Multifunctional hybrid bioprobes and their preparation methods and applications

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 spectroscopy, such as 2x amplification, high selectivity, high sensitivity, no photobleaching effect, interference resistance, high speed, and damage-free detection, surface-enhanced Raman scattering (SERS) spectroscopy has shown great potential for application in optical detection fields such as in vitro diagnostics and liquid biopsy. Currently, typical SERS bioprobes are divided into noble metal probes, which have the advantage of high detection sensitivity, and semiconductor bioprobes, which have functional advantages such as high signal stability and optical, electrical, and magnetic response. To combine the advantages of both, noble metals and functional semiconductor materials are typically composited using chemical synthesis methods. However, this method is relatively complex and it is difficult to obtain highly uniform composites, severely limiting the application and development of noble metal and semiconductor materials in the field of SERS. Summary of the Invention

[0003] The present invention addresses the shortcomings of the prior art by providing a multifunctional hybrid bioprobe and its preparation method and application.

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

[0005] The multifunctional hybrid 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, even 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 a hemilayer, a tetrahedron, a hexahedron, an octahedron, a dodecahedron, a hollow cage, a circular granule, and a rod.

[0009] Preferably, the noble metal SERS bioprobe comprises a noble metal material having SERS properties, and the noble metal material includes, but is not limited to, one or more of a single material and a composite material such as 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, and 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, and 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 Fe and one or more of Zn, Co, Ni, Cr, and Mn, which has a higher SERS enhancement effect on the magnetic semiconductor SERS bioprobe constructed from the magnetic metal oxide formed by doping with Fe oxide, thereby helping to improve 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 method for preparing the magnetic metal oxide comprises the steps of mixing a solution of one or more of zinc salt, cobalt salt, nickel salt, chromium salt, and manganese salt with an iron salt, and reacting it 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 these methods do not have SERS performance. Through research into the present invention, it has been found that the magnetic metal oxide nanoparticles prepared by the method of the present invention do have SERS performance.

[0016] Iron salts include, but are not limited to, one or more of iron chloride, ferrous chloride, ferrous sulfate, ferrous sulfate, ammonium iron(II) sulfate, iron 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; and chromium salts include, but are not limited to, one or more of chromium chloride, chromium sulfate, and chromium nitrate. Examples of suitable organic solvents include, but are not limited to, manganese salts include, but are not limited to, one or more of manganese chloride, manganese sulfate, and manganese nitrate; basic inorganic substances include, but are not limited to, barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, and aqueous ammonia; long alkyl chain organic acids include, but are not limited to, oleic acid, stearic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid; and 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 the concentration of the iron salt 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 is 2 to 200 mmol / L.

[0018] The solution formed from 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, and the basic inorganic substance is added to 1 to 100 ml of the long alkyl chain organic acid and 1 to 100 ml of the polar organic solvent per gram of the basic inorganic substance.

[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 one or more of zinc salts, cobalt salts, nickel salts, chromium salts, and manganese salts 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, 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, or 10 hours, or a range between any two of these values. Optionally, the reaction temperature is 100, 200, 250, 300, 350, 400, 450, or 500°C, or a range between any two of these values.

[0022] Preferably, the phase inversion comprises adding the initial magnetic granules and citric acid to an organic solvent and stirring at room temperature for 1 to 50 hours. Optionally, the stirring time is any one of 5, 8, 10, 15, and 20 hours, or a range between any two of these values. Examples of organic solvents include chloroform, N,N-dimethylformamide, trichloromethane, carbon tetrachloride, formamide, DMSO, tetrahydrofuran, and 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 revealing 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, in that order.

[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 any substance having the properties of a biopolymer may be a biopolymer of the present invention.

[0027] Preferably, the target antibody protein is a protein or polypeptide that targets a tumor marker. Examples of tumor markers include tumor cells, protein markers, exosomes, and CtDNA. Examples of tumors include breast cancer, liver cancer, lung cancer, and esophageal cancer. Examples of the above proteins and polypeptides include folate antibody protein, Trop2 antibody protein, and GE11 polypeptide. However, the proteins and polypeptides are not limited to the above proteins and polypeptides. Any proteins and polypeptides that have tumor-targeting properties 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, but the target antibody proteins in the two types of probes are the same substance. Thus, the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe can 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 step of mixing one or more noble metal SERS bioprobes and one or more 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 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, allowing the multifunctional composite bioprobe to bind to a target substance in the test system, undergoing magnetic concentration and separation, and then determining the concentration of the target substance in the test system by Raman spectroscopy and / or fluorescence spectroscopy.

[0034] The wavelength of the excitation light used in measuring the Raman spectrum and / or fluorescence spectrum 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. Examples of the in vitro detector material include a sensor, a detector, and a spectral responder.

[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 noble metal SERS bioprobe has the property of high detection sensitivity, and the magnetic semiconductor SERS bioprobe has the property of magnetic concentration. The noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe target tumor cells, and can realize rapid concentration of the nanoprobe and tumor cells through the magnetic field, which helps to improve the detection sensitivity and accuracy of tumor cells. (2) Both noble metal SERS bioprobes and magnetic semiconductor SERS bioprobes can provide Raman and fluorescence spectral signals, which improves the accuracy of the detection system through dual-signal molecular mode. In addition, the magnetic semiconductor SERS bioprobe also has the ability to provide magnetic resonance imaging. Therefore, the combination of noble metal SERS bioprobes and magnetic semiconductor SERS bioprobes can realize the multimodal application of Raman, fluorescence, and nuclear magnetic resonance. (3) The noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe targeted and identified the same tumor cells, and Raman and / or fluorescence spectrum measurements showed that the test cells simultaneously exhibited two or more Raman and / or fluorescence signals, thereby ensuring the accuracy of tumor detection and eliminating the interference of blood cells in peripheral blood samples. (4) The noble metal SERS bioprobe and magnetic semiconductor SERS bioprobe provided by the present invention can be added to a test system by simple mixing and reacting, thereby avoiding the need to obtain a noble metal-semiconductor composite material through complex chemical synthesis. The process is simple, the equipment is simplified, the cost is low, and it is safe and feasible. (5) In the present invention, the addition of the functions of the noble metal SERS bioprobe and the magnetic semiconductor SERS bioprobe is realized in the simplest and most effective manner, thereby improving the sensitivity, accuracy, and magnetic concentration properties of 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 with one or more of Zn, Co, Ni, Cr, and Mn, and the magnetic metal oxide has a higher SERS enhancement effect. [Brief explanation of the drawings]

[0038] [Figure 1] 1 is a TEM chromatogram of the noble metal gold nanomaterial prepared in Example 1. [Figure 2] 1 is a TEM chromatogram of the noble metal gold nanomaterial prepared in Example 2. [Figure 3] 1 is a TEM chromatogram of the noble metal gold nanomaterial prepared in Example 3. [Figure 4] TEM chromatogram of ZnO magnetic nanoparticles prepared in Example 4. [Figure 5] TEM chromatogram of ZnO magnetic nanoparticles prepared in Example 5. [Figure 6] TEM chromatogram of ZnO.2FeO.8 magnetic nanoparticles prepared in Example 6. [Figure 7] 7a is a surface-enhanced Raman spectrogram corresponding to 4MBA molecules on gold nanoparticles in Example 1, and FIG. 7b is a surface-enhanced Raman spectrogram corresponding to crystal violet molecules on ZnO magnetic nanoparticles in Example 5. [Figure 8] 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 noble 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 Fe 3 O 4 magnetic nanoparticles of Comparative Example 1 with respect to methylene blue molecules. [Figure 10]FIG. 10 shows the concentrated capture of tumor cells by the ZnO.2Fe2.8O4-alizarin red-PDA-Trop2 antibody protein nanoprobe of Example 8. [Figure 11] 1 shows SERS spectrograms of the Au-IR783-rBSA-Trop2 antibody protein nanoprobe of Example 7 and the ZnO-Alizarin Red-PDA-Trop2 antibody protein nanoprobe of Example 8 after binding to MCF7 breast cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0039] The technical solutions of the present invention will be further described below with reference to specific examples and drawings. However, 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. Furthermore, 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 specified, all materials used in the examples of the present invention are 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 2 mL of 5 mM HAuCl4·4H2O solution was added to 7.85 mL of water and stirred at room temperature. 0.15 mL of 0.1 M glutathione was added dropwise and stirred for 10 minutes. The mixture was then placed in a dark water bath at 70°C for 24 hours, allowed to stand for 4 days, and allowed to cool naturally. After that, the sample was centrifuged to wash, washed three times with water and three times with ethanol, and finally dried in a 70°C oven for 12 hours. This resulted in the preparation of 3 nm gold nanoparticles, as shown in Figure 1.

[0041] <Example 2> Preparation of 10nm noble metal gold nanomaterials 1 mL of 50 mM HAuCl4·4H2O solution and 49 mL of water were added to a round-bottom flask and heated to a boil while stirring. 10 mL of 1% sodium citrate aqueous solution was quickly added and the mixture was heated to a boil for 10 minutes. After heating, the mixture was cooled to room temperature and allowed to stand for 4 days. After natural cooling, the sample was centrifuged to wash, washed three times with water and ethanol, and finally dried in a 70°C oven for 12 hours. This resulted in the preparation of 10 nm gold nanoparticles, as shown in Figure 2.

[0042] Example 3 Preparation of 40nm noble metal gold nanomaterials 1 mL of 50 mM HAuCl4·4H2O solution and 49 mL of water were added to a round-bottom flask and heated to a boil while stirring. 2 mL of 1% sodium citrate solution was quickly added and heated to a boil for 10 minutes. After heating, the mixture was cooled to room temperature and allowed to stand for 4 days. After natural cooling, the mixture was centrifuged to wash the sample, washed three times with water and ethanol, and finally dried in a 70°C oven for 12 hours. This resulted in the preparation of 40 nm gold nanoparticles, as shown in Figure 3.

[0043] Example 4 4nm Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles A solution was prepared by adding 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water. 1 g of sodium hydroxide was added to a mixture of oleic acid (10 mL) and ethanol (10 mL) and stirred until completely dissolved. 20 mL of ammonium iron(II) sulfate and zinc sulfate solution was added. After the mixture turned reddish-brown, it was transferred to a 50 mL reactor and heated at 230°C for 8 hours. After cooling, the reactor was removed, washed three times with ethanol by centrifugation, and dispersed 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 30 mL of a mixed solution of chloroform / DMF (v / v: 1 / 1), stirred for 12 hours, 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 cooling naturally, the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70 °C oven for 12 hours, 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 A solution was prepared by adding 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water. 1 g of sodium hydroxide was added to a mixture of oleic acid (10 mL) and ethanol (10 mL) and stirred until completely dissolved. 20 mL of ammonium iron(II) sulfate and zinc sulfate solution was added. After the mixture turned reddish-brown, it was transferred to a 50 mL reactor and heated at 230°C for 16 hours. After cooling, the reactor was removed, washed three times with ethanol by centrifugation, and dispersed 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 30 mL of a mixed solution of chloroform / DMF (v / v: 1 / 1), stirred for 12 hours, 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 cooling naturally, the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70 °C oven for 12 hours to obtain 7 nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles were prepared and are shown in Figure 5.

[0045] Example 6 10nm Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles A solution was prepared by adding 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water. 1 g of sodium hydroxide was added to a mixture of oleic acid (10 mL) and ethanol (10 mL) and stirred until completely dissolved. 20 mL of ammonium iron(II) sulfate and zinc sulfate solution was added. After the mixture turned reddish-brown, it was transferred to a 50 mL reactor and heated at 230°C for 24 hours. After cooling, the reactor was removed, washed three times with ethanol by centrifugation, and dispersed 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 30 mL of a mixed solution of chloroform / DMF (v / v: 1 / 1), stirred for 12 hours, 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 cooling naturally, the sample was washed by centrifugation, washed three times with water and ethanol, and finally dried in a 70 °C oven for 12 hours to obtain 10 nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles were prepared and are shown in Figure 6.

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

[0047] The surface of the noble 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 noble metal gold nanomaterial of Example 1 with the luminescent indicator CCK-8.

[0048] <Comparative Example 1> Preparation of Fe3O4 magnetic nanoparticles A solution was prepared by adding 2 mmol of ammonium iron(II) sulfate hexahydrate to 20 mL of ultrapure water. 1 g of sodium hydroxide was added to a mixture of 10 mL of oleic acid and 10 mL of ethanol and stirred until completely dissolved. 20 mL of ammonium iron(II) sulfate solution was then added. After the mixture turned reddish-brown, it was transferred to a 50 mL reactor and heated at 230°C for 16 hours. After cooling, the reactor was removed, washed three times with ethanol by centrifugation, and dispersed 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 30 mL of a mixed solution of chloroform / DMF (v / v: 1 / 1), stirred for 12 hours, centrifuged and washed three times with ethanol, and then dispersed in 20 mL of water to obtain the desired magnetic nanoparticles. After leaving to stand for 4 days and cooling naturally, the sample is centrifuged to wash, washed three times with water and ethanol, and finally dried in a 70°C oven for 12 hours, thereby preparing 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 were compared, and as SERS substrates, under the action of an excitation wavelength of 532 nm, the concentration of each was 1 × 10 -5SERS spectroscopy was performed on mol / L methylene blue molecules, and the resulting SERS spectrogram was shown in Figure 9. It was found that the Zn-doped nanoparticles had a higher SERS enhancement effect. The possible reason for this is that after Zn doping, more orbital energy levels are provided, which contributes to the charge transfer effect between the material and the molecule, thereby enhancing 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, and have shown higher detection sensitivity and accuracy.

[0050] Example 7 Preparation of Au-IR783-rBSA-Trop2 antibody protein nanoprobe (1) Preparation of nanomaterials of the precious metal gold Add 2 mL of 5 mM HAuCl4·4H2O solution to 7.85 mL of water and stir evenly at room temperature. Add 0.15 mL of 0.1 M glutathione dropwise and continue stirring for 10 minutes. Place in a dark water bath at 70°C for 24 hours, then leave to stand for 4 days. After natural cooling, the sample is centrifuged to wash, washed three times with water and ethanol, and finally dried in a 70°C oven for 12 hours to prepare 3 nm gold nanomaterials. (2) Preparation of Au-IR783 nanoparticles 1 mg of gold nanomaterials 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 to obtain 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 4 ml of the Au-IR783-rBSA solution was taken and magnetically attracted to obtain Au-IR783-rBSA nanoparticles. The supernatant was discarded, followed by the addition of 4 ml of Tris-HCl solution (10 mM, pH = 8.5), followed by the addition of 40 μg of Trop2 antibody protein. The mixture was stirred at room temperature for 12 hours, washed three times with PBS, and finally dispersed in 4 ml of PBS solution to obtain the Au-IR783-rBSA-Trop2 antibody protein bioprobe.

[0051] Example 8 Zn 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 A solution was prepared by adding 1.73 mmol of ammonium iron(II) sulfate hexahydrate and 0.534 mmol of zinc sulfate heptahydrate to 20 mL of ultrapure water. 1 g of sodium hydroxide was added to a mixture of 10 mL of oleic acid and 10 mL of ethanol and stirred until completely dissolved. 20 mL of ammonium iron(II) sulfate and zinc sulfate solution was added. After the mixture turned reddish-brown, it was transferred to a 50 mL reactor and heated at 230 °C for 16 h. After cooling, the reactor was removed, centrifuged and washed three times with ethanol, and dispersed 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 30 mL mixture of chloroform / DMF (v / v: 1 / 1), stirred for 12 h, centrifuged and washed three times with ethanol, and dispersed in 200 mL of water to obtain the ZnO nanoparticles. 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, and then stirred with a polytetrafluoroethylene rod for 20 min. 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, thereby obtaining 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 subjected to magnetic adsorption. 0.2 Fe 2.8 The O4-alizarin red-PDA nanoparticles were obtained, the supernatant was discarded, and then 4 ml of Tris-HCl solution (10 mM, pH = 8.5) was added, followed by the addition of 40 μg of Trop2 antibody protein. The mixture was stirred at room temperature for 12 h, washed three times with PBS, and finally dispersed in 4 ml of 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 2.8O4-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 for magnetic enrichment. The enrichment effect was as shown in Figure 10. 0.2 Fe 2.8 The excellent binding ability of the O4 nanoprobes allows tumor cells to be concentrated after magnetic concentration, and the filtered waste liquid is free of tumor cells. 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, followed by SERS spectrum measurement. The SERS spectrogram is shown in Figure 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, followed by SERS spectroscopy. The SERS spectrogram is shown in Figure 11. The Au-IR783-rBSA-Trop2 antibody protein nanoprobe of Example 7 and the Zn-IR783-rBSA-Trop2 antibody protein nanoprobe of Example 8 were co-cultured with MCF7 breast cancer cells, followed by SERS spectroscopy. 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 spectroscopy. The SERS spectrogram is shown in FIG.

[0054] As can be seen from Figure 11, when only a single probe was used to detect cancer cells, the SERS signal had fewer peaks, was unstable, and was less uniform, while when the Au-IR783-rBSA-Trop2 antibody protein nanoprobe and the Zn of Example 8 were used, the SERS signal was less uniform. 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 was stable, which helped to improve 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 exemplified, and any change in the 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 noted that the specific examples described in this specification are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention pertains may make various modifications or additions to the specific examples described, or replace them in a similar manner, and it is not necessary, and even impossible, to list all the embodiments herein. Any obvious changes or modifications derived from the essential spirit of the present invention still fall within the scope of protection of the present invention, and any interpretation of them as any additional limitations would deviate from the spirit of the present invention.

Claims

1. one or more of noble metal SERS bioprobes and one or more of magnetic semiconductor SERS bioprobes; The noble metal SERS bioprobe comprises a noble metal material having SERS capability, the noble metal material being one or more of gold, silver, palladium, and copper, or a composite material comprising one or more of gold, silver, palladium, and copper; the magnetic semiconductor SERS bioprobe comprises a magnetic metal oxide having SERS performance, the magnetic metal oxide being an oxide material containing one or more of Fe, Zn, Co, Ni, Cr, and Mn; The noble metal SERS bioprobe is a multifunctional composite bioprobe that 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 is a multifunctional composite bioprobe that sequentially comprises, from the inside to the outside, a magnetic metal oxide, a Raman / fluorescence signal molecule, a biopolymer, and a target antibody protein.

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. 2. The multifunctional composite bioprobe according to claim 1, wherein the particle size of the noble metal material is 1 to 100 nm, and the particle size of the magnetic metal oxide is 1 to 50 nm.

4. 2. The multifunctional composite bioprobe according to claim 1, wherein the magnetic metal oxide is an oxide material formed from 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 5. The multifunctional hybrid bioprobe according to claim 4, wherein x is 0<x<3.

6. Mixing one or more of the noble metal SERS bioprobes with one or more of the magnetic semiconductor SERS bioprobes in a liquid or solid state to obtain a multifunctional composite bioprobe; the magnetic semiconductor SERS bioprobe comprises a magnetic metal oxide having SERS performance, the magnetic metal oxide being an oxide material formed of one or more of Zn, Co, Ni, Cr, and Mn and Fe; The method for preparing the magnetic metal oxide according to claim 1, characterized in that it comprises 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 an 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.

7. The iron salts include one or more of iron chloride, ferrous chloride, ferrous sulfate, ferrous sulfate, ammonium iron (II) sulfate, ferrous nitrate, and ferrous nitrate; the zinc salts include one or more of zinc chloride, zinc sulfate, and zinc nitrate; the cobalt salts include one or more of cobalt chloride, cobalt sulfate, and cobalt nitrate; the nickel salts include one or more of nickel chloride, nickel sulfate, and nickel nitrate; the chromium salts include one or more of chromium chloride, chromium sulfate, and chromium nitrate; the manganese salts include one or more of manganese chloride, manganese sulfate, and manganese nitrate; the basic inorganic substance is one or more of barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide, and aqueous ammonia; The method for preparing a multifunctional composite 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 method for preparing a multifunctional composite bioprobe according to claim 6, wherein the reaction temperature is 200 to 500°C and the reaction time is 2 to 50 hours.

9. A method for preparing a multifunctional composite bioprobe as described in claim 6, characterized in that the phase inversion includes a step of adding initial magnetic granules and citric acid to an organic solvent and stirring at room temperature for 1 to 50 hours.

10. The method for preparing a multifunctional composite bioprobe according to claim 9, wherein the organic solvent is chloroform, N,N-dimethylformamide, trichloromethane, carbon tetrachloride, formamide, DMSO, tetrahydrofuran or pyridine.

11. 2. 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 the 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.

12. The application of the multifunctional composite bioprobe to in vitro detection described in claim 11, characterized in that after adding the multifunctional composite bioprobe to a test system, the method further includes the steps of binding the multifunctional composite bioprobe to a target substance in the test system, undergoing magnetic concentration and separation, and then determining the concentration of the target substance in the test system by Raman spectrum and / or fluorescence spectrum measurement.

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

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