Chelated magnesium ion electrostatic spinning membrane as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a chelated magnesium ion electrostatic spinning membrane and a preparation method and application thereof, and the chelated magnesium ion electrostatic spinning membrane comprises a modified polymer porous membrane and magnesium ions loaded on the modified polymer porous membrane; the modified polymer porous membrane is a polymer porous membrane loaded with ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl) ethylidene amino) prop-2-yl) dithioalkyl) ethylidene imino) ethyl) naphthalene-1-alcohol. The prepared chelated magnesium ion electrostatic spinning membrane can specifically adsorb magnesium ion related proteins, and has important meanings for researching proteins specifically bound with magnesium ions and exploring the relationship between magnesium ions and diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a chelated magnesium ion electrospinning membrane and a preparation method and application thereof. Background Art

[0002] Magnesium is a metal element that is closely related to important physiological activities such as growth, development, metabolic regulation and immunity. As a cofactor of more than 300 enzymes, it stabilizes the structure of enzymes, regulates the activity of enzymes, and participates in important life processes such as energy metabolism, DNA replication and repair, and protein synthesis.

[0003] Existing technologies commonly use materials such as magnetic nanoparticles, affinity columns, and silica matrices to enrich proteins. However, these materials suffer from nonspecific adsorption, long processing times, poor reusability, and high cost. Electrospinning is a process that forms nanofibers by stretching a polymer solution or melt through a high electric field. Electrospun nanofibers are widely used in biomedicine, drug delivery, and protein enrichment due to their high surface area, ability to incorporate specific functional groups for protein enrichment, low cost, and simple preparation. Summary of the Invention

[0004] The purpose of the present invention is to provide a chelated magnesium ion electrospinning membrane and its preparation method and application, so as to overcome the shortcomings of the existing technology. The chelated magnesium ion electrospinning membrane prepared by the present invention can specifically adsorb magnesium ion-related proteins, which is of great significance for studying proteins that specifically bind to magnesium ions and exploring the relationship between magnesium ions and diseases.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a chelated magnesium ion electrospinning membrane, comprising a modified polymer porous membrane and magnesium ions loaded on the modified polymer porous membrane; the modified polymer porous membrane is a polymer porous membrane loaded with ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalene-1-ol.

[0007] The inventive concept adopted by the present invention is:

[0008] The polymer porous membrane serves as a base material, providing a structural support that maintains the membrane's overall morphology and mechanical strength. Its porous structure increases the membrane's specific surface area, facilitating efficient magnesium ion binding and the capture of magnesium-associated proteins in the sample. ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalen-1-ol (labeled as compound LH2) contains functional groups that form stable coordination bonds with magnesium ions, such as hydroxyl (-OH) and ethylideneamino (-CH2NH), enabling selective chelation of magnesium ions. Loading this compound onto the polymer porous membrane imparts the membrane with the ability to selectively chelate magnesium ions and capture magnesium-associated proteins. The hydroxyl (-OH) and ethyleneamino (-CH2NH) groups in LH2 on the polymer porous membrane can provide lone pairs of electrons to form coordination bonds with magnesium ions, so that the chelated magnesium ions still have some unoccupied coordination sites (such as vacancies in 4-6 coordination), which can form coordination bonds with specific atoms or groups in proteins, such as carboxyl (-COO-), hydroxyl (-OH), amino and amide groups (-NH2, -CONH2).

[0009] In some other embodiments, the loading amount of the magnesium ions is 1.0-2.0 wt.%.

[0010] In some other embodiments, the loading amount of the ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propan-2-yl)disulfanyl)ethylimino)ethyl)naphthalen-1-ol is 10-30 wt.%.

[0011] In some other embodiments, the polymer porous membrane is a PVDF membrane, a polysulfone membrane, and a polyamide membrane.

[0012] In some other embodiments, the source of the magnesium ions is a soluble magnesium salt, and the soluble magnesium salt is any one or more of magnesium chloride, magnesium phosphate and magnesium sulfate.

[0013] In a second aspect, the present invention provides a method for preparing the chelated magnesium ion electrospinning membrane according to the first aspect, comprising the following steps:

[0014] Dissolving a polymer and ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalen-1-ol in a solvent to obtain an electrospinning solution, and performing electrospinning to obtain an electrospinning membrane;

[0015] The electrospun membrane is immersed in a soluble magnesium salt and mixed to obtain a chelated magnesium ion electrospun membrane.

[0016] In some other embodiments, the solvent is a mixed solvent consisting of N,N-dimethylacetamide and acetone, and the mixed volume ratio of N,N-dimethylacetamide and acetone is 1:(0.5-2);

[0017] Preferably, the mixing volume ratio of N,N-dimethylacetamide and acetone is 1:1;

[0018] The concentration of the electrospinning solution is 10-20wt%;

[0019] Preferably, the concentration of the electrospinning solution is 15 wt%.

[0020] In some other embodiments, the electrospinning conditions are: voltage of 15-20 kV, receiving distance of 15-20 cm, and propulsion speed of 0.5-1.5 mL / h;

[0021] Preferably, the electrospinning conditions are: voltage of 20 kV, receiving distance of 20 cm, and propulsion speed of 1.0 mL / h.

[0022] In a third aspect, the present invention provides use of the chelated magnesium ion electrospinning membrane described in the first aspect in extracting proteins that specifically bind to magnesium ions.

[0023] In a fourth aspect, the present invention provides a method for extracting proteins that specifically bind to magnesium ions, comprising mixing the magnesium ion chelated electrospinning membrane described in the first aspect with a protein solution, and eluting the membrane to obtain the specifically bound proteins.

[0024] Beneficial effects of the present invention:

[0025] The present invention creatively prepares a chelated magnesium ion electrospinning membrane and uses it to specifically adsorb magnesium ion-related proteins, which is of great significance for studying proteins that specifically bind to magnesium ions and exploring the relationship between magnesium ions and diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 The scanning electron microscope images in the embodiment of the present invention are shown; A is a PVDF-LH2 electrospinning membrane, and B is a PVDF-LH2 electrospinning membrane chelated with Mg 2+ ;

[0028] Figure 2 The results of the mapping in the embodiment of the present invention are shown in Figure 2; A is a PVDF-LH2 electrospinning membrane; B is a PVDF-LH2 electrospinning membrane chelated with Mg2+ , C is the element mapping result of PVDF-LH2 electrospinning membrane, D is the chelated Mg of PVDF-LH2 electrospinning membrane 2+ Element Mapping result;

[0029] Figure 3 This is the infrared test result in the embodiment of the present invention;

[0030] Figure 4 This is a Coomassie Brilliant Blue staining picture of the PVDF-LH2 electrospun membrane in an embodiment of the present invention; wherein, 1 is 20 μg of control protein, 2 is the remaining protein incubation solution, 3 is the solution after the first rinse, 4 is the solution after the second rinse of the material, 5 is the solution after the third rinse of the material, 6 is the solution after the fourth rinse, and 7 is the PVDF-LH2 electrospun membrane after adsorbing 293T cell protein and washing;

[0031] Figure 5 PVDF-LH2 electrospinning membrane chelated Mg in the embodiment of the present invention 2+ Figure 8 shows the Coomassie Brilliant Blue staining image, where 8 is 20 μg of control protein, 9 is the remaining protein incubation solution, 10 is the solution after the first rinse, 11 is the solution after the second rinse, 12 is the solution after the third rinse, 13 is the solution after the fourth rinse, and 14 is the PVDF-LH2 electrospun membrane after adsorbing 293T cell protein and being washed. DETAILED DESCRIPTION

[0032] Example 1

[0033] 1. Preparation of chelated magnesium ion electrospinning membrane

[0034] (1) Synthesis of compound LH2

[0035] According to the reference Selvan GT, Chitra V, Vmv EI, et al. Development of a fluorescent chemosensor towards sensing and separation of Mg 2+ ions inchlorophyll and hard water[J], 2018, 42(2): 902-909, compound 2-((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalen-1-ol (labeled as compound LH2) was synthesized. The synthesis equation of compound LH2 is:

[0036]

[0037] (2) Preparation of PVDF-LH2 electrospinning membrane

[0038] PVDF (1.2 g) and LH2 (0.3 g) were ultrasonically dissolved in a mixture of N,N-dimethylacetamide / acetone (v / v = 1:1) to prepare an electrospinning solution with a 15 wt.% LH2 content. The solution was stirred at 50°C for 6 h to fully dissolve. Spinning was performed using a high-voltage DC power supply (DW-P303-1ACDF). The solution was allowed to stand at room temperature for 1 h to defoam. After defoaming, a plastic syringe (10 mL) containing the solution was mounted on a micro-constant current syringe pump (LSP02-1B) and spinning was performed using the following parameters: voltage 20.0 kV, receiving distance 20.0 cm, propulsion speed 1.0 mL / h, and a distance of 20.0 cm between the needle tip and the flat receiver (covered with aluminum foil). After electrospinning, the prepared PVDF-LH2 electrospun membrane was air-dried at room temperature to evaporate any solvent that did not completely evaporate during electrospinning and stored for future use.

[0039] (3) Chelated Mg 2+ Preparation of PVDF-LH2 electrospinning membrane

[0040] The PVDF-LH2 electrospun membrane was cut into 1cm×2cm size and placed in a 5mL epoxy resin (EP) tube. Then, 1ml of 1mol / L MgCl2 solution was added and the tube was placed in a rotary mixer and rotated at room temperature for 24h at a speed of 10rpm / min. 1mL of wash buffer solution (composition: 7.80mmol / L NaH2PO4, 12.20mmol / L Na2HPO4, 0.50mol / L NaCl) was added and the tube was rotated in a rotary mixer at room temperature for 5min at a speed of 10rpm / min. The binding buffer solution was aspirated and the rotary mixing was repeated twice to remove unbound Mg. 2+ .

[0041] The prepared PVDF-LH2 electrospinning membrane and chelated Mg 2+ The PVDF-LH2 electrospun membrane was subjected to scanning electron microscopy and element mapping tests. The results are as follows Figure 1 and Figure 2 As shown, from Figure 1 (B) and Figure 1 (A) By comparison, it can be found that Mg 2+ It has been successfully loaded on PVDF-LH2 electrospinning membrane.

[0042] from Figure 2 (B) with Figure 2 (A) Comparison shows that Mg 2+By uniformly distributing on the PVDF-LH2 electrospinning membrane and chelating Mg 2+ Mg in PVDF-LH2 electrospun membranes 2+ The normalized mass fraction is 1.51% ( Figure 2 (D)), and Figure 2 Mg in (C) 2+ The normalized quality score is 0%.

[0043] Figure 3 The infrared test results of the spinning material showed that LH2 and PVDF were dissolved in a mixed solvent of N, N-dimethylacetamide / acetone, and LH2 was loaded on the polymer membrane through physical mixing.

[0044] 2. Preparation of Specific Binding Protein

[0045] (1) Protein extraction

[0046] Add appropriate amounts of cell lysis buffer (Western and IP cell lysis buffer (Biyuntian, P0013)) and protease inhibitors (Halt protease inhibitor cocktail, EDTA-free (Thermo Fisher, 87785)) to the 293T cell pellet. Incubate at 4°C for 30 min, gently inverting every 5 min to mix thoroughly. Centrifuge at 12,000 rpm for 10 min at 4°C. Collect the supernatant to obtain a protein solution. The protein concentration is approximately 6-8 mg / mL.

[0047] (2) Incubation protein

[0048] To chelate Mg 2+ Add 2-4 mg of protein solution to the PVDF-LH2 electrospun membrane. If the volume is insufficient, add lysis buffer (Western and IP cell lysis buffer (Biyuntian, P0013)) to 400 μl. Incubate in a rotary mixer at 4°C for 8-12 h at a rotation speed of 10 rpm / min.

[0049] (3) Washing unbound proteins

[0050] Add 400 μL of wash buffer (7.80 mmol / L NaH2PO4, 12.20 mmol / L Na2HPO4, 0.50 mol / L NaCl). Rotate the tube at 4°C for 15 seconds at 10 rpm / min. Aspirate the wash buffer. Add wash buffer again and repeat a total of four times.

[0051] (4) Elute specific binding protein

[0052] Add 200 μL of SDS-PAGE protein loading buffer 2X and incubate in a 100°C water bath for 10 min to obtain specifically bound proteins.

[0053] 3. Coomassie Brilliant Blue Staining

[0054] The conventional protein detection method was used to detect the PVDF-LH2 electrospun membrane (marked as material ①) and the Mg chelated 2+ The PVDF-LH2 electrospun membrane (marked as material ②) was stained with Coomassie Brilliant Blue G250 for 3 h and then rinsed with a decolorizing solution (composition: V (ethanol): V (glacial acetic acid): V (ultrapure water) = 1:1:8, glacial acetic acid (Xilong Science), ethanol (Cologne Chemical)).

[0055] Coomassie Brilliant Blue staining experiment, the specific process is as follows:

[0056] (1) Coomassie Brilliant Blue Staining of PVDF-LH2 Electrospun Membrane

[0057] 1: 20 μg control protein (untreated 293T cell protein sample)

[0058] 2: Material ① Remaining protein incubation solution

[0059] 3: Solution after the first rinse of material ①

[0060] 4: Solution after second flushing of material ①

[0061] 5: Solution after rinsing material ① for the third time

[0062] 6: Solution after rinsing material ① for the fourth time

[0063] 7: Material after adsorption with 293T cell protein sample and washing with Wash Buffer①

[0064] Figure 4 This is a Coomassie Brilliant Blue staining picture of the PVDF-LH2 electrospun membrane in the embodiment of the present invention; wherein, 1 is 20 μg of control protein, 2 is the remaining protein incubation solution, 3 is the solution after the first rinse, 4 is the solution after the second rinse, 5 is the solution after the third rinse, 6 is the solution after the fourth rinse, and 7 is the PVDF-LH2 electrospun membrane after adsorbing 293T cell protein and washing. Figure 4 It can be seen that unchelated Mg 2+ After the PVDF-LH2 electrospun membrane was reacted with the protein solution and stained with Coomassie Brilliant Blue, the band of solution 7 was lighter, which may be the non-specific protein adsorbed by the membrane.

[0065] (2) PVDF-LH2 electrospinning membrane chelating Mg 2+Coomassie blue staining

[0066] 8: 20 μg control protein (untreated 293T cell protein sample)

[0067] 9: Material ② remaining protein incubation solution

[0068] 10: Solution after the first flush of material ②

[0069] 11: Solution after second flushing of material ②

[0070] 12: Solution after rinsing material ② for the third time

[0071] 13: Solution after rinsing material ② for the 4th time

[0072] 14: Material after adsorption with 293T cell protein sample and washing with Wash Buffer②

[0073] Figure 5 PVDF-LH2 electrospinning membrane chelated Mg in the embodiment of the present invention 2+ The Coomassie Brilliant Blue staining picture shows that 8 is 20 μg of control protein, 9 is the remaining protein incubation solution, 10 is the solution after the first rinse, 11 is the solution after the second rinse, 12 is the solution after the third rinse, 13 is the solution after the fourth rinse, and 14 is the PVDF-LH2 electrospun membrane after adsorbing 293T cell protein and washing. Figure 5 It can be seen that chelated Mg 2+ After the PVDF-LH2 electrospun membrane was reacted with the protein solution and stained with Coomassie Brilliant Blue, the protein bands in solution 14 were significantly increased, clearer and stronger, indicating that magnesium ions were successfully chelated with LH2 on the PVDF-LH2 electrospun membrane and could adsorb magnesium ion affinity proteins.

[0074] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A chelated magnesium ion electrospinning membrane, characterized in that: The invention comprises a modified polymer porous membrane and magnesium ions loaded on the modified polymer porous membrane; the modified polymer porous membrane is a polymer porous membrane loaded with ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylenamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalene-1-ol.

2. The chelated magnesium ion electrospinning membrane according to claim 1, characterized in that: The loading amount of the magnesium ions is 1.0-2.0 wt.%.

3. The chelated magnesium ion electrospinning membrane according to claim 1, characterized in that: The loading amount of the ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalen-1-ol is 10-30 wt.%.

4. The chelated magnesium ion electrospinning membrane according to claim 1, characterized in that: The polymer porous membrane is a PVDF membrane, a polysulfone membrane and a polyamide membrane.

5. The chelated magnesium ion electrospinning membrane according to claim 2, characterized in that: The source of the magnesium ions is a soluble magnesium salt, and the soluble magnesium salt is any one or more of magnesium chloride, magnesium phosphate and magnesium sulfate.

6. A method for preparing a chelated magnesium ion electrospinning membrane according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dissolving a polymer and ((Z)-1-(2-(2-(1-((Z)-1-(2-hydroxynaphthyl)ethylideneamino)propane-2-yl)disulfanyl)ethylimino)ethyl)naphthalen-1-ol in a solvent to obtain an electrospinning solution, and performing electrospinning to obtain an electrospinning membrane; The electrospun membrane is immersed in a soluble magnesium salt and mixed to obtain a chelated magnesium ion electrospun membrane.

7. The method for preparing the chelated magnesium ion electrospinning membrane according to claim 6, characterized in that: The solvent is a mixed solvent composed of N, N-dimethylacetamide and acetone, and the mixed volume ratio of N, N-dimethylacetamide and acetone is 1: (0.5-2); Preferably, the mixing volume ratio of N,N-dimethylacetamide and acetone is 1:1; The concentration of the electrospinning solution is 10-20wt%; Preferably, the concentration of the electrospinning solution is 15 wt %.

8. The method for preparing the chelated magnesium ion electrospinning membrane according to claim 6, characterized in that: The electrospinning conditions are: voltage of 15-20 kV, receiving distance of 15-20 cm, and propulsion speed of 0.5-1.5 mL / h; Preferably, the electrospinning conditions are: voltage of 20 kV, receiving distance of 20 cm, and propulsion speed of 1.0 mL / h.

9. Use of the chelated magnesium ion electrospinning membrane according to any one of claims 1 to 5 in extracting proteins that specifically bind to magnesium ions.

10. A method for extracting a protein that specifically binds to magnesium ions, characterized in that: The chelated magnesium ion electrospinning membrane according to any one of claims 1 to 5 is mixed with a protein solution and eluted to obtain the specifically bound protein.

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