A "kiwifruit"-type magnetic fluorescent nanospheres and its preparation method

Abstract

The present invention discloses a "kiwifruit"-type magnetic fluorescent nanosphere and a preparation method thereof. The preparation method is as follows: hydrolyze a silica shell layer on the surface of Fe3O4 nanoparticles in a bicontinuous microemulsion, then perform thiolation modification, assemble CdSe / CdS / ZnS quantum dots (QDs) to form a Fe3O4@dSiO2 / rQDs microsphere assembly, subsequently carry out a hydrolysis and condensation reaction through OTMS to realize the phase transfer process of the microsphere assembly from the organic phase to the aqueous phase, and then grow and deposit silica by the Stöber method to encapsulate the pores of the silica shell layer of the microsphere assembly, thereby preparing the "kiwifruit"-type magnetic fluorescent nanosphere. The present invention uses a magnetic dendritic mesoporous silica microsphere as a carrier. Firstly, due to its large number of pores providing a large amount of internal space for the assembly of quantum dots, compared with fluorescent microspheres with quantum dots embedded in the outer layer, it has a larger specific surface area, can load more quantum dots, and has a stronger fluorescence intensity.

Description

Technical field

[0001] The present invention relates to a "kiwifruit"-type magnetic fluorescent nanomicrosphere and a preparation method thereof. Background technique

[0002] Nanobioconjugate tags are key elements in the development of nanobiosensors, which involve conjugating biomolecules to artificial nanostructures, combining the excellent recognition properties of biomolecules with the signal generation and signal enhancement capabilities of artificial nanostructures. Currently, various types of biomolecules have been used as biorecognition elements in biosensing, including antibodies (Abs), aptamers, DNA oligonucleotides / peptide nucleic acids (PNAs) / locked nucleic acids (LNAs) probes, molecular beacons, and even whole cells. Among them, Abs are the most widely used recognition elements in biosensors, especially in rapid detection. For example, lateral flow immunoassays (LFIAs), a paper-based point-of-care testing platform, utilize the specific recognition ability of antigen-antibody to detect targets quickly and sensitively, and are one of the main platforms for antibody-based immunoassays. Due to the low cost, simple operation, and fast detection speed of LFIAs, and the ability to directly obtain detection results with the naked eye, they have received extensive attention and are currently the most widely used POC sensors in diagnostics (such as pregnancy testing, COVID-19 monitoring), the environment (such as pesticide, heavy metal, and bacteria monitoring), and food safety applications (such as foodborne allergens and pathogens).

[0003] Currently, most commercial LFIA technologies mainly rely on the use of colloidal gold or dye beads as markers for qualitative or semi - quantitative colorimetric detection, which can rapidly generate optical signals directly observable by the naked eye. However, these are not applicable to the situation where the target analyte exists at ultra - low concentrations, mainly due to their low molar absorption coefficients, which means that a large accumulation of nanoparticles is required on the test line to generate a measurable optical signal, thus limiting their further application in fields requiring high sensitivity and quantitative detection. Currently, fluorescence detection has become a common method to improve the sensitivity of LFIAs. Quantum dots (QDs) have become one of the most commonly used markers due to their narrow emission peaks and high quantum yields. In such a system, the absorbance or emission of the T - line can be measured to calculate the binding concentration of the analyte, and the emitted light is usually measured. The reason for this is that fluorescence measurements are more sensitive, mainly due to the way they are measured. Absorbance is measured against a bright background, enabling the detection of low - intensity light. However, many biological applications often occur in very complex matrices (serum, whole blood, cerebrospinal fluid, etc.), which may significantly affect the properties of the nanoparticles. Superparamagnetic magnetic nanospheres (MNPs) are often used for separation and enrichment in detection due to their excellent magnetic properties. They can not only improve the sensitivity of immunoassays through enrichment, but also reduce matrix interference, improve accuracy, and expand the application of LFIAs in the detection of targets in complex biological matrices. Based on these excellent properties, integrating both QDs and MNPs into a single nanobiosensor to prepare a magnetic fluorescent nano - quantum dot microsphere will significantly improve the sensitivity and accuracy of the test strip in detection.

[0004] Fluorescent / magnetic nanospheres based on nanoparticles (NPs, including QDs and MNPs) are mainly constructed by four methods: (Ⅰ) embedding NPs into nanospheres; (II) doping NPs during the formation of nanospheres; (Ⅲ) assembling NPs on the surface of nanospheres; (Ⅳ) in - situ generating NPs within the pores of nanospheres. Insin et al. prepared core - shell magnetic fluorescent microspheres with silica spheres as the core and QDs and Fe2O3 embedded in the outer layer by using a polymerization reaction of silica spheres with Fe2O3 and QDs in a stober system. Huang et al. prepared magnetic fluorescent microspheres by covalently coupling QDs on the surface of magnetic beads; Song et al. used polystyrene / acrylamide polymer microspheres as templates, mixed QDs and Fe2O3 nanoparticles in a chloroform / butanol mixture, and prepared magnetic fluorescent microspheres by ultrasonic assembly for 30 min. Wang et al. used Fe3O4 as the core, coated a layer of dendritic mesoporous silica on its surface, then used this radially magnetic dendritic mesoporous silica nanosphere as a template, loaded QDs in its pores, and finally through The method continues to coat silica, and finally a kind of magnetic fluorescent nanospheres is prepared. However, in the synthesis process of these magnetic fluorescent nanoparticles, there are problems such as the uncontrollable amount of MNPs and QDs embedded or assembled, or the too small loading amount, resulting in random magnetic strength, and the too thick silicon layer coated on the outer layer of the magnetic core greatly shielding the magnetism of the magnetic fluorescent microspheres, etc.

[0005] Due to the uncertainty of the number of particles of MNPs and QDs in this randomly embedded or assembled magnetic fluorescent nanoparticles, the fluorescence intensity and magnetic intensity are unstable, which may affect the analytical performance of the magnetic fluorescent nanoparticles. And this kind of magnetic dendritic mesoporous silica nanospheres with a radial shape centered on magnetic nanoparticles can regulate its magnetic properties by adjusting the thickness of the outer silica. The present invention uses a magnetic dendritic mesoporous silica composite nanomaterial with the thickness of the outer dendritic silicon regulated as a carrier. Utilizing its vertical central radial pore channels and a large amount of internal space, it can provide a large number of loading surfaces for functional nanobuilding blocks, thereby assembling QDs into its pore channels, and finally through The method encapsulates the pore channels to synthesize a kind of "kiwifruit"-type magnetic fluorescent nanospheres. Summary of the Invention

[0006] Aiming at the above technical problems existing in the prior art, the purpose of the present invention is to provide a kind of "kiwifruit"-type magnetic fluorescent microspheres composed of Fe3O4, quantum dots, and dendritic silica microsphere templates, and to provide a preparation method of magnetic fluorescent microspheres with excellent fluorescence performance, good biocompatibility, and adjustable silicon layer size and pore size, using a dendritic silica-coated Fe3O4 composite nanomaterial as a carrier and loading quantum dot signal building blocks.

[0007] In the prior art, immunochromatographic test strips have been widely used in point-of-care testing in fields such as environmental monitoring, food detection, and in vitro diagnosis due to their advantages of simple operation, rapidity, portability, and low cost. Currently, commercially available immunochromatographic test strips mainly rely on colloidal gold colorimetric test strips, which mostly suffer from low sensitivity and poor quantification ability, and are insufficient to meet the requirements of in vitro diagnosis for high sensitivity. Secondly, this technology still faces challenges in detecting trace analytes in complex samples. The matrix interference effect of complex samples and the low concentration of analytes in the samples are the main factors restricting the application of immunochromatographic detection. QDs and MNPs each have their unique advantages in test strip detection. QDs have high fluorescence intensity, while MNPs can effectively enrich and purify analytes, which is particularly important for the analysis of trace analytes in complex samples. Currently, detecting trace analytes in complex samples is the main problem faced by current test strip detection. Therefore, how to utilize the advantages of the two materials in test strip detection is an important and key research topic. The present invention is to use Fe3O4 as the magnetic core and coat a layer of dendrimer mesoporous silica with adjustable size on its outer layer, aiming to reduce the magnetic shielding effect of the silica layer on the magnetic core by regulating the silica layer thickness of the dendrimer mesoporous silica, thereby significantly increasing its magnetism, facilitating magnetic enrichment and recovery, and using the internal space of the dendrimer mesoporous silica to achieve high-density loading of quantum dots, so as to improve its fluorescence performance. Finally, through a method to encapsulate the pores to prepare a "kiwifruit"-type magnetic fluorescent nanosphere with high luminescence performance.

[0008] The technical solution adopted by the present invention is as follows:

[0009] The preparation method of the "kiwifruit"-type magnetic fluorescent nanosphere disclosed by the present invention is: using a radially magnetic dendritic mesoporous silica nanosphere as a template and loading QDs at a high density in its pores, aiming to provide a preparation method for a magnetic fluorescent material with uniform size, high fluorescence intensity, and good biocompatibility. First, superparamagnetic Fe3O4 with the required size and good dispersibility was prepared by a solvothermal method to ensure its good magnetic properties and the smooth preparation of the next template. Subsequently, Fe3O4 was synthesized into a radially magnetic dendritic mesoporous silica nanosphere (Fe3O4@dSiO2) in a bicontinuous microemulsion, and then surface-modified with mercapto silane (MPS). The oil-phase QDs were loaded under ultrasonic assistance by using the coordination effect of QD-SH to obtain a microsphere assembly Fe3O4@dSiO2 / QDs. Then, the phase transfer process of the Fe3O4@dSiO2 / QDs microsphere assembly from the organic phase to the aqueous phase was realized by hydrolysis and condensation of octyltrimethoxysilane. Then, a silica shell layer was grown on the surface by hydrolysis of tetraethyl orthosilicate to prepare a "kiwifruit"-type magnetic fluorescent nanosphere (FMS).

[0010] The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere is characterized by preparing superparamagnetic nano-Fe3O4, and then hydrolyzing a silica shell layer on the surface of Fe3O4 nanoparticles in a bicontinuous microemulsion to form Fe3O4@dSiO2 microspheres; then performing mercapto modification on the surface of Fe3O4@dSiO2, using CdSe / CdS / ZnS quantum dots QDs as the quantum dots to be assembled, and loading the quantum dots by the coordination of the mercapto groups on Fe3O4@dSiO2 with the metals in the quantum dots to realize the assembly of the quantum dots to form an Fe3O4@dSiO2 / rQDs microsphere assembly. Subsequently, hydrolysis and condensation reaction occurs on the surface of the Fe3O4@dSiO2 / rQDs microsphere assembly by n-octyltrimethoxysilane OTMS to obtain Fe3O4@dSiO2 / rQDs modified with organosilane on the surface. Then, a silica shell layer is grown by hydrolysis on its surface with tetraethyl orthosilicate TEOS (that is, by growing and depositing silica by this method to encapsulate the pores of the silica shell layer of the microsphere assembly), and then successively performing amino modification with 3-aminopropyltriethoxysilane APTES and carboxyl modification with succinic anhydride to complete the preparation.

[0011] The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere is characterized in that the preparation method of the superparamagnetic nano-Fe3O4 is as follows: Dissolve FeCl3·6H2O and sodium citrate dihydrate in ethylene glycol, add a mixed solution of water and sodium acetate and stir evenly, and then react at 180 - 220 °C for 8 - 12 h, cool, separate out the precipitate, and wash the precipitate several times with ethanol and water; the volume ratio of ethylene glycol to water in the mixed solution is 40 - 45:1, the mass ratio of FeCl3·6H2O, sodium citrate dihydrate and sodium acetate is 1:0.3 - 0.5:2 - 2.5, and the mass-volume concentration of FeCl3·6H2O in the mixed solution is 0.02 - 0.03 g / mL.

[0012] The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere is characterized in that the preparation method of Fe3O4@dSiO2 includes the following steps:

[0013] 1) Dissolve CTAB and urea in water to form an aqueous phase, dissolve n-pentanol and TEOS in cyclohexane to form an oil phase, add the oil phase and superparamagnetic nano-Fe3O4 to the aqueous phase, and stir vigorously to mix the aqueous phase and the oil phase to form a bicontinuous microemulsion. Subsequently, the whole mixed solution is refluxed in a water bath at 65 - 75 °C for 14 - 18 h to react to form Fe3O4@dSiO2 microspheres;

[0014] 2) After the reaction is completed, the precipitate is collected by magnetic separation and washed several times with ethanol; then, the precipitate is refluxed in acetone to remove CTAB in the pores of silica; finally, Fe3O4@dSiO2 is collected by centrifugation, washed several times with ethanol, and stored in ethanol for later use.

[0015] The preparation method of the "kiwifruit"-type magnetic fluorescent nanospheres, characterized in that in step 1), the volume ratio of the aqueous phase to the oil phase is 1:0.8 - 1.2, preferably 1:1; the mass-volume concentrations of CTAB and urea in the aqueous phase are 0.06 - 0.07 g / mL and 0.015 - 0.025 g / mL respectively; the feeding ratio of the superparamagnetic nano-Fe3O4 to TEOS is 0.1 g:0.5 - 0.6 mL, preferably 0.1 g:0.6 mL; the volume ratio of n-pentanol to TEOS is 0.6 - 1:1, preferably 0.83 - 0.85:1; the concentration of the superparamagnetic nano-Fe3O4 in the aqueous phase is 2 - 2.5 mg / mL.

[0016] The preparation method of the "kiwifruit"-type magnetic fluorescent nanospheres, characterized in that the preparation method of the Fe3O4@dSiO2 / rQDs microsphere assembly includes the following steps:

[0017] S1: Add ammonia water and (3-mercaptopropyl)trimethoxysilane to the ethanol solution of Fe3O4@dSiO2 microspheres, then stir and react at room temperature, centrifuge to collect the precipitate, wash to obtain mercapto-functionalized Fe3O4@dSiO2;

[0018] S2: Take the mercapto-functionalized Fe3O4@dSiO2 and add it to the solution of CdSe / CdS / ZnS quantum dots QDs, sonicate to be uniform, then centrifuge to remove the supernatant, wash to remove the excess QDs, and obtain the Fe3O4@dSiO2 / rQDs microsphere assembly.

[0019] The preparation method of the "kiwifruit"-type magnetic fluorescent nanospheres, characterized in that in step S1, the Fe3O4@dSiO2 microspheres account for 0.5 - 1.0% of the total mass of the reaction system, the volume fraction of the ammonia water in the total reaction system is 1.2 - 1.5%, the volume fraction of the (3-mercaptopropyl)trimethoxysilane in the total reaction system is 0.8 - 1.2%, and the total stirring reaction time is 10 - 14 h; in step S2, the concentration of the CdSe / CdS / ZnS quantum dots QDs solution is 8 - 12 mg / mL, and the mass ratio of the mercapto-functionalized Fe3O4@dSiO2 to the CdSe / CdS / ZnS quantum dots QDs is 1:0.8 - 1.2.

[0020] The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere is characterized in that hydrolysis and condensation reaction occurs on the surface of the Fe3O4@dSiO2 / rQDs microsphere assembly through n-octyltrimethoxysilane (OTMS). The specific process is as follows:

[0021] Step A: Add n-octyltrimethoxysilane (OTMS) to the Fe3O4@dSiO2 / rQDs microsphere assembly. After ultrasonic mixing, add methanol and ammonia water. Ultrasonic mixing for 20 - 60 min is carried out to transfer the microsphere assembly from the oil phase to the water phase. After centrifugation, remove the supernatant, and wash once with methanol to remove the excess OTMS;

[0022] In Step A, the volume ratio of n-octyltrimethoxysilane (OTMS), methanol, and ammonia water is 1:70 - 80:1.5 - 2;

[0023] Step B: Further add ultrapure water and ammonia water with a volume ratio of 450 - 550:1 to the precipitate, stir and react at room temperature for 15 - 20 h. After the reaction ends, wash several times with ethanol to obtain Fe3O4@dSiO2 / rQDs modified with surface organosilane.

[0024] The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere is characterized in that the processes of growing a silica shell layer, amino modification, and carboxyl modification include the following steps:

[0025] (1) Add the Fe3O4@dSiO2 / rQDs modified with surface organosilane to the system and stir and react to obtain Fe3O4@dSiO2 / rQDs coated with silica. Then wash the product with ethanol;

[0026] (2) Disperse the Fe3O4@dSiO2 / rQDs coated with silica in ethanol, then add ammonia water and 3-aminopropyltriethoxysilane. After stirring and reacting at room temperature for 10 - 15 h, centrifuge and purify with ethanol to obtain amino-modified microspheres. Then add them to an organic solution containing succinic anhydride and stir at room temperature for 20 - 30 h. After centrifugation and washing several times with ethanol, carboxyl-modified microspheres are obtained, and the preparation is completed.

[0027] The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere is characterized in that the reaction system method is as follows: Disperse the reactants in a mixture of ethanol, water, and ammonia water. Based on the total volume of the mixture, add 0.7 - 1.0 μL / mL of tetraethyl orthosilicate every 1 h, and add it in several times. The number of addition times can be 2 - 8 times, and it is further preferably 3 - 4 times.

[0028] Compared with the prior art, the beneficial effects obtained by the present invention are:

[0029] 1. A magnetic dendritic mesoporous silica is prepared by an improved reverse microemulsion method. Using well-dispersed Fe3O4 as the magnetic core, by adjusting the amounts of fatty alcohol (n-pentanol) and TEOS, the thickness and pore diameter of the silica shell on the surface of Fe3O4@dSiO2 can be regulated. By adjusting the thickness of the dendritic mesoporous silica microspheres on the surface of Fe3O4, the magnetic properties of the template can be regulated. When processing sample solutions with complex matrices, the interference matrix can be maximally removed and the time for magnetic separation of the sample can be shortened, thus improving the detection efficiency.

[0030] 2. The present invention provides a method for preparing magnetic fluorescent microspheres based on oil-phase quantum dots, using radially magnetic dendritic mesoporous silica nanospheres (Fe3O4@dSiO2) as carriers. The prepared microspheres have the characteristics of good stability, good magnetism, excellent fluorescence performance, and easy synthesis. By loading oil-phase quantum dots into the pores of the dendritic mesoporous silica, the purpose of fluorescence enhancement can be achieved. Using magnetic dendritic mesoporous silica microspheres as carriers, firstly, due to their large number of pores providing a large amount of internal space for the assembly of quantum dots, compared with fluorescent microspheres with quantum dots embedded in the outer layer, they have a larger specific surface area, can load more quantum dots, and have stronger fluorescence intensity.

[0031] 3. Since a large number of quantum dots can be uniformly loaded on the template through the coordination of thiol-metal, there is no need to modify the surface of the quantum dots, thus maximizing the retention of the fluorescence characteristics of the quantum dots. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the synthesis process of the magnetic fluorescent microspheres (FMS) of the present invention and their subsequent carboxylation modification;

[0033] Figure 2 Comparison results of scanning electron microscope images of magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in step 2 of Example 1, magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in step 2 of Example 2, magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in step 2 of Example 3, and magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in step 2 of Example 4;

[0034] Figure 3Comparison results of transmission electron microscope images of the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 1, the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 2, the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 3, and the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 4;

[0035] Figure 4 Comparison results of transmission electron microscope images of Fe3O4 synthesized in Step 1 of Example 4, the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2, Fe3O4@dSiO2 / rQDs synthesized in Step 3, and FMS synthesized in Step 3;

[0036] Figure 5 Comparison results of scanning electron microscope images of Fe3O4 synthesized in Step 1 of Example 4, the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2, Fe3O4@dSiO2 / rQDs synthesized in Step 3, and FMS synthesized in Step 3;

[0037] Figure 6 Fluorescence spectra and photographs (insets) of rQDs and FMS obtained in Example 4.

[0038] Figure 7 Hysteresis curves of Fe3O4, Fe3O4@dSiO2, and FMS obtained in Example 4, and photographs of the FMS aqueous dispersion under magnetic attraction;

[0039] Figure 8 Results of the relationship between the rQDs loading ratio and the amount of rQDs added for Fe3O4@dSiO2 synthesized in Step 2 of Example 5. Detailed implementation manners

[0040] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0041] Example 1:

[0042] 1. Synthesis of Fe3O4

[0043] Some modifications were made to the synthesis of Fe3O4. Specifically, first, 0.81 g of FeCl3·6H2O and 0.318 g of sodium citrate dihydrate were dissolved in 30 mL of ethylene glycol solution, then 0.7 mL of water and 1.8 g of sodium acetate were added and stirred for another 30 minutes. Finally, the solution was placed in a 50 mL reaction kettle and reacted at 200 °C for 10 h. After the reaction kettle cooled down, the obtained precipitate was washed three times with ethanol and water respectively and stored in water for later use.

[0044] 2. Synthesis of Fe3O4@dSiO2 Template

[0045] First, 3 g of CTAB and 0.9 g of urea were dissolved in 45 mL of water and placed under mechanical stirring. Then, 100 mg of Fe3O4 and 45 mL of a cyclohexane solution containing 0.3 mL of n-pentanol and 0.5 mL of TEOS were added, and the mixture was vigorously stirred for 1 h. Subsequently, the entire mixed solution was refluxed in a 70 °C water bath for 16 h. After the reaction ended, the precipitate was collected by magnetic separation and washed three times with ethanol. Then, the precipitate was refluxed in an 80 °C acetone solution for 48 h to completely remove CTAB in the dendritic silica pores. Finally, Fe3O4@dSiO2 was collected by centrifugation, washed three times with ethanol, and stored in 20 mL of ethanol solution for later use.

[0046] 3. Synthesis of Magnetic Fluorescent Nanospheres (FMS)

[0047] First, 0.25 mL of ammonia water and 0.2 mL of (3-mercaptopropyl)trimethoxysilane (MPTMS) were added to 20 mL of the above-prepared Fe3O4@dSiO2 ethanol solution (containing approximately 120 mg of Fe3O4@dSiO2), and the mixture was stirred at 300 rpm at room temperature for 12 h to prepare thiolated Fe3O4@dSiO2. After the reaction ended, the precipitate was collected by centrifugation and washed three times with ethanol. Subsequently, 10 mg of the Fe3O4@dSiO2 precipitate was added to 1.0 mL of a 10 mg / mL fluorescent CdSe / CdS / ZnS QDs (rQDs) chloroform solution, and the mixture was sonicated for 5 minutes to obtain a uniformly dispersed solution. The Fe3O4@dSiO2 / rQDs precipitate was obtained by centrifugation at 10000 rpm for 5 minutes and washed once with chloroform to remove the excess rQDs.

[0048] Next, under ultrasonic conditions, 16 mg of the obtained Fe3O4@dSiO2 / rQDs precipitate was dispersed in 100 μL of octyltrimethoxysilane (OTMS), and then a mixture containing 7.5 mL of methanol and 187.5 μL of ammonia water was added, and sonication was continued for 30 minutes. The precipitate was collected by centrifugation at 10000 rpm and washed once with methanol to remove the excess OTMS. Subsequently, it was dispersed in a mixture of 16.5 mL of water and 33 μL of ammonia water and stirred for 18 h to deposit a thin silica layer. After centrifugation, 20 mg of the obtained precipitate was redispersed in a mixture of 10 mL of ethanol, 2.5 mL of water, and 312.5 μL of ammonia water, and 10 μL of TEOS was injected into it every 1 h to continue to coat silica to encapsulate the pores of the silica shell of FMS. After 3 h, a total of 30 μL of TEOS was added, and then FMS was collected by centrifugation and washed three times with ethanol.

[0049] 4. Preparation of Carboxylated FMS Microspheres

[0050] The carboxylation of FMS was carried out according to the following steps. First, the obtained FMS precipitate (20 mg) was redispersed in a mixed solution of 20 mL of ethanol, 0.5 mL of ammonia water, and 20 μL of 3-aminopropyltriethoxysilane (APTES), and stirred at room temperature for 12 h to prepare amino-functionalized FMS. After the reaction, the precipitate was centrifuged, washed with ethanol, and then redispersed in 10 mL of N,N-dimethylformamide (DMF) solution containing 0.1 mol / L succinic anhydride, and stirred at room temperature for 24 h. Finally, the carboxylated FMS was collected by centrifugation, washed 3 times with ethanol, and finally dispersed in ultrapure water for standby.

[0051] The scanning electron microscope image and transmission electron microscope image of the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 1 are shown in Figure 2 2(a) in Figure 3 and Figure 2 (a) It can be seen that the pore diameter of Fe3O4@dSiO2 is about 5 - 8 nm, Figure 3 (a) It can be seen that the shell thickness of Fe3O4@dSiO2 is about 27 nm, and the size is about 254 nm.

[0052] Example 2: The operation of Example 2 was repeated as in Example 1, except that "in the synthesis of the Fe3O4@dSiO2 template in Step 2, the amount of n-pentanol was adjusted to 0.4 mL", and the rest of the operations were the same as in Example 1.

[0053] The scanning electron microscope image and transmission electron microscope image of the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 2 are shown in Figure 2 2(b) in Figure 3 and Figure 2 (b) It can be seen that the pore diameter of Fe3O4@dSiO2 is about 8 - 10 nm, Figure 3 (b) It can be seen that the shell thickness of Fe3O4@dSiO2 is about 30 nm, and the size is about 260 nm.

[0054] Example 3: The operation of Example 3 was repeated as in Example 1, except that "in the synthesis of the Fe3O4@dSiO2 template in Step 2, the amount of n-pentanol was adjusted to 0.5 mL", and the rest of the operations were the same as in Example 1.

[0055] The scanning electron microscope image and transmission electron microscope image of the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 3 are shown in Figure 2 2(c) in Figure 3as shown in 3(c) Figure 2 (c) It can be seen that the pore diameter of Fe3O4@dSiO2 is about 10 - 15 nm. Figure 3 (a) It can be seen that the shell thickness of Fe3O4@dSiO2 is about 57 nm and the size is about 314 nm.

[0056] Example 4: The operations of Example 4 were repeated as in Example 3, with the only differences being the following two points:

[0057] Change 1: In the synthesis of the Fe3O4@dSiO2 template in Step 2, the amount of TEOS was adjusted to 0.6 mL.

[0058] Change 2: In Step 3, when encapsulating the pores of the silica shell of FMS by continuously coating silica through the method, 40 uL of TEOS was injected in total over 4 h. All other operations were the same as in Example 1.

[0059] The scanning electron microscope images and transmission electron microscope images of the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2 of Example 4 are respectively as shown in Figure 2 2(d) in Figure 3 and 3(d) in Figure 2 (d) It can be seen that the pore diameter of Fe3O4@dSiO2 is about 20 - 30 nm. Figure 3 (d) It can be seen that the shell thickness of Fe3O4@dSiO2 is about 80 nm and the size is about 360 nm.

[0060] The transmission electron microscope images of Fe3O4 synthesized in Step 1 of Example 4, the magnetic dendritic silica microspheres (Fe3O4@dSiO2) synthesized in Step 2, Fe3O4@dSiO2 / rQDs synthesized in Step 3, and FMS synthesized in Step 3 are respectively as shown in Figure 4 sub - figures a, b, c, and d in Figure 4 (a) It can be seen that the size of Fe3O4 is about 200 nm. From Figure 4 (b) It can be seen that the thickness of the silicon layer is about 80 nm. From Figure 4 (c) It can be seen that with the loading of rQDs, the pores of Fe3O4@dSiO2 are filled and full. From Figure 4 (d) It can be seen that the pores of the silicon layer are basically encapsulated.

[0061] The scanning electron microscope image of Fe3O4 synthesized in Step 1 of Example 4 is as shown in Figure 5 (a), with a size of about 200 nm. The scanning electron microscope image of the magnetic dendritic silica microspheres (Fe3O4@dSiO2) 4 synthesized in Step 2 of Example 4 is as shown in Figure 5(b), the pores of the microspheres in the picture were measured by Image J, and the pore diameter was measured to be approximately 20 - 30 nm. The scanning electron microscope image of Fe3O4@dSiO2 / rQDs synthesized in step 3 of Example 4 is as shown in Figure 5 (c), with the loading of rQDs, the pores of Fe3O4@dSiO2 become smaller. The scanning electron microscope image of FMS synthesized in step 3 of Example 4 is as shown in Figure 5 (d), with a size of approximately 380 nm.

[0062] In summary, with the increase in the amount of n-pentanol used, the pore diameter of Fe3O4@dSiO2 synthesized in Examples 1 - 3 gradually increases. With the increase in the amount of TEOS used, the shell thickness of Fe3O4@dSiO2 synthesized in Examples 1 and 4 becomes thicker. In the Fe3O4@dSiO2 template prepared in Example 1, the silicon layer thickness is approximately 27 nm, and the pore diameter is approximately 5 - 8 nm. In the Fe3O4@dSiO2 template prepared in Example 2, the silicon layer thickness is approximately 30 nm, and the pore diameter is approximately 8 - 10 nm. In the Fe3O4@dSiO2 template prepared in Example 3, the silicon layer thickness is approximately 54 nm, and the pore diameter is approximately 10 - 15 nm. In the Fe3O4@dSiO2 template prepared in Example 4, the silicon layer thickness is approximately 80 nm, and the pore diameter is approximately 20 - 30 nm.

[0063] The hysteresis curves of Fe3O4, Fe3O4@dSiO2, and FMS obtained in Example 4, as well as the photo of the FMS aqueous dispersion under magnetic attraction, are as shown in Figure 7 shown. The fluorescence spectra and photos of rQDs and FMS obtained in Example 4 are as shown in Figure 6 shown. It can be seen that the coincidence degree of the two curves is relatively high, indicating that the optical properties of rQDs are well maintained during the synthesis of the nanocomposite FMS.

[0064] The rQDs loading ratios of the Fe3O4@dSiO2 / rQDs products prepared in Examples 1 - 4 were tested according to the method of Example 5, and the test results were 29%, 31%, 41%, and 66.2% respectively. It can be seen that with the increase in the silicon layer thickness and pore diameter in the Fe3O4@dSiO2 template, the loading ratio of rQDs during the assembly process can be improved to a certain extent. However, the increase in the silicon layer thickness will affect the magnetism of the product. Therefore, in this application, the preferred amount of TEOS used is 0.6 mL.

[0065] Example 5

[0066] The preparation process of magnetic fluorescent nanosphere synthesis (FMS) in Step 3 of Example 5 was repeated as in Example 4, except that "the concentration of 1 mL of chloroform solution of fluorescent CdSe / CdS / ZnS QDs (rQDs) was changed to 2, 4, 6, 8, 10, and 12 mg / mL respectively", and finally Fe3O4@dSiO2 / rQDs products with rQDs feeding ratios of 20%, 40%, 60%, 80%, 100%, and 120% were obtained. Among them, the rQDs feeding ratio = mass of rQDs added / mass of Fe3O4@dSiO2 × 100%, that is, the percentage of the mass ratio of the guest rQDs to the mass of the host Fe3O4@dSiO2.

[0067] For the relationship between the rQDs loading ratio and the amount of rQDs added of Fe3O4@dSiO2 synthesized in Step 3 of Example 5, see Figure 8 . Figure 8 The inset in it is a photo of the supernatant of assembled rQDs (upper) and Fe3O4@dSiO2 / rQDs nanospheres redispersed in chloroform (lower). The inset (upper) is the result obtained by adding the Fe3O4@dSiO2 / rQDs products prepared at different rQDs feeding ratios to chloroform at a concentration of 10 mg / mL, centrifuging at 8000 rpm for 5 min, and taking the supernatant and filling it into a glass bottle. The inset (lower) is a photo result of the precipitate mixture obtained by redispersing the centrifuged precipitate in a chloroform solution with the same volume as the supernatant and filling it into a glass bottle.

[0068] From Figure 8 it can be seen that: when the mass ratio of the guest rQDs to the host Fe3O4@dSiO2 is less than 60%, the supernatant is almost colorless and the assembly efficiency is close to 100%. By changing the mass ratio of the guest to the loaded host during the assembly process, the amount of rQDs on the dendritic silica wall can be adjusted, and the loading ratio can be simply controlled within the range of 0 - 66.2%. Among them, the rQDs loading ratio = amount of QDs loaded / mass of Fe3O4@dSiO2 × 100%, and the amount of QDs loaded is calculated by the fluorescence intensity of the quantum dots before and after assembly.

[0069] The content described in this specification is only a list of the implementation forms of the inventive concept, and the protection scope of the present invention should not be regarded as limited to the specific forms stated in the embodiments.

Claims

1. A method for preparing a "kiwifruit"-type magnetic fluorescent nanospheres, characterized in that Superparamagnetic nano-Fe3O4 was prepared, and then a silica shell layer was hydrolyzed on the surface of Fe3O4 nanoparticles in a bicontinuous microemulsion to form Fe3O4@dSiO2 microspheres; then the surface of Fe3O4@dSiO2 was modified with mercapto groups, and CdSe / CdS / ZnS quantum dots QDs were used as the quantum dots to be assembled. The quantum dots were loaded by the coordination of the mercapto groups on Fe3O4@dSiO2 with the metal in the quantum dots to achieve the assembly of the quantum dots to form Fe3O4@dSiO2 / rQDs microsphere assemblies. Subsequently, hydrolysis and condensation reactions occurred on the surface of the Fe3O4@dSiO2 / rQDs microsphere assemblies by octyltrimethoxysilane OTMS to achieve the phase transfer process of the Fe3O4@dSiO2 / QDs microsphere assemblies from the organic phase to the aqueous phase, and Fe3O4@dSiO2 / rQDs modified with organosilicon on the surface was obtained. Then, the pores of the silica shell layer of the microsphere assemblies were encapsulated by growing and depositing silica by the Stöber method to prepare "kiwifruit"-type magnetic fluorescent nanomicrospheres.

2. The preparation method of a "kiwifruit"-type magnetic fluorescent nanomicrosphere according to claim 1, characterized in that The preparation method of the superparamagnetic nano-Fe3O4 is as follows: FeCl3·6H2O and sodium citrate dihydrate are dissolved in ethylene glycol. After adding a mixed solution of water and sodium acetate and stirring evenly, the reaction is carried out at 180 - 220 °C for 8 - 12 h, cooled, and the precipitate is separated. The precipitate is washed several times with ethanol and water; the volume ratio of ethylene glycol to water in the mixed solution is 40 - 45:1, the mass ratio of FeCl3·6H2O, sodium citrate dihydrate and sodium acetate is 1:0.3 - 0.5:2 - 2.5, and the mass-volume concentration of FeCl3·6H2O in the mixed solution is 0.02 - 0.03 g / mL.

3. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere as claimed in claim 1, characterized in that The preparation method of the Fe3O4@dSiO2 includes the following steps: 1) CTAB and urea are dissolved in water to form an aqueous phase, and n-pentanol and TEOS are dissolved in cyclohexane to form an oil phase. The oil phase and superparamagnetic nano-Fe3O4 are added to the aqueous phase, and stirred vigorously to mix the aqueous phase and the oil phase to form a bicontinuous microemulsion. Subsequently, the entire mixed solution is refluxed in a water bath at 65 - 75 °C for 14 - 18 h to react to form Fe3O4@dSiO2 microspheres; 2) After the reaction is completed, the precipitate is collected by magnetic separation and washed several times with ethanol; then, the precipitate is refluxed in acetone to remove CTAB in the silica pores; finally, Fe3O4@dSiO2 is collected by centrifugation, washed several times with ethanol, and stored in ethanol for standby.

4. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere according to claim 3, characterized in that In step 1), the volume ratio of the aqueous phase to the oil phase is 1:0.8 - 1.2; the mass-volume concentrations of CTAB and urea in the aqueous phase are 0.06 - 0.07 g / mL and 0.015 - 0.025 g / mL respectively; The feeding ratio of the superparamagnetic nano-Fe3O4 to TEOS is 0.1 g:0.5 - 0.6 mL; The volume ratio of n-pentanol to TEOS is 0.6 - 1:1; The concentration of superparamagnetic nano-Fe3O4 in the aqueous phase is 2 - 2.5 mg / mL.

5. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere according to claim 4, characterized in that In step 1), the volume ratio of the aqueous phase to the oil phase is 1:1; The feeding ratio of the superparamagnetic nano-Fe3O4 to TEOS is 0.1 g: 0.6 mL; The volume ratio of n-pentanol to TEOS is 0.83 - 0.85:

1.

6. The preparation method of a "kiwifruit"-type magnetic fluorescent nanomicrosphere according to claim 1, characterized in that The preparation method of the Fe3O4@dSiO2 / rQDs microsphere assembly includes the following steps: S1: Add ammonia water and (3-mercaptopropyl)trimethoxysilane to the ethanol solution of Fe3O4@dSiO2 microspheres, then stir and react at room temperature, centrifuge to collect the precipitate, wash to obtain mercapto-functionalized Fe3O4@dSiO2; S2: Take the mercapto-functionalized Fe3O4@dSiO2 and add it to the CdSe / CdS / ZnS quantum dot QDs solution, ultrasonically mix evenly, then centrifuge to remove the supernatant, and wash to remove the excess QDs to obtain the Fe3O4@dSiO2 / rQDs microsphere assembly.

7. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere according to claim 6, characterized in that In step S1, the Fe3O4@dSiO2 microspheres account for 0.5 - 1.0% of the total mass of the reaction system, the ammonia water accounts for 1.2 - 1.5% of the total volume of the reaction system, the (3-mercaptopropyl)trimethoxysilane accounts for 0.8 - 1.2% of the total volume of the reaction system, and the total stirring reaction time is 10 - 14 h; in step S2, the concentration of the CdSe / CdS / ZnS quantum dot QDs solution is 8 - 12 mg / mL, and the mass ratio of the mercapto-functionalized Fe3O4@dSiO2 to the CdSe / CdS / ZnS quantum dot QDs is 1:0.8 - 1.

2.

8. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere according to claim 1, characterized in that The hydrolysis and condensation reaction of n-octyltrimethoxysilane OTMS occurs on the surface of the Fe3O4@dSiO2 / rQDs microsphere assembly, and the specific process is as follows: Step A: Add n-octyltrimethoxysilane OTMS to the Fe3O4@dSiO2 / rQDs microsphere assembly, ultrasonically mix evenly, then add methanol and ammonia water, ultrasonically mix for 20 - 60 min to transfer the microsphere assembly from the oil phase to the water phase, centrifuge and remove the supernatant, and wash once with methanol to remove the excess OTMS; In step A, the volume ratio of n-octyltrimethoxysilane OTMS, methanol and ammonia water is 1:70 - 80:1.5 - 2; Step B: Further add ultrapure water and ammonia water with a volume ratio of 450 - 550:1 to the precipitate, stir and react at room temperature for 15 - 20 h, and after the reaction, wash several times with ethanol to obtain Fe3O4@dSiO2 / rQDs modified with organosilicon on the surface.

9. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere according to claim 1, characterized in that After the silica is grown and deposited by the Stöber method, it also includes the operation processes of amino modification and carboxyl modification, specifically as follows: Disperse the Fe3O4@dSiO2 / rQDs after being coated with silica in ethanol, then add ammonia water and 3-aminopropyltriethoxysilane, stir and react at room temperature for 10 - 15 h, then centrifuge and purify with ethanol to obtain the amino-modified microspheres, and then add them to the organic solution containing succinic anhydride, stir at room temperature for 20 - 30 h, centrifuge and wash several times with ethanol to obtain the carboxyl-modified microspheres, that is, the preparation is completed.

10. The preparation method of a "kiwifruit"-type magnetic fluorescent nanosphere according to claim 1, characterized in that The Stöber method is as follows: Disperse the Fe3O4@dSiO2 / rQDs with surface organosilicon modification on the reactant in a mixture of ethanol, water, and ammonia water. Based on the total volume of the mixture, add 0.7 - 1.0 μL / mL of tetraethyl orthosilicate to it every 1 h, and add it in several portions.

11. A "kiwifruit"-type magnetic fluorescent nanospheres prepared by the method according to any one of claims 1 - 10.

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