O solution was added to 7.85mL water, stirred uniformly at room temperature, 0.15mL 0.1M glutathione was added dropwise and stirred for 10min, then placed in a dark water bath at 70℃ for 24h, allowed to stand for 4 days, naturally cooled, centrifuged to wash the sample, washed three times with water and ethanol, and finally dried in a 70℃ oven for 12h, thus preparing a 3nm gold nanomaterial, as shown in Figure 1.

[0041] <Example 2> Preparation of 10nm noble metal gold nanomaterials 1mL 50mM HAuCl4·4H2O solution and 49mL water were added to a round-bottom flask, heated to boiling while stirring, 10mL 1% sodium citrate aqueous solution was quickly added, and then heated to boiling for 10min. Heating was stopped and the mixture was cooled to room temperature, then allowed to stand for 4 days, naturally cooled, and then centrifuged to wash the sample. It was then washed three times with water and ethanol, and finally dried in a 70℃ oven for 12h. Thus, a 10nm gold nanomaterial was prepared, as shown in Figure 2.

[0042] <Example 3> Preparation of 40nm noble metal gold nanomaterials 1mL 50mM HAuCl4·4H2O solution and 49mL water were added to a round-bottom flask, heated to boiling while stirring, 2mL 1% sodium citrate aqueous solution was quickly added, and the mixture was heated to boiling for 10min. Heating was stopped and the mixture was cooled to room temperature, then left to stand for 4 days, naturally cooled, and the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70℃ oven for 12h. Thus, a 40nm gold nanomaterial was prepared, as shown in Figure 3.

[0043] <Example 4> 4 nm Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles Add 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water to prepare a solution. Add 1 g of sodium hydroxide to a mixture of oleic acid (10 mL) and ethanol (10 mL) and stir until completely dissolved, then add 20 mL of ammonium iron(II) sulfate and zinc sulfate solution. After the mixture turns reddish brown, transfer it to a 50 mL reactor and heat at 230 °C for 8 h. After cooling the reactor, remove it, wash it with ethanol by centrifugation three times, and disperse it in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Next, 2 g of citric acid and 20 mL of oil-soluble magnetic nanoparticles were added to a mixed solution of 30 mL of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed with ethanol by centrifugation three times, and dispersed in 20 mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70 °C dryer for 12 h, obtaining 4 nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles were prepared and are shown in Figure 4.

[0044] <Example 5> 7nm Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles Add 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water to prepare a solution. Add 1 g of sodium hydroxide to a mixture of oleic acid (10 mL) and ethanol (10 mL) and stir until completely dissolved, then add 20 mL of ammonium iron(II) sulfate and zinc sulfate solution. After the mixture turns reddish brown, transfer it to a 50 mL reaction kettle and heat it at 230 °C for 16 h. After cooling the reaction kettle, remove it, wash it with ethanol by centrifugation three times, and disperse it in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Next, 2 g of citric acid and 20 mL of oil-soluble magnetic nanoparticles were added to a mixed solution of 30 mL of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed by centrifugation with ethanol three times, and dispersed in 20 mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70 °C oven for 12 h, obtaining the 7 nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles were prepared and are shown in Figure 5.

[0045] <Example 6> 10 nm Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles Add 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water to prepare a solution. Add 1 g of sodium hydroxide to a mixture of oleic acid (10 mL) and ethanol (10 mL) and stir until completely dissolved, then add 20 mL of ammonium iron(II) sulfate and zinc sulfate solution. After the mixture turns reddish brown, transfer it to a 50 mL reaction kettle and heat it at 230 °C for 24 h. After cooling the reaction kettle, remove it, wash it with ethanol by centrifugation three times, and disperse it in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Next, 2 g of citric acid and 20 mL of oil-soluble magnetic nanoparticles were added to a mixed solution of 30 mL of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed with ethanol by centrifugation three times, and dispersed in 20 mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70 °C oven for 12 h, obtaining the 10 nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles were prepared and are shown in FIG.

[0046] Using the precious metal gold nanomaterial of Example 1 and the magnetic nanoparticles of Example 5 as SERS substrates, SERS spectrum measurements were carried out for different concentrations of 4-mercaptobenzoic acid (4MBA) and crystal violet molecules (CV) under the action of an excitation wavelength of 633 nm. As shown in Figure 7, the precious metal gold nanomaterial of Example 1 has high SERS detection imaging performance for low concentrations of 4MBA molecules, and the magnetic nanoparticles of Example 5 have high SERS detection imaging performance for low concentrations of crystal violet molecules.

[0047] The surfaces of the precious metal gold nanomaterial of Example 1 and the magnetic nanoparticles of Example 5 were modified with the luminescent indicator CCK-8 and characterized, respectively, and the fluorescence emission patterns of the materials were obtained. Figure 8(a) is a fluorescence spectrogram of the surface modification of the magnetic nanoparticles of Example 5 with the luminescent indicator CCK-8, and Figure 8(b) is a fluorescence spectrogram of the surface modification of the precious metal gold nanomaterial of Example 1 with the luminescent indicator CCK-8.

[0048] <Comparative Example 1> Preparation of Fe3O4 magnetic nanoparticles Add 2 mmol of ammonium iron(II) sulfate hexahydrate to 20 mL of ultrapure water to prepare a solution. Add 1 g of sodium hydroxide to a mixture of 10 mL of oleic acid and 10 mL of ethanol, stir until completely dissolved, then add 20 mL of ammonium iron(II) sulfate solution. After the mixture turns reddish brown, transfer it to a 50 mL reactor and heat at 230 °C for 16 h. After cooling the reactor, remove it, wash it with ethanol by centrifugation three times, and disperse it in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Next, 2 g of citric acid and 20 mL of oil-soluble magnetic nanoparticles are added to a mixed solution of 30 mL of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, washed by centrifugation with ethanol three times, and dispersed in 20 mL of water to obtain the desired magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample is washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70°C oven for 12 h, thereby preparing the Fe3O4 magnetic nanoparticles.

[0049] The SERS detection imaging capabilities of the magnetic nanoparticles of Example 5 and the Fe3O4 magnetic nanoparticles of Comparative Example 1 are compared, and as SERS substrates, the concentration of each is 1×10 -5SERS spectrum measurement was performed on mol / L methylene blue molecules, and the SERS spectrogram was generated, as shown in Figure 9. It can be seen that the Zn-doped nanoparticles have a higher SERS enhancement effect, and the possible reason is that after doping with Zn, more orbital energy levels are provided, which contributes to the charge transfer effect between the material and the molecule, and enhances the SERS effect. Zn 0.2 Fe 2.8 Both the magnetic semiconductor SERS bioprobe formed by O4 magnetic nanoparticles and the noble metal SERS bioprobe have been applied to in vitro detection, with higher detection sensitivity and higher accuracy.

[0050] <Example 7> Preparation of Au-IR783-rBSA-Trop2 antibody protein nanoprobe (1) Preparation of precious metal gold nanomaterials 2mL of 5mM HAuCl4·4H2O solution was added to 7.85mL of water, stirred uniformly at room temperature, 0.15mL of 0.1M glutathione was added dropwise and stirred for 10min, then placed in a dark water bath at 70℃ for 24h, allowed to stand for 4 days, naturally cooled, centrifuged to wash the sample, washed three times with water and ethanol, and finally dried in a 70℃ oven for 12h, thus preparing 3nm gold nanomaterials. (2) Preparation of Au-IR783 nanoparticles 1 mg of gold nanomaterial was added to 15 ml of 0.05 mmol / L IR783 ethanol solution, stirred with a polytetrafluoroethylene rod for 2 h, thoroughly washed with deionized water, and finally dispersed in 18 ml of deionized water, thus obtaining an Au-IR783 solution. (3) Preparation of Au-IR783-rBSA nanoparticles After mixing 18 ml of Au-IR783 solution, 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 rBSA 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, thereby obtaining Zn 0.2 Fe 2.8An O4-IR780-rBSA solution is prepared. (4) Preparation of Au-IR783-rBSA-Trop2 antibody protein nanoparticles Take 4ml Au-IR783-rBSA solution and obtain Au-IR783-rBSA nanoparticles by magnetic adsorption, discard the supernatant, then add 4ml Tris-HCl solution (10mM, pH=8.5), then add 40μg Trop2 antibody protein, stir at room temperature for 12h, wash with PBS three times, and finally disperse in 4ml PBS solution, thus obtaining Au-IR783-rBSA-Trop2 antibody protein bioprobe.

[0051] <Example 8> Zinc 0.2 Fe 2.8 Preparation of O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe (1)Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles Add 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water to prepare a solution. Add 1 g of sodium hydroxide to a mixture of 10 mL of oleic acid and 10 mL of ethanol, stir until completely dissolved, then add 20 mL of ammonium iron(II) sulfate and zinc sulfate solution, and after the color of the mixture turns reddish brown, transfer it to a 50 mL reaction kettle and heat it at 230 °C for 16 h. After cooling the reaction kettle, it is taken out, centrifuged and washed three times with ethanol, and dispersed in 20 mL of cyclohexane to obtain the desired oil-soluble magnetic nanoparticles. Then, add 2 g of citric acid and 20 mL of oil-soluble magnetic nanoparticles to a mixture of 30 mL of chloroform / DMF (v / v: 1 / 1), stir for 12 h, centrifuged and washed three times with ethanol, and dispersed in 200 mL of water to obtain Zn 0.2 Fe 2.8 Obtain the O4 magnetic nanoparticle solution. (2) Zn 0.2 Fe 2.8 Preparation of O4-Alizarin Red nanoparticles 150 μl of 1 mmol / L alizarin red ethanol solution was added to 15 ml of magnetic nanoparticle solution (solvent is water, concentration is 0.21 mg / ml), stirred with a polytetrafluoroethylene rod for 2 h, thoroughly washed with deionized water, and finally dispersed in 18 ml of deionized water, thereby obtaining Zn 0.2 Fe 2.8 An O4-alizarin red solution is obtained. (3) Zn 0.2 Fe 2.8 Preparation of O4-Alizarin Red-PDA nanoparticles 18ml Zn 0.2 Fe 2.8 O4-alizarin red solution (solvent is water, concentration is 0.19 mg / ml) was mixed with 8 ml CH3CH2OH and 600 μl NH3·H2O, then stirred with a polytetrafluoroethylene rod for 20 min, followed by slowly adding 2 ml of polydopamine solution (40 mg / ml). After 5 h, it was thoroughly washed with deionized water and dispersed in 8 ml of deionized water, whereby Zn 0.2 Fe 2.8 An O4-Alizarin Red-PDA nanoparticle solution is prepared. (4) Zn 0.2 Fe 2.8 Preparation of O4-Alizarin Red-PDA-Trop2 antibody protein nanoparticles 4ml Zn 0.2 Fe 2.8 The O4-alizarin red-PDA nanoparticle solution was taken and magnetically attracted to Zn 0.2 Fe 2.8 The O4-alizarin red-PDA nanoparticles were obtained, the supernatant was discarded, 4 ml Tris-HCl solution (10 mM, pH = 8.5) was added, and then 40 μg Trop2 antibody protein was added. The mixture was stirred at room temperature for 12 h, washed three times with PBS, and finally dispersed in 4 ml PBS solution, thereby obtaining Zn 0.2 Fe 2.8 An O4-Alizarin Red-PDA-40 μg Trop2 antibody protein solution is obtained.

[0052] Zn in Example 8 0.2 Fe 2.8The O4-alizarin red-PDA-40μg Trop2 antibody protein nanoprobe was co-cultured with tumor cells, and then combined with the magnetic enrichment module of the circulating tumor cell detection device to perform magnetic enrichment. The enrichment effect was as shown in Figure 10. 0.2 Fe 2.8 The ZnO nanoprobe has excellent binding ability, and after magnetic concentration, tumor cells are concentrated, and there are no tumor cells in the filtrated waste liquid. 0.2 Fe 2.8 This indicates that the O4 nanoprobe has good enrichment and capture ability for tumor cells.

[0053] The Au-IR783-rBSA-Trop2 antibody protein nanoprobe of Example 7 was co-cultured with MCF7 breast cancer cells alone, and then SERS spectrum measurement was performed. The SERS spectrogram is shown in FIG. 11. The Zn of Example 8 0.2 Fe 2.8 The O4-alizarin red-PDA-Trop2 antibody protein nanoprobe was co-cultured with MCF7 breast cancer cells alone, and then SERS spectrum measurement was performed. The SERS spectrogram is shown in FIG. 11. The Au-IR783-rBSA-Trop2 antibody protein nanoprobe of Example 7 and the Zn 0.2 Fe 2.8 The O4-alizarin red-PDA-Trop2 antibody protein nanoprobe was co-cultured with MCF7 breast cancer cells, followed by magnetic enrichment and separation, followed by SERS spectrum measurement, and the SERS spectrogram is shown in FIG.

[0054] As can be seen from FIG. 11, when only a single probe is used to detect cancer cells, the SERS signal has few peaks, is unstable, and is less uniform, while the Au-IR783-rBSA-Trop2 antibody protein nanoprobe and the Zn of Example 8 are used to detect cancer cells. 0.2 Fe 2.8 When O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe was used to jointly detect cancer cells, the SERS signal had many peaks and the signal was stable, which was helpful in improving the detection sensitivity and accuracy.

[0055] Each aspect, embodiment, and feature of the present invention is to be considered in all respects as illustrative and not limiting of the invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses may occur to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0056] In the preparation method of the present invention, the order of each step is not limited to the order illustrated, and any change in order of each step made by a person skilled in the art without requiring creative effort is also included within the scope of protection of the present invention. In addition, two or more steps or operations can be performed simultaneously.

[0057] Finally, it should be explained that the specific examples described in this specification are merely for the purpose of illustrating the present invention, and do not limit the embodiments of the present invention. Those skilled in the art to which the present invention belongs may make various modifications or supplements to the specific examples described, or replace them in a similar manner, and it is not necessary to list all the embodiments here, and even cannot list all of them. Any obvious changes or modifications derived from the substantial spirit of the present invention still belong to the protection scope of the present invention, and interpreting them as any additional limitations will all deviate from the spirit of the present invention.

Claims

1. A multifunctional hybrid bioprobe comprising one or more types of noble metal SERS bioprobes and one or more types of magnetic semiconductor SERS bioprobes.

2. 2. The multifunctional composite bioprobe according to claim 1, wherein the particle size of the noble metal SERS bioprobe is 0.1 nm to 10,000 nm, and the particle size of the magnetic semiconductor SERS bioprobe is 0.1 nm to 10,000 nm.

3. The noble metal SERS bioprobe comprises a noble metal material having SERS capability, the noble metal material comprising one or more of gold, silver, palladium, and copper, or a composite material comprising one or more of gold, silver, palladium, and copper; The multifunctional composite bioprobe according to claim 1, characterized in that the magnetic semiconductor SERS bioprobe comprises a magnetic metal oxide having SERS performance, and the magnetic metal oxide comprises one or more of oxide materials including one or more of Fe, Zn, Co, Ni, Cr, and Mn.

4. 4. The multifunctional composite bioprobe according to claim 3, wherein the magnetic metal oxide is an oxide material formed of one or more of Zn, Co, Ni, Cr, and Mn and Fe.

5. The magnetic metal oxide is Zn ｘ Fe ３－ｘ O ４ 4. The multifunctional hybrid bioprobe according to claim 3, wherein x is 0<x<3.

6. The multifunctional composite bioprobe of claim 4, characterized in that the method for preparing the magnetic metal oxide includes the steps of mixing and reacting a solution of one or more of zinc salt, cobalt salt, nickel salt, chromium salt, and manganese salt with a solution formed from a basic inorganic substance, a long alkyl chain organic acid, and a polar organic solvent to obtain initial magnetic granules, and then performing phase inversion to obtain magnetic metal oxide nanoparticles.

7. The basic inorganic substance is one or more of barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, and aqueous ammonia; 7. The multifunctional hybrid bioprobe according to claim 6, wherein the long alkyl chain organic acid is one or more of oleic acid, stearic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid.

8. The multifunctional composite bioprobe according to claim 6, wherein the phase inversion comprises a step of adding the initial magnetic granules and citric acid to an organic solvent and stirring at room temperature for 1 to 50 hours.

9. The multifunctional composite bioprobe of claim 1, characterized in that the noble metal SERS bioprobe sequentially comprises, from the inside to the outside, a noble metal material, a Raman / fluorescence signal molecule, a biopolymer and a target antibody protein, and the magnetic semiconductor SERS bioprobe sequentially comprises, from the inside to the outside, a magnetic metal oxide, a Raman / fluorescence signal molecule, a biopolymer and a target antibody protein.

10. A method for preparing the multifunctional composite bioprobe according to claim 1, characterized in that it comprises a step of mixing one or more of the noble metal SERS bioprobes with one or more of the magnetic semiconductor SERS bioprobes in liquid or solid form to obtain a multifunctional composite bioprobe.

11. The application of the multifunctional composite bioprobe according to claim 1 to in vitro detection, characterized in that it comprises a step of mixing one or more of the noble metal SERS bioprobes and one or more of the magnetic semiconductor SERS bioprobes in liquid or solid form, and then adding them to a test system, or a step of sequentially adding one or more of the noble metal SERS bioprobes and one or more of the magnetic semiconductor SERS bioprobes in liquid or solid form to a test system.

12. The application of the multifunctional composite bioprobe described in claim 11 to in vitro detection, further comprising the steps of: adding the multifunctional composite bioprobe to a test system; binding the multifunctional composite bioprobe to a target substance in the test system; conducting magnetic concentration and separation; and determining the concentration of the target substance in the test system by Raman spectrum and / or fluorescence spectrum measurement.

13. 13. An in vitro detection device comprising the multifunctional composite bioprobe according to claim 1.

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