Magnetic photosensitive nanosphere for magnetically driven cluster immunodetection and preparation method of magnetic photosensitive nanosphere

Abstract

The invention provides a magnetic photosensitive nanosphere for magnetic drive cluster immunodetection and a preparation method of the magnetic photosensitive nanosphere. The preparation method comprises the following steps: preparing a Fe3O4 nanosphere; the preparation method comprises the following steps: preparing Fe3O4 (at) mSiO2 nanospheres; the preparation method comprises the following steps: preparing Fe3O4 (at) mSiO2-NH2 nanospheres; the Fe3O4 (at) mSiO2-NH2 nanospheres, HATU, Ce6 and DIPEA are subjected to a coupling reaction in DMF for 20-28 h, a product is separated through a magnet, and after cleaning, Fe3O4 (at) mSiO2-Ce6 nanospheres are obtained; and activating the Fe3O4 (at) mSiO2-Ce6 nanosphere by EDC / NHS, coupling the activated Fe3O4 (at) mSiO2-Ce6 nanosphere with an antibody, and closing a non-specific site to obtain the antibody-modified magnetic donor sphere. By adopting the technical scheme provided by the invention, the donor sphere is high in photosensitive efficiency, and can be combined with a magnetic control control platform, so that the incubation time is shortened, and meanwhile, non-specific adsorption is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of detection materials, and particularly relates to a magnetic photosensitive nanosphere for magnetic-driven cluster immunoassay and a preparation method thereof. Background Art

[0002] Compared with traditional methods such as chemiluminescence immunoassay and fluorescence immunoassay, the photoinduced chemiluminescence immunoassay has the following two obvious advantages: one is homogeneous reaction, eliminating the washing step; the other is that the emission wavelength is shorter than the excitation wavelength, avoiding the interference of background fluorescence.

[0003] Currently, the nanoprobes used in the photoinduced chemiluminescence immunoassay include donor spheres and acceptor spheres. The donor spheres are nanospheres with a particle size of about 200 nm, loaded with photosensitizers, and singlet oxygen is generated under the excitation of a laser with a wavelength of 680 nm. The acceptor spheres are nanospheres with a particle size of about 200 nm, loaded with chemiluminescent agents and fluorescent agents. When the acceptor spheres come into contact with singlet oxygen, the chemiluminescent agents on the acceptor spheres are oxidized by singlet oxygen, and the generated excited-state products excite the fluorescent agents through the chemiluminescence energy resonance transfer (CRET) process. When the fluorescent agents transition back to the ground state, red light with a wavelength of 615 nm is emitted. If two different monoclonal antibodies of the antigen to be detected are respectively modified on the acceptor spheres and donor spheres. When a sample containing the antigen to be detected is incubated with the donor spheres and acceptor spheres in a sample pool, a "donor sphere - antibody 1 - antigen - antibody 2 - acceptor sphere" sandwich complex will be formed. When the sample pool is irradiated with a laser with a wavelength of 680 nm, in the sandwich complex, the singlet oxygen generated by the donor spheres diffuses to the acceptor spheres and reacts with the acceptor spheres to emit light. Because the diffusion distance of singlet oxygen in water is only 200 nm, the nanospheres that do not form the sandwich complex do not participate in the luminescence. Therefore, the luminescence intensity of the sample is proportional to the content of the sandwich complex, and the content of the antigen to be detected can be measured in this way, and the washing step is eliminated.

[0004] Currently, there are the following problems in the photoinduced chemiluminescence immunoassay: 1) The photosensitization efficiency of the donor spheres is not high enough to effectively utilize the excitation light energy; 2) The incubation time is relatively long during detection; 3) Nonspecific adsorption is difficult to avoid, resulting in relatively low detection accuracy. Summary of the Invention

[0005] In view of the above technical problems, the present invention discloses a magnetic photosensitive nanosphere for magnetic-driven cluster immunoassay and a preparation method thereof, which have high photosensitization efficiency of the donor spheres, shorten the incubation time during detection, and reduce the problem of nonspecific adsorption.

[0006] For this, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of a magnetic photosensitive nanosphere for magnetic-driven cluster immunoassay includes the following steps:

[0008] Step S1: Prepare Fe3O4 nanospheres;

[0009] Step S2: Preparation of Fe3O4@mSiO2 nanospheres: Disperse the Fe3O4 nanospheres, an amine weak base, and CTAB (cetyltrimethylammonium bromide) in water, heat to 80 - 85 °C under a protective gas atmosphere, add tetraethyl orthosilicate (TEOS) dropwise, stir and react for 4 - 6 h, remove CTAB by magnetic separation to obtain Fe3O4@mSiO2 nanospheres coated with mesoporous SiO2; wherein, the molar ratio of TEOS to Fe3O4 nanospheres is 1.2 - 7:1; the amine weak base is K b = 1×10 -7 ~10×10 -7 amine, which can provide a weakly basic environment.

[0010] Step S3: Preparation of Fe3O4@mSiO2-NH2 nanospheres: Stir and incubate the Fe3O4@mSiO2 nanospheres with APTES ((3-aminopropyl)triethoxysilane) in a solvent for 18 - 24 h, separate the product with a magnet, and obtain amino-functionalized Fe3O4@mSiO2-NH2 nanospheres after washing; further, the solvent is ethanol.

[0011] Step S4: Preparation of Fe3O4@mSiO2-Ce6 nanospheres: Couple the Fe3O4@mSiO2-NH2 nanospheres with HATU (2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate), Ce6, and DIPEA (diisopropylethylamine) in DMF for 20 - 28 h, separate the product with a magnet, and obtain Fe3O4@mSiO2-Ce6 nanospheres after washing;

[0012] Step S5: Antibody modification: Activate the Fe3O4@mSiO2-Ce6 nanospheres with EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) / NHS (N-hydroxysuccinimide), couple with an antibody, and block non-specific sites to obtain antibody-modified magnetic donor spheres.

[0013] The Fe3O4@mSiO2-Ce6 magnetic donor spheres prepared by the technical solution of the present invention can be combined with a magnetic control manipulation platform in a photochemiluminescence immunoassay. With the magnetism of Fe3O4, the magnetic donor spheres can form magnetic clusters, thus realizing magnetic control manipulation. During the detection process, this combination method can reduce the incubation time, speed up the detection speed, while reducing non-specific adsorption and improving the accuracy and efficiency of the detection. Among them, Ce6 with a relatively high singlet oxygen generation efficiency is selected as the photosensitizer, which preferably solves the problem of relatively low photosensitization efficiency of the system.

[0014] As a further improvement of the present invention, step S1 includes: stirring and dispersing FeCl3, PEG 400, and sodium acetate in ethylene glycol, then transferring the obtained dispersion to a hydrothermal reaction kettle, carrying out a solvothermal reaction at 180 - 220 °C for 6 h, separating the product with a magnet after cooling, and obtaining Fe3O4 nanospheres after washing; wherein, the molar ratio of FeCl3, PEG 400, and sodium acetate is 4 - 5:1:15 - 20.

[0015] As a further improvement of the present invention, the weak amine base is at least one of triethanolamine, diethanolamine, triisopropanolamine, diisopropanolamine, and N - methyldiethanolamine.

[0016] As a further improvement of the present invention, in step S2, the molar ratio of tetraethyl orthosilicate to Fe3O4 nanospheres is 1.2 - 7:1. The thickness of the SiO2 shell layer of the product depends on the amount of TEOS added. When the amount of TEOS added is 1.17 mL, the thickness of the SiO2 shell layer of the obtained Fe3O4@mSiO2 nanospheres is about 30 nm.

[0017] As a further improvement of the present invention, in step S2, the thickness of the SiO2 shell layer of the Fe3O4@mSiO2 nanospheres is 20 - 50 nm. Further, the thickness of the SiO2 shell layer is 25 - 35 nm.

[0018] As a further improvement of the present invention, in step S2, the protective gas is nitrogen.

[0019] As a further improvement of the present invention, in step S2, the amount of triethanolamine in terms of molar amount is 2.5 - 3.5 times that of Fe3O4 nanospheres, and the amount of cetyltrimethylammonium bromide in terms of molar amount is 1.1 - 1.5 times that of Fe3O4 nanospheres.

[0020] As a further improvement of the present invention, in step S2, the removal of CTAB includes: ultrasonically dispersing the product in an ethanol solution of ammonium acetate, extracting at a rotation speed of 30 - 50 r / min for more than 10 h, extracting at least twice in total, then separating the product with a magnet and washing.

[0021] As a further improvement of the present invention, in step S3, the aminosilane is APTES ((3 - aminopropyl)triethoxysilane). Further, for each mg of Fe3O4@mSiO2 nanospheres, the dosage of APTES is 0.005 - 0.02 mL.

[0022] As a further improvement of the present invention, in step S4, the conditions for the coupling reaction are incubation at room temperature with a rotation speed of 40 r / min.

[0023] As a further improvement of the present invention, the addition amount of Ce6 in step S4 is 50-70% of the mass of the Fe3O4@mSiO2-NH2 nanospheres.

[0024] As a further improvement of the present invention, in step S5, the magnetic donor spheres are dispersed in MES buffer solution, EDC and NHS are added, incubated on a turntable for 1 h, separated by a magnet, the precipitate is dispersed in PBS buffer solution, then the antibody to be loaded is added, after incubating on the turntable for 4 h, 100 mg of BSA and 30 μL of ethanolamine are added, continue to incubate on the turntable for 1 h, then the product is separated by a magnet, washed 3 times with PBS, and the product is dispersed in PBS to obtain antibody-modified magnetic donor spheres.

[0025] As a further improvement of the present invention, the masses of EDC and NHS in step S5 are 1.3-1.6 times and 1.5-1.8 times the mass of the Fe3O4@mSiO2-Ce6 nanospheres, respectively. Further, the masses of EDC and NHS in step S5 are 1.5 times and 1.6 times the mass of the Fe3O4@mSiO2-Ce6 nanospheres, respectively.

[0026] As a further improvement of the present invention, the coupling concentration of the antibody in step S5 is 0.1-0.5 mg / mL, that is, the concentration of the antibody in the solution is 0.1-0.5 mg / mL. Further, the coupling concentration of the antibody is 0.25 mg / mL.

[0027] The present invention also discloses a magnetic photosensitive nanosphere for magnetic drive cluster immunoassay, which is prepared by the preparation method of the magnetic photosensitive nanosphere for magnetic drive cluster immunoassay described in any one of the above. The structure of the magnetic photosensitive nanosphere sequentially includes an Fe3O4 magnetic core, a mesoporous SiO2 shell layer, a Ce6 photosensitizer, and an antibody modified on the surface from the inside to the outside. Under the irradiation of a 680 nm laser, the singlet oxygen generation efficiency of the magnetic photosensitive nanosphere is detected by an SOSG fluorescence probe, and the fluorescence intensity is increased to 1.5-2.0 times the initial value within 10 minutes.

[0028] The present invention also discloses the application of the magnetic photosensitive nanosphere for magnetic drive cluster immunoassay described above in magnetic control chemiluminescence immunoassay.

[0029] As a further improvement of the present invention, through a magnetic control manipulation platform, the Fe3O4@mSiO2-Ce6 magnetic donor spheres are formed into magnetic clusters by using the magnetism of Fe3O4 to achieve magnetic control manipulation, reduce the incubation time, accelerate the detection speed and reduce non-specific adsorption. By adopting this technical solution, magnetic clustering is realized through a magnetic control manipulation platform, the incubation time is shortened to 1-2 h, and non-specific adsorption is reduced.

[0030] Furthermore, the magnetic photosensitive nanospheres of the present invention can be integrated into a magnetically controlled detection platform for highly sensitive and rapid detection of tumor markers, pathogens, etc.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] First, magnetic control characteristics: The magnetic photosensitive nanospheres prepared in the present invention contain Fe3O4, making them magnetic and capable of forming magnetic clusters, which can then be combined with a magnetically controlled manipulation platform, solving the problem that existing photoinduced chemiluminescence immunoassay microspheres cannot be combined with a magnetically controlled manipulation platform, and providing the possibility for flexible operation during the detection process.

[0033] Second, high-efficiency photosensitive performance: Ce6 with a relatively high singlet oxygen generation efficiency is selected as the photosensitizer, significantly improving the photosensitive efficiency of the system, enhancing the detection sensitivity, and contributing to more accurate detection of target substances.

[0034] Third, improved detection efficiency: In photoinduced chemiluminescence immunoassay, the combination of magnetic photosensitive nanospheres with a magnetically controlled manipulation platform can reduce the incubation time, accelerate the detection speed, and at the same time reduce non-specific adsorption, improving the overall detection efficiency and accuracy, and providing a better solution for practical applications. Description of the Drawings

[0035] Figure 1 is a schematic diagram of the preparation process of the magnetic photosensitive nanospheres for magnetically driven cluster immunoassay according to an embodiment of the present invention.

[0036] Figure 2 is a TEM image of the Fe3O4@mSiO2 nanospheres prepared according to an embodiment of the present invention. Detailed Embodiments

[0037] The following further details the preferred embodiments of the present invention.

[0038] Example 1

[0039] A magnetic photosensitive nanosphere for magnetically driven cluster immunoassay, as Figure 1 shown, and its preparation method includes the following steps:

[0040] Step S1: Preparation of Fe3O4 nanospheres

[0041] 1.62 g of FeCl3·6H2O, 0.5 g of PEG 400, and 1.8 g of sodium acetate were stirred and dispersed in 30 mL of ethylene glycol. Then, the dispersion was transferred to a 100 mL hydrothermal reactor, and solvent thermal reaction was carried out at 200 °C for 6 h. After cooling, the reactor was opened, and the product was separated by a magnet. The product was washed 3 times with ethanol and water respectively, and finally dispersed in water to obtain an aqueous dispersion of Fe3O4 nanospheres.

[0042] Step S2: Preparation of Fe3O4@mSiO2 nanospheres

[0043] In a 1 L flat-bottomed cylindrical three-necked flask, 450 mg of Fe3O4 nanospheres, 0.9 g of triethanolamine (TEOA), and 0.9 g of cetyltrimethylammonium bromide (CTAB) were dispersed in 450 mL of water. After ultrasonic treatment for 30 min, the flask was placed on a hot plate for heating, and at the same time, mechanical stirring was carried out at a speed of 350 r / min, and N2 (2 L / min) was introduced to reduce oxidation. After the solution temperature was stabilized at about 80 °C, 0 - 2.7 mL of tetraethyl orthosilicate (TEOS) was added dropwise, and the mixture was continuously stirred and heated at this speed and temperature under N2 protection for 5 h. The product was separated by a magnet and washed 3 times with ethanol. To remove CTAB in the mesoporous SiO2 pores, the product was ultrasonically dispersed in 30 mL of an ethanol solution of ammonium acetate (CH3COONH4) with a concentration of 10 g / L, and extracted with a rotary table at a speed of 40 r / min for 12 h, and extracted twice in total. Then, the product was separated by a magnet and washed 3 times with ethanol, and finally the product was dispersed in water. The thickness of the SiO2 shell layer of the product depends on the addition amount of TEOS. When the addition amount of TEOS is 1.17 mL, the TEM image of the obtained Fe3O4@mSiO2 nanospheres is as Figure 2 shown, and the thickness of the SiO2 shell layer is about 30 nm.

[0044] Step S3: Preparation of Fe3O4@mSiO2-NH2 nanospheres

[0045] 150 mg of Fe3O4@mSiO2 nanospheres from which CTAB had been removed were ultrasonically dispersed in 60 mL of ethanol, 0.75 mL of APTES was added, and the mixture was incubated with a rotary table at a speed of 40 r / min for 24 h. Then, the product was separated by a magnet and washed 3 times with DMF, and finally the product was dispersed in water.

[0046] Step S4: Preparation of Fe3O4@mSiO2-Ce6 nanospheres

[0047] Disperse 70 mg of Fe3O4@mSiO2-NH2 nanospheres in 35 mL of DMF, add 26.6 mg of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 42 mg of Ce6, and 73.15 μL of diisopropylethylamine (DIPEA), incubate at room temperature with a turntable at a rotation speed of 40 r / min for 24 h, then separate the product with a magnet, wash the product 3 times with DMF and water respectively, and finally disperse it in water to obtain an aqueous dispersion of Fe3O4@mSiO2-Ce6 magnetic donor spheres (i.e., MDB).

[0048] The photosensitivity of Fe3O4@mSiO2-Ce6 nanospheres (MDB) was characterized using the SOSG fluorescent probe. Mix the SOSG fluorescent probe with the nanospheres and measure the fluorescence intensity; measure the fluorescence intensity again after irradiating with a 680 nm laser for a period of time. The results are shown in Table 1. In the table, MDB&SOSG represents the mixture of the SOSG fluorescent probe and Fe3O4@mSiO2-Ce6 nanospheres, and SOSG is the control sample with only the SOSG fluorescent probe. It can be seen that due to its photosensitive characteristics, singlet oxygen is generated after laser irradiation to oxidize the SOSG fluorescent probe, resulting in fluorescence in the system.

[0049] Table 1 Characterization of the singlet oxygen generation performance of Fe3O4@mSiO2-Ce6 nanospheres

[0050] Group Initial fluorescence intensity Fluorescence intensity after 5 min Fluorescence intensity after 10 min MDB&SOSG 5039.8 7222.7 9780.7 SOSG 16.0 16.5 15.7

[0051] Step S5: Modification of the antibody

[0052] Disperse 5 mg of magnetic donor spheres in 5 mL of MES buffer solution, add 7.5 mg of EDC and 8 mg of NHS, incubate on a turntable for 1 h, separate with a magnet, disperse the precipitate in 5 mL of PBS buffer solution, then add 1.25 mg of the antibody to be loaded, incubate on a turntable for 4 h, add 100 mg of BSA and 30 μL of ethanolamine, continue to incubate on a turntable for 1 h, then separate the product with a magnet, wash 3 times with PBS, and disperse the product in PBS to obtain antibody-modified magnetic donor spheres.

[0053] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should all be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay, characterized in that: The steps include: Step S1, preparing Fe3O4 nanospheres; Step S2, preparation of Fe3O4@mSiO2 nanospheres: dispersing the Fe3O4 nanospheres, amine weak base, and CTAB in water, heating to 80-85°C under a protective gas atmosphere, dropping tetraethyl orthosilicate, stirring and reacting for 4-6 hours, removing CTAB after magnetic separation, and obtaining mesoporous SiO2-coated Fe3O4@mSiO2 nanospheres; wherein the molar ratio of tetraethyl orthosilicate to Fe3O4 nanospheres is 1.2-7:1, and the amine weak base is K b =1×10 -7 ~10×10 -7 Amines; Step S3, preparation of Fe3O4@mSiO2-NH2 nanospheres: stirring and incubating the Fe3O4@mSiO2 nanospheres and aminosilane in a solvent for 18-24 hours, separating the products with a magnet, and washing to obtain amino-functionalized Fe3O4@mSiO2-NH2 nanospheres; Step S4, preparation of Fe3O4@mSiO2-Ce6 nanospheres: coupling reaction of the Fe3O4@mSiO2-NH2 nanospheres with HATU, Ce6, and DIPEA in DMF for 20-28 hours, separating the products with a magnet, and washing to obtain Fe3O4@mSiO2-Ce6 nanospheres; Step S5, antibody modification: the Fe3O4@mSiO2-Ce6 nanospheres are activated by EDC / NHS, coupled with antibodies, and non-specific sites are blocked to obtain antibody-modified magnetic donor spheres.

2. The method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 1, characterized in that: Step S1 comprises: stirring and dispersing FeCl3, PEG 400 and sodium acetate in ethylene glycol, then transferring the obtained dispersion into a hydrothermal reactor, performing a solvent thermal reaction at 180-220° C. for 6 hours, separating the product with a magnet after cooling, and obtaining Fe3O4 nanospheres after washing; wherein the molar ratio of FeCl3, PEG 400 and sodium acetate is 4-5:1:15-20.

3. The method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 2, characterized in that: In step S2, the amine weak base is at least one of triethanolamine, diethanolamine, triisopropanolamine, diisopropanolamine, and N-methyldiethanolamine; The molar ratio of TEOS to Fe3O4 nanospheres is 1.2-7:1; the thickness of the SiO2 shell of the Fe3O4@mSiO2 nanospheres is 20-50nm.

4. The method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 3, characterized in that: In step S2, the amount of triethanolamine is 2.5-3.5 times that of the Fe3O4 nanospheres, and the amount of hexadecyltrimethylammonium bromide is 1.1-1.5 times that of the Fe3O4 nanospheres; In step S2, the removal of CTAB includes: ultrasonically dispersing the product in an ethanol solution of ammonium acetate, extracting at a speed of 30-50 r / min for more than 10 hours, extracting at least twice in total, and then separating the product with a magnet and washing it.

5. The method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 1, characterized in that: In step S3, the amount of APTES used is 0.005-0.02 mL per mg of Fe3O4@mSiO2 nanospheres.

6. The method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 1, characterized in that: The amount of Ce6 added in step S4 is 50-70% of the mass of the Fe3O4@mSiO2-NH2 nanospheres.

7. The method for preparing magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 1, characterized in that: The conjugated concentration of the antibody in step S5 is 0.1-0.5 mg / mL.

8. A magnetic photosensitive nanosphere for magnetic driven cluster immune detection, characterized in that: The magnetic photosensitive nanospheres are prepared by the preparation method for magnetically driven cluster immunoassay as described in any one of claims 1 to 7.

9. The use of magnetic photosensitive nanospheres for magnetic driven cluster immunoassay as claimed in claim 8, characterized in that: Used in magnetronized photochemiluminescence immunoassay.

10. The use of magnetic photosensitive nanospheres for magnetic driven cluster immunoassay according to claim 9, characterized in that: Through the magnetic control platform, the magnetism of Fe3O4 is used to make Fe3O4@mSiO2-Ce6 magnetic donor spheres form magnetic clusters, realizing magnetic control, reducing incubation time, speeding up detection and reducing nonspecific adsorption.

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