Multifunctional composite biological probe and preparation method and application thereof

Abstract

The application belongs to the technical field of life science, and relates to a multifunctional composite biological probe and a preparation method and application thereof.The multifunctional composite biological probe comprises one or more than one of a noble metal SERS biological probe and one or more than one of a magnetic semiconductor SERS biological probe.The application realizes the functional superposition of the noble metal SERS biological probe and the magnetic semiconductor SERS biological probe in the simplest and most effective way, improves the sensitivity and accuracy of the detection method, and endows the detection method with the magnetic enrichment characteristic.

Description

Technical Field

[0001] This invention belongs to the field of life science technology, and relates to a multifunctional composite biological probe, its preparation method and application. Background Technology

[0002] 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. It shows immense application potential in optical detection fields, such as in vitro diagnostics and liquid biopsy. Currently, conventional SERS biological probes are divided into noble metal probes and semiconductor biological probes. The former offers high detection sensitivity, while the latter provides functional advantages, such as good signal stability and photoelectromagnetic response. To combine the advantages of both, noble metals and functional semiconductor materials are typically synthesized using chemical methods. However, this method is relatively complex and it is difficult to obtain composite materials with good homogeneity, severely limiting the application and development of noble metals and semiconductor materials in the SERS field. Summary of the Invention

[0003] This invention addresses the shortcomings of existing technologies by providing a multifunctional composite biological probe, its preparation method, and its applications.

[0004] One objective of this invention is achieved through the following technical solution:

[0005] A multifunctional composite biological probe, comprising one or more noble metal SERS biological probes and one or more magnetic semiconductor SERS biological probes.

[0006] Preferably, the particle size of the noble metal SERS biological probe is 0.1 nm to 10000 nm; more preferably, the particle size is 0.1 to 1000 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 magnetic semiconductor SERS biological probe has a particle size of 0.1 nm to 10000 nm; more preferably, the particle size is 0.1 to 1000 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 morphology of the noble metal SERS biological probe and the magnetic semiconductor SERS biological probe includes, but is not limited to, any one of the following: lamellar, tetrahedral, hexahedral, octahedral, dodecahedral, hollow cage-like, spherical granular, and rod-like.

[0009] Preferably, the precious metal SERS bioprobe includes a precious metal material with SERS properties. The precious metal material includes, but is not limited to, one or more of the following: single materials such as gold, silver, palladium, and copper, and composite materials. 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. Optionally, the particle size of the precious metal material is any one value or a range between any two values ​​from 0.5, 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500.

[0011] Preferably, the magnetic semiconductor SERS biological probe comprises a magnetic metal oxide with SERS properties. The magnetic metal oxide includes, but is not limited to, one or more oxide materials corresponding to Fe, Zn, Co, Ni, Cr, and Mn, and one or more composite materials containing the above materials. The aforementioned materials are: one or more oxide materials corresponding to Fe, Co, Ni, Cr, and Mn.

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

[0013] Preferably, the magnetic metal oxide is one or more of Zn, Co, Ni, Cr, and Mn combined with Fe oxide. Magnetic semiconductor SERS bioprobes constructed based on magnetic metal oxides formed by doping Fe oxides with one or more of Zn, Co, Ni, Cr, and Mn exhibit superior SERS enhancement effects, which is beneficial for improving detection sensitivity and accuracy.

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

[0015] Preferably, the method for preparing the magnetic metal oxide includes the following steps: mixing a solution of one or more of zinc salts, cobalt salts, nickel salts, chromium salts, manganese salts, and iron salts with a solution formed by an alkaline inorganic substance, a long alkyl chain organic acid, and a polar organic solvent; reacting to obtain initial magnetic particles; and then performing phase inversion to obtain magnetic metal oxide nanoparticles. The magnetic metal oxide nanoparticles prepared in this way exhibit SERS properties.

[0016] Iron salts include, but are not limited to, one or more of ferric chloride, ferrous chloride, ferrous sulfate, ferrous sulfate, ferrous ammonium sulfate, ferric 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; manganese salts include, but are not limited to, one or more of manganese chloride, manganese sulfate, and manganese nitrate; 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, methanol, ethanol, isopropanol, and other highly polar organic solvents.

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

[0018] 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.

[0019] Preferably, the volume ratio of a solution of one or more of zinc salts, cobalt salts, nickel salts, chromium salts, manganese salts, and iron salts to a solution formed by an alkaline 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, manganese salts, and iron salts 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.

[0021] Preferably, the reaction time is any one of 1, 2, 3, 5, 8, or 10 hours, or a range between any two values. Optionally, the reaction temperature is any one of 100, 200, 250, 300, 350, 400, 450, or 500 °C, or a range between any two values.

[0022] 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 1–50 h. Optionally, the stirring time is any one of 5, 8, 10, 15, or 20 h, or a range between any two values. Examples of organic solvents include chloroform, N,N-dimethylformamide, chloroform, carbon tetrachloride, formamide, DMSO, tetrahydrofuran, and pyridine.

[0023] By using a phase inversion reaction, nanoparticles are converted from the oil phase to the aqueous phase, thereby improving their dispersibility in water and enhancing their SERS performance.

[0024] Preferably, the noble metal SERS bioprobe comprises, from the inside out, a noble metal material, a Raman / fluorescent signal molecule, a biomolecule, and a targeting antibody protein; the magnetic semiconductor SERS bioprobe comprises, from the inside out, a magnetic metal oxide, a Raman / fluorescent signal molecule, a biomolecule, and a targeting antibody protein.

[0025] Preferably, the Raman / fluorescence signal molecule is a substance that has 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, Nell blue, and methylene blue.

[0026] Preferably, the biomacromolecules include, but are not limited to, one or more of polydopamine, dopamine hydrochloride, bovine serum albumin, reduced bovine serum albumin, and polyethylene glycol. Any biomacromolecule with biomacromolecule characteristics can be the biomacromolecule of the present invention.

[0027] Preferably, the targeted antibody protein is a protein or polypeptide that targets tumor markers. Tumor markers can include tumor cells, protein markers, exosomes, CtDNA, etc. Tumors can include breast cancer, liver cancer, lung cancer, esophageal cancer, etc. The proteins and polypeptides mentioned above can include folic acid antibody protein, Trop2 antibody protein, and GE11 polypeptide, etc., but are not limited to the above proteins and polypeptides. Any protein or polypeptide with tumor-targeting properties can be the targeted antibody protein of the present invention.

[0028] The Raman / fluorescent signal molecules and biological macromolecules in noble metal SERS biological probes and magnetic semiconductor SERS biological probes can be the same or different; however, the targeting antibody protein in both probes is the same substance. Thus, noble metal SERS biological probes and magnetic semiconductor SERS biological probes can target the same tumor markers.

[0029] Another objective of this invention is achieved through the following technical solution:

[0030] The preparation method of the multifunctional composite biological probe includes the following steps: mixing one or more of noble metal SERS biological probes with one or more of magnetic semiconductor SERS biological probes in a liquid or solid manner to obtain the multifunctional composite biological probe.

[0031] Another objective of this invention is achieved through the following technical solution:

[0032] The application of the multifunctional composite biological probe in in vitro detection includes the following steps: one or more of the noble metal SERS biological probes and one or more of the magnetic semiconductor SERS biological probes are mixed in liquid or solid form and then added to the test system; or one or more of the noble metal SERS biological probes and one or more of the magnetic semiconductor SERS biological probes are added to the test system in liquid or solid form sequentially.

[0033] Preferably, the application further includes: after adding the multifunctional composite biological probe to the test system, the multifunctional composite biological probe binds to the target substance in the test system, and after magnetic enrichment and separation, the concentration of the target substance in the test system is determined by Raman spectroscopy and / or fluorescence spectroscopy.

[0034] The excitation wavelength used in Raman spectroscopy and / or fluorescence spectroscopy detection is 266–1064 nm; preferably, the lower limit of the excitation wavelength is 266 nm, and the upper limit is selected from any one of 325, 488, 514, 532, 633, 647, 785, and 1064 nm; preferably, the excitation wavelength 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 objective of this invention is achieved through the following technical solution:

[0036] An in vitro detection device includes the aforementioned multifunctional composite biological probe. Examples of such in vitro detection devices include sensors, detectors, and spectral responders.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) In the multifunctional composite biological probe of the present invention, the noble metal SERS biological probe has the characteristic of high detection sensitivity, and the magnetic semiconductor SERS biological probe has the characteristic of magnetic enrichment. The noble metal SERS biological probe and the magnetic semiconductor SERS biological probe target tumor cells. The magnetic field can realize the rapid enrichment of nanoprobes and tumor cells, which is beneficial to improve the detection sensitivity and accuracy of tumor cells.

[0039] (2) Both noble metal SERS biological probes and magnetic semiconductor SERS biological probes can provide Raman spectral signals and fluorescence spectral signals, and improve the accuracy of the detection system through dual signal molecular modes; at the same time, the magnetic semiconductor SERS biological probes also have the ability to provide magnetic resonance imaging. Therefore, the combined use of noble metal SERS biological probes and magnetic semiconductor SERS biological probes can realize multimodal applications of Raman, fluorescence and nuclear magnetic resonance.

[0040] (3) Noble metal SERS bioprobes and magnetic semiconductor SERS bioprobes target and identify the same tumor cell. By Raman spectroscopy and / or fluorescence spectroscopy, the cells to be tested show two or more Raman and / or fluorescence signals at the same time, which can ensure the accuracy of tumor detection and eliminate false positive interference from blood cells in peripheral blood samples.

[0041] (4) The noble metal SERS biological probe and magnetic semiconductor SERS biological probe provided by the present invention are added to the test system in a simple mixing manner to react, avoiding the need to obtain noble metal-semiconductor composite materials through complex chemical synthesis. It has the characteristics of simple process, simplified equipment, low cost and safety.

[0042] (5) This invention achieves the functional superposition of noble metal SERS biological probe and magnetic semiconductor SERS biological probe in the simplest and most effective way, thereby improving the sensitivity and accuracy of the detection method and endowing the detection method with magnetic enrichment characteristics.

[0043] (6) The magnetic metal oxide in the magnetic semiconductor SERS bioprobe of the present invention is preferably one or more of Zn, Co, Ni, Cr, Mn and Fe oxide materials, which have a better SERS enhancement effect. Attached Figure Description

[0044] Figure 1 The TEM image of the noble metal gold nanomaterial prepared in Example 1;

[0045] Figure 2 The TEM image of the noble metal gold nanomaterial prepared in Example 2;

[0046] Figure 3 The TEM image of the noble metal gold nanomaterial prepared in Example 3;

[0047] Figure 4 Zn prepared in Example 4 0.2 Fe 2.8 TEM images of O4 magnetic nanoparticles;

[0048] Figure 5 Zn prepared in Example 5 0.2 Fe2.8 TEM images of O4 magnetic nanoparticles;

[0049] Figure 6 Zn prepared in Example 6 0.2 Fe 2.8 TEM images of O4 magnetic nanoparticles;

[0050] Figure 7 The surface-enhanced Raman spectra of 4MBA molecules on gold nanoparticles in Example 1 and Zn in Example 5 are shown. 0.2 Fe 2.8 Surface-enhanced Raman spectrum of crystal violet molecules on O4 magnetic nanoparticles; Figure 8 The fluorescence spectra of CCK-8, a surface-modified luminescent indicator of magnetic nanoparticles in Example 5, and the fluorescence spectra of CCK-8, a surface-modified luminescent indicator of noble metal gold nanomaterials in Example 1, are shown below.

[0051] Figure 9 The SERS spectra of the magnetic nanoparticles of Example 5 and the Fe3O4 magnetic nanoparticles of Comparative Example 1 with methylene blue molecules are shown.

[0052] Figure 10 Zn in Example 8 0.2 Fe 2.8 Enrichment and capture diagram of tumor cells by O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe;

[0053] Figure 11 The Au-IR783-rBSA-Trop2 antibody protein nanoprobe from Example 7 and the Zn from Example 8 0.2 Fe 2.8 SERS spectrum of O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe after binding to MCF7 breast cancer cells. Detailed Implementation

[0054] 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.

[0055] Example 1

[0056] Preparation of 3nm noble metal gold nanomaterials

[0057] 2 mL of 5 mM HAuCl4·4H2O solution was added to 7.85 mL of water and stirred at room temperature until homogeneous. Then, 0.15 mL of 0.1 M glutathione was added dropwise and stirring continued for 10 min. The mixture was then incubated in a 70°C water bath in the dark for 24 h. After standing for 4 days and allowing to cool naturally, the sample was centrifuged and washed three times each with water and ethanol. Finally, it was dried in a 70°C oven for 12 h to obtain 3 nm gold nanomaterials. Figure 1 As shown.

[0058] Example 2

[0059] Preparation of 10nm noble metal gold nanomaterials

[0060] 1 mL of 50 mM HAuCl4·4H2O solution and 49 mL of water were added to a round-bottom flask, stirred, and heated to boiling. 10 mL of 1% sodium citrate aqueous solution was quickly added, and heating was continued for 10 min. Heating was then stopped and the mixture was cooled to room temperature. The mixture was then allowed to stand for 4 days, allowed to cool naturally, centrifuged, and washed three times each with water and ethanol. Finally, it was dried in a 70°C oven for 12 h to obtain 10 nm gold nanomaterials. Figure 2 As shown.

[0061] Example 3

[0062] Preparation of 40nm noble metal gold nanomaterials

[0063] 1 mL of 50 mM HAuCl4·4H2O solution and 49 mL of water were added to a round-bottom flask, stirred, and heated to boiling. 2 mL of 1% sodium citrate aqueous solution was quickly added, and heating was continued for 10 min. Heating was then stopped and the mixture was cooled to room temperature. The mixture was then allowed to stand for 4 days, allowed to cool naturally, centrifuged, and washed three times each with water and ethanol. Finally, it was dried in a 70°C oven for 12 h to prepare 40 nm gold nanomaterials. Figure 3 As shown.

[0064] Example 4

[0065] Preparation of 4nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles

[0066] A solution was prepared by adding 1.73 mmol of ferrous ammonium 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. 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 8 hours. 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, 2g of citric acid and 20mL of oil-soluble magnetic nanoparticles were added to 30mL of a chloroform / DMF (v / v: 1 / 1) mixed solution, stirred for 12h, washed three times by centrifugation with ethanol, and dispersed in 20mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample was washed by centrifugation, three times each with water and ethanol, and finally dried in a 70℃ oven for 12h to obtain 4nm Zn. 0.2 Fe 2.8 O4 magnetic nanoparticles, such as Figure 4 As shown.

[0067] Example 5

[0068] Preparation of 7nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles

[0069] A solution was prepared by adding 1.73 mmol of ferrous ammonium 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. 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 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, 2g of citric acid and 20mL of oil-soluble magnetic nanoparticles were added to 30mL of a chloroform / DMF (v / v: 1 / 1) mixed solution, stirred for 12h, washed three times by centrifugation with ethanol, and dispersed in 20mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample was centrifuged and washed three times each with water and ethanol. Finally, it was dried in a 70℃ oven for 12h to obtain 7nm Zn. 0.2 Fe 2.8 O4 magnetic nanoparticles, such as Figure 5 As shown.

[0070] Example 6

[0071] Preparation of 10nm Zn 0.2 Fe 2.8 O4 magnetic nanoparticles

[0072] A solution was prepared by adding 1.73 mmol of ferrous ammonium 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. 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 24 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, 2g of citric acid and 20mL of oil-soluble magnetic nanoparticles were added to 30mL of a chloroform / DMF (v / v: 1 / 1) mixed solution, stirred for 12h, washed three times by centrifugation with ethanol, and dispersed in 20mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and naturally cooling, the sample was centrifuged and washed three times each with water and ethanol. Finally, it was dried in a 70℃ oven for 12h to obtain 10nm Zn. 0.2 Fe 2.8 O4 magnetic nanoparticles, such as Figure 6 As shown.

[0073] Using the noble metal gold nanomaterials of Example 1 and the magnetic nanoparticles of Example 5 as SERS substrates, SERS spectra of different concentrations of carboxymethylthiophenol (4MBA) and crystal violet (CV) were detected under an excitation wavelength of 633 nm. The results are as follows. Figure 7 As shown, where Figure 7 (a) is the surface-enhanced Raman spectrum of 4MBA molecules on gold nanoparticles in Example 1. Figure 7 (b) Zn from Example 5 0.2 Fe 2.8 The surface-enhanced Raman spectrum corresponding to crystal violet molecules on O4 magnetic nanoparticles; the noble metal gold nanomaterial of Example 1 has excellent SERS detection and imaging performance for low concentrations of 4MBA molecules; the magnetic nanoparticles of Example 5 have excellent SERS detection and imaging performance for low concentrations of crystal violet molecules.

[0074] The surfaces of the noble metal gold nanomaterials of Example 1 and the magnetic nanoparticles of Example 5 were modified with the luminescent indicator CCK-8, and the fluorescence emission patterns of the materials were obtained, as shown in the figure. Figure 8 As shown, where, Figure 8 (a) is the fluorescence spectrum of CCK-8, a magnetic nanoparticle surface-modified luminescent indicator from Example 5. Figure 8 (b) is the fluorescence spectrum of CCK-8, a surface-modified luminescent indicator of precious metal gold nanomaterials in Example 1.

[0075] Comparative Example 1

[0076] Preparation of Fe3O4 magnetic nanoparticles

[0077] 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 20 mL of water to obtain the desired water-soluble magnetic nanoparticles. After standing for 4 days and natural cooling, the sample was centrifuged and washed three times each with water and ethanol, and finally dried in a 70 °C oven for 12 h to prepare Fe3O4 magnetic nanoparticles.

[0078] Comparing the SERS detection and imaging capabilities of the magnetic nanoparticles of Example 5 and the Fe3O4 magnetic nanoparticles of Comparative Example 1, the magnetic nanoparticles were used as SERS substrates under an excitation wavelength of 532 nm to detect magnetic nanoparticles with 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 9 As shown, Zn-doped nanoparticles exhibit a better SERS enhancement effect. This is likely because Zn doping provides more orbital energy levels, which is beneficial for charge transfer between the material and molecules, thereby improving the SERS effect. Based on Zn... 0.2 Fe 2.8 Magnetic semiconductor SERS bioprobes formed from O4 magnetic nanoparticles, together with noble metal SERS bioprobes, can be used for in vitro detection, resulting in higher detection sensitivity and better accuracy.

[0079] Example 7

[0080] Preparation of Au-IR783-rBSA-Trop2 antibody protein nanoprobe

[0081] (1) Preparation of noble metal gold nanomaterials

[0082] 2 mL of 5 mM HAuCl4·4H2O solution was added to 7.85 mL of water and stirred at room temperature until homogeneous. 0.15 mL of 0.1 M glutathione was added dropwise and stirring was continued for 10 min. The mixture was then placed in a 70 °C water bath in the dark for 24 h. After standing for 4 days and cooling naturally, the sample was centrifuged and washed three times each with water and ethanol. Finally, it was dried in a 70 °C oven for 12 h to obtain 3 nm gold nanomaterials.

[0083] (2) Preparation of Au-IR783 nanoparticles

[0084] 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 to obtain Au-IR783 solution.

[0085] (3) Preparation of Au-IR783-rBSA nanoparticles

[0086] 18 ml of Au-IR783 solution, 8 ml of CH3CH2OH, and 600 μl of NH3·H2O were mixed and stirred with a polytetrafluoroethylene rod for 20 min. 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 to prepare Zn. 0.2 Fe 2.8 O4-IR780-rBSA solution.

[0087] (4) Preparation of Au-IR783-rBSA-Trop2 antibody protein nanoparticles

[0088] Take 4 ml of Au-IR783-rBSA solution, and use a magnet to adsorb Au-IR783-rBSA nanoparticles. Discard the supernatant, then add 4 ml of Tris-HCl solution (10 mM, pH = 8.5), followed by 40 μg of Trop2 antibody protein. Stir at room temperature for 12 h, wash three times with PBS, and finally disperse in 4 ml of PBS solution to obtain the Au-IR783-rBSA-Trop2 antibody protein bioprobe.

[0089] Example 8

[0090] Preparation of Zn 0.2 Fe 2.8 O4-Alizarin Red-PDA-Trop2 Antibody Protein Nanoprobe

[0091] (1)Zn 0.2 Fe 2.8 Preparation of O4 magnetic nanoparticles

[0092] 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 Zn. 0.2 Fe 2.8 O4 magnetic nanoparticle solution.

[0093] (2)Zn 0.2 Fe 2.8 Preparation of O4-Alizarin Red Nanoparticles

[0094] Add 150 μl of 1 mmol / L alizarin red ethanol solution to 15 mL of magnetic nanoparticle solution (solvent: water, concentration: 0.21 mg / mL), stir with a polytetrafluoroethylene rod for 2 h, wash thoroughly with deionized water, and finally disperse in 18 mL of deionized water to obtain Zn. 0.2 Fe 2.8 O4-Alizarin Red Solution.

[0095] (3)Zn 0.2 Fe 2.8 Preparation of O4-Alizarin Red-PDA Nanoparticles

[0096] 18ml Zn 0.2 Fe 2.8 ZnO4-Alizarin Red solution (solvent: water, concentration: 0.19 mg / ml) was mixed with 8 ml of CH3CH2OH and 600 μl of NH3·H2O, 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 ZnO4-Alizarin Red. 0.2 Fe 2.8 O4-Alizarin Red-PDA Nanoparticle Solution.

[0097] (4)Zn 0.2 Fe 2.8 Preparation of O4-Alizarin Red-PDA-Trop2 Antibody Protein Nanoparticles

[0098] Take 4ml of Zn 0.2 Fe 2.8Zn was obtained by magnetic adsorption from O4-Alizarin Red-PDA nanoparticle solution. 0.2 Fe 2.8 O4-Alizarin Red-PDA nanoparticles were collected, the supernatant was discarded, and then 4 ml of Tris-HCl solution (10 mM, pH = 8.5) was added. Following this, 40 μg of Trop2 antibody protein was added, and the mixture was stirred at room temperature for 12 h. The nanoparticles were washed three times with PBS and finally dispersed in 4 ml of PBS to obtain Zn. 0.2 Fe 2.8 O4-Alizarin Red-PDA-40μg Trop2 antibody protein solution.

[0099] Zn from Example 8 0.2 Fe 2.8 O4-Alizarin Red-PDA-40μg Trop2 antibody protein nanoprobe was co-incubated with tumor cells, and then magnetically enriched using the magnetic enrichment module of a circulating tumor cell detection device. The enrichment effect was as follows: Figure 10 As shown, tumor cells and Zn 0.2 Fe 2.8 The O4 nanoprobe exhibits 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 nanoprobes have a good ability to enrich and capture tumor cells.

[0100] The Au-IR783-rBSA-Trop2 antibody protein nanoprobe from Example 7 was co-incubated with MCF7 breast cancer cells, followed by SERS spectroscopy detection. The SERS spectrum is shown below. Figure 11 As shown; Zn in Example 8 0.2 Fe 2.8 O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe was co-incubated with MCF7 breast cancer cells alone, followed by SERS spectroscopy detection. The SERS spectrum is shown below. Figure 11 As shown; the Au-IR783-rBSA-Trop2 antibody protein nanoprobe from Example 7 and the Zn from Example 8 were used. 0.2 Fe 2.8 O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobe was co-incubated with MCF7 breast cancer cells, magnetically enriched and separated, and then subjected to SERS spectroscopy detection. The SERS spectrum is shown below. Figure 11 As shown.

[0101] from Figure 11 As can be seen, using only a single probe to detect cancer cells results in fewer and unstable SERS signal peaks with poor uniformity; while using Au-IR783-rBSA-Trop2 antibody protein nanoprobes and the Zn from Example 8... 0.2Fe 2.8 The O4-Alizarin Red-PDA-Trop2 antibody protein nanoprobes are used together to detect cancer cells. The SERS signal has multiple peaks and the signal is stable, which helps to improve the detection sensitivity and accuracy.

[0102] 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.

[0103] 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.

[0104] 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 multifunctional complex biological probe, characterized by, one or more of the noble metal SERS biological probes and one or more of the magnetic semiconductor SERS biological probes; the noble metal SERS biological probes comprise a noble metal material with SERS performance, the noble metal material comprising one or more of gold, silver, palladium, copper and a composite material containing gold, silver, palladium or copper; The magnetic semiconductor SERS biological probe comprises a magnetic metal oxide with SERS performance, the magnetic metal oxide is Zn x Fe 3-x O4, wherein 0 < x < 3; The Zn x Fe 3-x The preparation method of Zn x Fe 3-x O4 nanoparticles the phase inversion comprises the following steps: adding the initial magnetic particles and citric acid into a mixed solution of chloroform and DMF, stirring at room temperature for 1-50 h.

2. The multifunctional composite bioprobe according to claim 1, wherein the particle size of the noble metal SERS biological probes is 0.1 nm-10000 nm, and the particle size of the magnetic semiconductor SERS biological probes is 0.1 nm-10000 nm.

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

4. The multifunctional complex biological probe according to claim 1, wherein the iron salt is one or more of ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, ferrous ammonium sulfate, ferric nitrate and ferrous nitrate; the zinc salt is one or more of zinc chloride, zinc sulfate and zinc nitrate; the basic inorganic substance is one or more of barium hydroxide, potassium hydroxide, calcium hydroxide, sodium hydroxide and ammonia water; the long-chain alkyl organic acid is one or more of oleic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid and dodecanoic acid; and the polar organic solvent is one or more of methanol, ethanol and isopropanol.

5. The multifunctional composite bioprobe according to claim 1, wherein the noble metal SERS biological probes comprise, from inside to outside, a noble metal material, a Raman / fluorescent signal molecule, a biological macromolecule and a targeting antibody protein; and the magnetic semiconductor SERS biological probes comprise, from inside to outside, a magnetic metal oxide, a Raman / fluorescent signal molecule, a biological macromolecule and a targeting antibody protein.

6. The multifunctional composite bioprobe according to claim 5, wherein the Raman / fluorescent signal molecule is one or more of IR783, IR780, 3,3'-diethylthiatri-carbocyanine iodide, rhodamine, crystal violet, alizarin red, Nile blue and methylene blue; the biological macromolecule is one or more of polydopamine, dopamine hydrochloride, bovine serum albumin, reduced bovine serum albumin and polyethylene glycol; the targeting antibody protein is a protein or polypeptide targeting a tumor marker, and the tumor marker is a tumor cell, a protein marker, an exosome or CtDNA.

7. The method for preparing a multifunctional composite biological probe as described in claim 1, characterized in that, comprises the following steps: one or more of the noble metal SERS biological probes and one or more of the magnetic semiconductor SERS biological probes are mixed in liquid or solid form to obtain a multifunctional composite biological probe.

8. The use of a multifunctional complex biological probe according to claim 1 in in vitro detection, characterized by, comprises the following steps: one or more of the noble metal SERS biological probes and one or more of the magnetic semiconductor SERS biological probes are mixed in liquid or solid form and then added to the system to be tested; or one or more of the noble metal SERS biological probes and one or more of the magnetic semiconductor SERS biological probes are added to the system to be tested in liquid or solid form in sequence.

9. Use of a multifunctional composite biological probe according to claim 8 in in vitro detection, characterized by, further comprises the following steps: After the multifunctional composite biological probe is added into the system to be detected, the multifunctional composite biological probe is combined with the target in the system to be detected, and then the target is enriched and separated by magnetism, and the concentration of the target in the system to be detected is determined by Raman spectrum and / or fluorescence spectrum detection.

10. An in vitro detection device, characterized in that The multifunctional composite biological probe comprises the multifunctional composite biological probe of claim 1.

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