CN112661964B - Double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere and preparation method and application thereof - Google Patents
Double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere as well as a preparation method and application thereof, belonging to the technical field of nano material preparation and nano medicine. The magnetic nanospheres modified by amphiphilic functional groups are used as carriers, testosterone is used as template molecules, 3-aminopropyl triethoxysilane and phenyl trimethoxysilane are used as bifunctional monomers, and a method combining two-step fixed templates and surface imprinting is adopted to prepare the bifunctional monomer testosterone molecularly imprinted magnetic nanospheres with double imprinted layers. The bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres prepared by the method have the advantages of uniform particle size, good structural stability, high selectivity, rich functional monomer groups and good biological safety; the method is a novel method for preparing the testosterone molecularly imprinted polymer with high adsorption capacity and high selectivity, and can remarkably improve the efficiency of castration treatment on the prostate cancer by sufficiently adsorbing and removing the testosterone in the cells, thereby providing a new idea for endocrine treatment of the prostate cancer.
Description
Technical Field
The invention belongs to the technical field of nano material preparation and nano medicine, and relates to a bifunctional monomer double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere as well as a preparation method and application thereof.
Background
The prostatic cancer is one of common malignant tumors of the male urinary system, the incidence rate of the prostatic cancer in China shows an obvious continuous growth trend, and the prostatic cancer is becoming the urinary malignant tumor which seriously affects the health of the male in China. The prostate is an androgen-dependent organ, the growth and metastasis of which are closely dependent on androgen, which enters cells after being secreted to be combined with androgen receptors and then enters cell nuclei to promote gene expression, thereby promoting the proliferation and metastasis of prostate cancer. The action mechanism of the method for treating the prostate cancer in clinical application mainly aims at the production of the androgen and the blocking of the combination process of the androgen and the androgen receptor, so that the pathological symptoms of the prostate cancer are relieved. However, due to the complexity of the activation of the signaling pathway, there is no method available at this stage that completely blocks the action of androgens at the local cellular level, nor is there a method that completely strips testosterone fundamentally. Therefore, the development of a new material which can freely enter cells to remove androgen in the prostatic cancer tissues has very important research significance for providing a new idea for the endocrine treatment of prostatic cancer.
The molecular imprinting technology is a technology for preparing a cross-linked polymer which has a fixed cavity and a fixed shape and has a specific binding site for target molecules by selecting the target molecules (organic micromolecules and biological macromolecules). The prepared Molecularly Imprinted Polymer (MIPs) is a high-efficiency separation material which has high affinity to target molecules and mainly adsorbs specifically and selectively. Several reports of preparing MIPs by using endocrine disruptors such as testosterone as template molecules exist at home and abroad, but the problems of low adsorption capacity and the like generally exist.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the bifunctional monomer double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere, and the preparation method and the application thereof, so as to solve the problem of low adsorption capacity of a testosterone molecularly imprinted polymer and realize the adsorption and removal of testosterone at a cellular level.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a preparation method of a bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere, which comprises the following steps:
s1: the magnetic nano particles modified by amphiphilic functional groups are used as a carrier and are uniformly mixed with template molecules testosterone in a mixed solution of ethanol and water, the hydrophilic end of the amphiphilic functional groups increases the dispersibility of the carrier, the hydrophobic end preliminarily fixes the template molecules through hydrophobic interaction, and the acting force between the template molecules is enhanced to prepare a carrier-template compound;
s2: sequentially adding ammonia water, the bifunctional monomer 3-aminopropyltriethoxysilane, the phenyltrimethoxysilane and the cross-linking agent ethyl orthosilicate into the carrier-template compound in the step S1, and realizing the re-fixation of template molecules through non-covalent interaction to obtain a solid polymer;
s3: and (4) separating, eluting and drying the solid polymer in the step S2 to obtain the bifunctional monomer single-imprinted-layer testosterone molecularly imprinted magnetic nanosphere.
S4: and (5) repeating the steps S1 and S2 on the bifunctional monomer single imprinting layer testosterone molecularly imprinted magnetic nanosphere in the step S3, and separating, eluting and drying to obtain the bifunctional monomer double imprinting layer testosterone molecularly imprinted magnetic nanosphere, so that the adsorption capacity and selectivity of the imprinted polymer are greatly increased.
Preferably, the volume ratio of the ethanol to the water in the step S1 is (1-3): (7-9).
Further preferably, the volume ratio of ethanol to water is 2: 8.
Preferably, the carrier-template complex in step S1 is prepared by modifying magnetic nanospheres modified with amphiphilic functional groups, testosterone and a solvent according to a ratio of (100-500) mg: (15-60) mg: (30-100) mL, and reacting for 30-100 min.
Preferably, in step S2, the solid polymer is prepared by mixing ammonia water, 3-aminopropyltriethoxysilane, phenyltrimethoxysilane and tetraethoxysilane in a ratio of (3-6) mL: (100-400) μ L: (100-400) μ L: (2-5) mL of the complex is sequentially added into the carrier-template complex solution prepared in the step S1, mixed and reacted for 6-10 hours, and then separated, washed and dried to obtain the complex.
Preferably, the solution for eluting the solid polymer in step S3 is prepared by mixing absolute ethyl alcohol and acetic acid in a volume ratio of (90-95): (5-10) mixing to obtain a mixed solution.
The invention also discloses the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared by the preparation method, and the particle size of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere is 500-700 nm.
Preferably, the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is 40.82-48.31 mg/g.
The invention also discloses application of the double-functional monomer double-imprinting layer testosterone molecular imprinting magnetic nanosphere as a testosterone adsorbent.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere disclosed by the invention has the following advantages: (1) the magnetic nanospheres modified by amphiphilic functional groups are used as carriers, the dispersibility of the carriers is enhanced by the hydrophilic ends, the interaction between the carriers and testosterone molecules is enhanced by hydrophobic alkyl chains, the adsorption capacity is remarkably increased, template molecules are preliminarily fixed through hydrophobic interaction, and the acting force between the template molecules is enhanced; (2) the magnetic nanoparticles modified by amphiphilic functional groups are used as a carrier, and the application of the magnetic nanoparticles can realize the rapid separation of solid and liquid under an external magnetic field and has the effects of magnetic targeting and the like; (3) 3-aminopropyltriethoxysilane and phenyltrimethoxysilane are adopted as the bifunctional monomers, so that the interaction and selectivity of a target and the functional monomer are enhanced, and the stability of imprinted pores and the reusability of imprinted polymers are improved; (4) the two hydrophilic functional monomers are cheap and easy to obtain, and the experimental method is simple and easy to realize; (5) the double-imprinting layer obviously increases the number of imprinting adsorption sites and improves the selectivity of target molecules.
The bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres prepared by the method have the advantages of rich functional monomer groups, good water dispersibility, good structural stability, strong magnetic response, uniform particle size and good biological safety, and show high binding capacity, fast adsorption rate, good regeneration capacity and high selectivity on target molecule testosterone.
The double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere can be used as an androgen testosterone adsorbent and can also be applied to prostate cancer castration treatment. Because the functional monomer groups are rich, the number of the imprinting layers is optimized, and testosterone in cells can be sufficiently and selectively adsorbed and removed, the efficiency of castration treatment on the prostatic cancer can be obviously improved, and a new thought is provided for endocrine treatment of the prostatic cancer, so that the method has very important research significance.
Drawings
FIG. 1 is a transmission electron microscope image of a bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere prepared in example 1 of the present invention;
fig. 2 is an adsorption curve of a bifunctional monomer single-imprinted layer testosterone molecular imprinting/non-imprinted magnetic nanosphere and a bifunctional monomer double-imprinted layer testosterone molecular imprinting/non-imprinted magnetic nanosphere prepared in example 1 of the present invention;
FIG. 3 is a drawing showing the selective adsorption of testosterone and its competitors by the bifunctional monomeric single-imprinted layer testosterone molecularly imprinted/non-imprinted magnetic nanospheres and the bifunctional monomeric double-imprinted layer testosterone molecularly imprinted/non-imprinted magnetic nanospheres prepared in example 1 of the present invention;
FIG. 4 is a light mirror image of the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanospheres prepared in example 1 of the present invention after being co-cultured with cells for 24h (carrier concentrations are 0, 10, 20, 40. mu.g/mL, respectively).
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention discloses a preparation method of a bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanosphere, which comprises the following steps of firstly, placing magnetic nanoparticles modified by amphiphilic functional groups and testosterone into an alcohol-water solution for reaction, and fixing through a template to prepare a template-carrier compound; secondly, polymerizing and fixing template molecules again by taking 3-aminopropyltriethoxysilane and phenyltrimethoxysilane as bifunctional monomers; then adding a cross-linking agent of ethyl orthosilicate for polymerization to obtain a solid polymer; then, separating the solid polymer by an external magnetic field, eluting and drying the separated solid polymer to obtain the double-function monomer single-imprinting layer testosterone molecularly imprinted magnetic nanospheres; and finally, polymerizing the 3-aminopropyltriethoxysilane and phenyltrimethoxysilane double-function monomers, fixing template molecules again, eluting the solid polymer obtained again, and drying to obtain the double-function monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere.
Example 1
The preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere comprises the following steps:
step one, placing 100mg of magnetic nanoparticles modified by amphiphilic functional groups, 15mg of testosterone and 30mL of alcohol-water mixed solution (1: 9) in a reaction container to react for 30min to form a carrier-template compound; then adding 3mL of ammonia water, 100 mu L of 3-aminopropyltriethoxysilane, 100 mu L of phenyltrimethoxysilane and 2mL of ethyl orthosilicate for reacting for 6h, washing the reaction product to be neutral by ultrapure water after the reaction is finished, and drying for 3h at 30 ℃ and 0.05 MPa;
and step two, eluting the dried solid polymer in the step one by using a mixed solution of absolute ethyl alcohol and acetic acid with a volume ratio of 90:10, and washing the eluted solid polymer to be neutral by using ultrapure water. And then, drying the obtained product at 30 ℃ under 0.05MPa for 3h to obtain the bifunctional monomer single-imprinted-layer testosterone molecularly imprinted magnetic nanospheres (S-MMIPs). Besides, the method is adopted to prepare the bifunctional monomer single-imprinted layer testosterone non-molecularly imprinted magnetic nanospheres (S-MNIPs) by the same preparation polymerization method except that the template molecule testosterone is not added.
And step three, repeating the step one and the step two, fixing template molecules again, polymerizing and eluting to form a second imprinted layer, and finally preparing the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanospheres (D-MMIPs). Besides, the bifunctional monomer double-imprinted layer testosterone non-molecularly imprinted magnetic nanospheres (D-MNIPs) are prepared by the same preparation polymerization method except that the template molecule testosterone is not added. As shown in the figure I, the particle size of the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere is about 600 nm.
The adsorption performance of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared in the embodiment 1 is detected as follows:
(1) adding 10mg of the double-function monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere into 10mL of testosterone solution with the concentration of 100 mu g/mL, oscillating for 50min at room temperature, and separating out supernatant through an external magnetic field;
(2) measuring the concentration of testosterone in the supernatant obtained in the step (1) by using HPLC, and then calculating the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres to testosterone;
the concentration of testosterone in the supernatant was determined to be 59.18. mu.g/mL.
The formula for calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is as follows:
in the formula CeThe concentration of testosterone in the supernatant is shown;
through calculation, the adsorption amount of the testosterone molecularly imprinted magnetic nanospheres of the double-function monomer double-imprinted layer to testosterone is as follows: 40.82 mg/g.
The bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres prepared in example 1 are subjected to isothermal adsorption experiment verification. As shown in fig. 2, with the change of the concentration of testosterone, the adsorption amount of testosterone of the imprinting nanospheres gradually tends to be balanced after being increased, and the adsorption amount of the polymer of the double imprinting layers is far higher than that of the single imprinting layer; meanwhile, selectivity experiments show that the selectivity of the polymer of the double imprinting layer is much higher than that of the polymer of the single imprinting layer (figure 3), and the excellent adsorption performance and selectivity of the polymer of the double imprinting layer are proved. The nanospheres with different concentrations are co-cultured with prostate cancer cells, so that the nanoparticles can freely enter the cells (figure 4), and the castration treatment of the prostate cancer can be further realized.
Example 2
The preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere comprises the following steps:
step one, placing 200mg of magnetic nanoparticles modified by amphiphilic functional groups, 25mg of testosterone and 40mL of alcohol-water mixed solution (1: 9) in a reaction container to react for 40min to form a carrier-template compound; then adding 3.5mL of ammonia water, 150 mu L of 3-aminopropyltriethoxysilane, 150 mu L of phenyltrimethoxysilane and 2.5mL of tetraethoxysilane for reaction for 7h, washing the reaction product to be neutral by ultrapure water after the reaction is finished, and drying for 3h at 30 ℃ and 0.05 MPa;
and step two, eluting the dried solid polymer in the step one by using a mixed solution of absolute ethyl alcohol and acetic acid with a volume ratio of 91:9, and washing the eluted solid polymer to be neutral by using ultrapure water. And then, drying the obtained product at 30 ℃ under 0.05MPa for 3h to obtain the bifunctional monomer single-imprinted layer testosterone molecularly imprinted magnetic nanospheres.
And step three, repeating the step one and the step two, fixing template molecules again, polymerizing and eluting to form a second imprinted layer, and finally preparing the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere.
The adsorption performance of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared in the embodiment 2 is detected as follows:
(1) adding 10mg of the double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere into 10mL of testosterone aqueous solution with the concentration of 100 mu g/mL, oscillating for 50min at room temperature, and separating out supernatant through an external magnetic field;
(2) measuring the concentration of testosterone in the supernatant obtained in the step (1) by using HPLC, and then calculating the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres to testosterone;
the measured concentration of testosterone in the supernatant was 57.70. mu.g/mL.
The formula for calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is as follows:
in the formula CeThe concentration of testosterone in the supernatant is shown;
through calculation, the adsorption amount of the testosterone molecularly imprinted magnetic nanospheres of the double-function monomer double-imprinted layer to testosterone is as follows: 42.30 mg/g.
Example 3
The preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere comprises the following steps:
step one, placing 300mg of magnetic nanoparticles modified by amphiphilic functional groups, 35mg of testosterone and 60mL of alcohol-water mixed solution (2: 8) in a reaction container to react for 60min to form a carrier-template compound; then adding 4mL of ammonia water, 200 mu L of 3-aminopropyltriethoxysilane, 200 mu L of phenyltrimethoxysilane and 3mL of tetraethoxysilane to react for 8h, after the reaction is finished, washing the reaction product to be neutral by using ultrapure water, and drying for 3h at 30 ℃ and 0.05 MPa;
and step two, eluting the dried solid polymer in the step one by using a mixed solution of absolute ethyl alcohol and acetic acid with a volume ratio of 92:8, and washing the eluted solid polymer to be neutral by using ultrapure water. And then, drying the obtained product at 30 ℃ under 0.05MPa for 3h to obtain the bifunctional monomer single-imprinted layer testosterone molecularly imprinted magnetic nanospheres.
And step three, repeating the step one and the step two, fixing template molecules again, polymerizing and eluting to form a second imprinted layer, and finally preparing the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere.
The adsorption performance of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared in the embodiment 3 is detected as follows:
(1) adding 10mg of the double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere into 10mL of testosterone aqueous solution with the concentration of 100 mu g/mL, oscillating for 50min at room temperature, and separating out supernatant through an external magnetic field;
(2) measuring the concentration of testosterone in the supernatant obtained in the step (1) by using HPLC, and then calculating the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres to testosterone;
the concentration of testosterone in the supernatant was measured to be 56.23. mu.g/mL.
The formula for calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is as follows:
in the formula CeThe concentration of testosterone in the supernatant is shown;
through calculation, the adsorption amount of the testosterone molecularly imprinted magnetic nanospheres of the double-function monomer double-imprinted layer to testosterone is as follows: 43.77 mg/g.
Example 4
The preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere comprises the following steps:
step one, placing 400mg of magnetic nanoparticles modified by amphiphilic functional groups, 45mg of testosterone and 70mL of alcohol-water mixed solution (2: 8) in a reaction container to react for 70min to form a carrier-template compound; then adding 4.5mL of ammonia water, 250 mu L of 3-aminopropyltriethoxysilane, 250 mu L of phenyltrimethoxysilane and 3.5mL of tetraethoxysilane for reaction for 6h, washing the reaction product to be neutral by ultrapure water after the reaction is finished, and drying for 3h at 30 ℃ and 0.05 MPa;
and step two, eluting the dried solid polymer in the step one by using a mixed solution of absolute ethyl alcohol and acetic acid with a volume ratio of 93:7, and washing the eluted solid polymer to be neutral by using ultrapure water. And then, drying the obtained product at 30 ℃ under 0.05MPa for 3h to obtain the bifunctional monomer single-imprinted layer testosterone molecularly imprinted magnetic nanospheres.
And step three, repeating the step one and the step two, fixing template molecules again, polymerizing and eluting to form a second imprinted layer, and finally preparing the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere.
The adsorption performance of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared in the embodiment 4 is detected as follows:
(1) adding 10mg of the double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere into 10mL of testosterone aqueous solution with the concentration of 100 mu g/mL, oscillating for 50min at room temperature, and separating out supernatant through an external magnetic field;
(2) measuring the concentration of testosterone in the supernatant obtained in the step (1) by using HPLC, and then calculating the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres to testosterone;
the concentration of testosterone in the supernatant was measured to be 54.37. mu.g/mL.
The formula for calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is as follows:
in the formula CeThe concentration of testosterone in the supernatant is shown;
through calculation, the adsorption amount of the testosterone molecularly imprinted magnetic nanospheres of the double-function monomer double-imprinted layer to testosterone is as follows: 45.63 mg/g.
Example 5
The preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere comprises the following steps:
step one, placing 500mg of magnetic nanoparticles modified by amphiphilic functional groups, 55mg of testosterone and 90mL of alcohol-water mixed solution (3: 7) in a reaction container to react for 90min to form a carrier-template compound; then adding 5mL of ammonia water, 300 mu L of 3-aminopropyltriethoxysilane, 300 mu L of phenyltrimethoxysilane and 4mL of tetraethoxysilane to react for 10h, after the reaction is finished, washing the reaction product to be neutral by using ultrapure water, and drying for 3h at 30 ℃ and 0.05 MPa;
and step two, eluting the dried solid polymer in the step one by using a mixed solution of absolute ethyl alcohol and acetic acid with a volume ratio of 94:6, and washing the eluted solid polymer to be neutral by using ultrapure water. And then, drying the obtained product at 30 ℃ under 0.05MPa for 3h to obtain the bifunctional monomer single-imprinted layer testosterone molecularly imprinted magnetic nanospheres.
And step three, repeating the step one and the step two, fixing template molecules again, polymerizing and eluting to form a second imprinting layer, and finally obtaining the testosterone molecularly imprinted magnetic nanosphere with the double-functional monomer double-imprinting layer.
The adsorption performance of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared in the embodiment 5 is detected as follows:
(1) adding 10mg of the double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere into 10mL of testosterone aqueous solution with the concentration of 100 mu g/mL, oscillating for 50min at room temperature, and separating out supernatant through an external magnetic field;
(2) measuring the concentration of testosterone in the supernatant obtained in the step (1) by using HPLC, and then calculating the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres to testosterone;
the testosterone concentration in the supernatant was determined to be 52.89 μ g/mL.
The formula for calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is as follows:
in the formula CeThe concentration of testosterone in the supernatant is shown;
through calculation, the adsorption amount of the testosterone molecularly imprinted magnetic nanospheres of the double-function monomer double-imprinted layer to testosterone is as follows: 47.11 mg/g.
Example 6
The preparation method of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere comprises the following steps:
step one, placing 500mg of magnetic nanoparticles modified by amphiphilic functional groups, 60mg of testosterone and 100mL of alcohol-water mixed solution (3: 7) in a reaction container to react for 100min to form a carrier-template compound; then 6mL of ammonia water, 400 μ L of 3-aminopropyltriethoxysilane, 400 μ L of phenyltrimethoxysilane and 5mL of ethyl orthosilicate are added for reaction for 10h, after the reaction is finished, the reaction product is washed to be neutral by ultrapure water, and then is dried for 3h at 30 ℃ and 0.05 MPa;
and step two, eluting the dried solid polymer in the step one by using a mixed solution of absolute ethyl alcohol and acetic acid with a volume ratio of 95:5, and washing the eluted solid polymer to be neutral by using ultrapure water. And then, drying the obtained product at 30 ℃ under 0.05MPa for 3h to obtain the bifunctional monomer single-imprinted layer testosterone molecularly imprinted magnetic nanospheres.
And step three, repeating the step one and the step two, fixing template molecules again, polymerizing and eluting to form a second imprinted layer, and finally preparing the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere.
The adsorption performance of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere prepared in the embodiment 6 is detected as follows:
(1) adding 10mg of the double-functional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere into 10mL of testosterone aqueous solution with the concentration of 100 mu g/mL, oscillating for 50min at room temperature, and separating out supernatant through an external magnetic field;
(2) measuring the concentration of testosterone in the supernatant obtained in the step (1) by using HPLC, and then calculating the adsorption amount of the bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres to testosterone;
the testosterone concentration in the supernatant was determined to be 51.69 μ g/mL.
The formula for calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres to testosterone is as follows:
in the formula CeThe concentration of testosterone in the supernatant is shown;
through calculation, the adsorption amount of the testosterone molecularly imprinted magnetic nanospheres of the double-function monomer double-imprinted layer to testosterone is as follows: 48.31 mg/g.
TABLE 1 comparison of adsorption amounts of the bifunctional monomer double-imprinted-layer testosterone molecularly imprinted magnetic nanospheres to testosterone under different preparation conditions
The adsorption capacity of the prepared testosterone molecularly imprinted magnetic nanospheres with the double-function monomer double-imprinted layers on testosterone by adopting different preparation schemes is higher than 40mg/g, so that the interaction of MIPs on testosterone is enhanced due to the introduction of the double-function monomer; and a double-imprinting-layer preparation strategy is adopted to effectively increase the specific surface area of the carrier and increase the number of imprinting sites. Compared with the adsorption performance of the existing testosterone imprinted polymer, the preparation strategy greatly enhances the selective adsorption capacity of testosterone.
In addition, the selectivity of the prepared testosterone molecularly imprinted magnetic nanosphere with the double-function monomer double-imprinted layer is researched, and the result is as follows:
the selective adsorption of the prepared magnetic nanospheres on testosterone is researched by taking testosterone propionate, methyltestosterone, estradiol and progesterone which are structural analogues of testosterone as competitors. Respectively adding 10mg of the double-function monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres into 10mL aqueous solution of testosterone and a competitive substance thereof with the concentration of 100 mu g/mL, oscillating for 30min at room temperature, and separating out supernatant through an external magnetic field; and measuring the concentration of testosterone and a competitor thereof in the obtained supernatant by using HPLC, and then calculating the adsorption quantity of the bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere on the testosterone and the competitor thereof by using the following formula.
In the formula CeIs the concentration of testosterone or its competitor in the supernatant.
Through calculation, the adsorption amounts of the prepared magnetic nanoparticles to testosterone, testosterone propionate, methyltestosterone, estradiol and progesterone are respectively as follows: 42.3, 2.56, 3.51, 1.60 and 1.31mg/g, it can be seen that the prepared magnetic nanospheres adsorb testosterone about 20 times more than their competitors. Therefore, the prepared bifunctional monomer double-imprinting layer testosterone molecular imprinting magnetic nanospheres have high selective adsorption capacity on testosterone.
In summary, the preparation method of the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere disclosed by the invention adopts the magnetic nanosphere modified by the amphiphilic functional group as the carrier, testosterone as the template molecule, and 3-aminopropyl triethoxysilane and phenyl trimethoxysilane as the bifunctional monomer, and adopts the method of combining the two-step fixed template and the surface imprinting to prepare the bifunctional monomer double-imprinted layer testosterone molecularly imprinted magnetic nanosphere. The amphiphilic functional group modification enriches the abundant functional group types on the surface of the magnetic nanosphere, not only enhances the water dispersibility of the carrier, but also enhances the interaction with the template molecule testosterone, thereby realizing better template fixing effect. And secondly, 3-aminopropyltriethoxysilane and phenyltrimethoxysilane are jointly used as functional monomers, so that the adsorption capacity of the imprinted polymer on testosterone and the material stability are improved. In addition, the double-imprinting layer imprinted polymer is prepared by optimizing the preparation conditions, so that the number of imprinting sites is greatly increased, and the adsorption capacity of the double-imprinting layer imprinted polymer is far higher than that of a single-imprinting layer polymer; and through the comparison of the imprinting factor and the selection factor, the double-imprinting layer imprinting polymer also shows high recognition specificity and selectivity. The invention enhances the interaction between the carrier and the target testosterone by developing a novel double-functional monomer and combining a double-imprinting layer strategy, increases the stability of imprinting holes, obviously increases the number of imprinting sites of the testosterone, and has positive promotion effect on improving the adsorption capacity of MIPs.
The bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres prepared by the method combine the magnetic effect and the nano effect of a substance, combine a magnetic nano material with a molecularly imprinted technology, and prepare the core-shell structured molecularly imprinted nanoparticles by adopting a fixed template and a surface imprinting method, wherein the core-shell structured molecularly imprinted nanoparticles have the advantages of large specific surface area, regularly and uniformly distributed imprinted sites, high mass transfer rate, superparamagnetism, high stability and the like besides the specific recognition performance on an adsorbed substance, and can realize the selective extraction and separation of a target substance. The bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanospheres obtained by the method have the advantages of good biological safety, high adsorption capacity, good selectivity, high stability, fast mass transfer, easiness in separation and the like, and are a method for removing testosterone in prostate cancer cells with high selectivity.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (9)
1. A preparation method of a bifunctional monomer double-imprinting layer testosterone molecularly imprinted magnetic nanosphere is characterized by comprising the following steps:
s1: the magnetic nano particles modified by amphiphilic functional groups are used as a carrier, testosterone is used as a template molecule, and the carrier-template compound is prepared by uniformly mixing the carrier-template molecule and the testosterone in a solvent; wherein the dosage ratio of the carrier, the template molecule and the solvent is (100-500) mg: (15-60) mg: (30-100) mL; the solvent is ethanol and water in a volume ratio of (1-3): (7-9) preparing a mixed solution;
s2: sequentially adding ammonia water, the bifunctional monomer 3-aminopropyltriethoxysilane, the phenyltrimethoxysilane and the cross-linking agent ethyl orthosilicate into the carrier-template compound, and uniformly mixing to obtain a solid polymer; wherein the dosage ratio of ammonia water, 3-aminopropyltriethoxysilane, phenyltrimethoxysilane to ethyl orthosilicate is (3-6) mL: (100-400) μ L: (100-400) μ L: (2-5) mL;
s3: separating, eluting and drying the solid polymer to obtain the double-function monomer single-imprinting-layer testosterone molecularly imprinted magnetic nanospheres;
s4: and (3) repeating the operations of S1-S3 by taking the prepared bifunctional monomer single-imprinting-layer testosterone molecularly imprinted magnetic nanospheres as a carrier to prepare the bifunctional monomer double-imprinting-layer testosterone molecularly imprinted magnetic nanospheres.
2. The preparation method of the bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere according to claim 1, wherein in S1: the mixing time is 30-100 min.
3. The preparation method of the bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere according to claim 1, wherein in S2: the mixing time is 6-10 h.
4. The preparation method of the bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere according to claim 1, wherein in S3: the solid polymer is eluted by adopting absolute ethyl alcohol and acetic acid according to the volume ratio of (90-95): (5-10) in the presence of a solvent.
5. The bifunctional monomer double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere prepared by the preparation method of any one of claims 1 to 4, wherein the particle size of the bifunctional monomer double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere is 500-700 nm.
6. The bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere according to claim 5, wherein the adsorption amount of the bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere to testosterone is 40.82-48.31 mg/g.
7. The use of the bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere of claim 5 or 6 as a testosterone adsorbent.
8. The use of the bifunctional monomeric double-imprinted-layer testosterone molecularly imprinted magnetic nanosphere of claim 5 or 6 in the preparation of a medicament for treating prostate cancer.
9. The use of claim 8, wherein the medicament is a medicament for selectively removing testosterone from prostate cancer cells.
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