Aptamer modified polydopamine coated magnetic composite material, preparation and application thereof
By synthesizing magnetic graphene oxide composite materials using a solvothermal method and coating them with polydopamine to generate gold nanoparticles, which are then bonded to nucleic acid aptamers, this method solves the problems of cumbersome operation and insufficient selectivity in existing recombinant protein purification methods, and achieves efficient and simple enrichment of recombinant proteins.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN MEDICAL UNIVERSITY
- Filing Date
- 2024-03-13
- Publication Date
- 2026-05-01
AI Technical Summary
Existing recombinant protein purification methods are cumbersome to operate, have limited enrichment capacity, and lack selectivity and specificity. Nucleic acid aptamer immobilization capacity is low and the preparation process is complicated.
A magnetic graphene oxide composite material was synthesized in one step using a solvothermal method. Polydopamine was coated by dopamine oxidative polymerization, and gold nanoparticles were generated in situ on the material surface. Finally, nucleic acid aptamers were bonded by the reaction of Au and thiol groups to increase the immobilization capacity of nucleic acid aptamers.
The preparation process was simplified, the hydrophilicity of the material and the loading capacity of nucleic acid aptamers were improved, and the enrichment of recombinant proteins with high selectivity and high specificity was achieved, which has good practical value.
Smart Images
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Abstract
Description
A magnetic composite material modified with nucleic acid aptamer and coated with polydopamine, its preparation and application Technical Field
[0001] This invention belongs to the field of inorganic materials and analytical technology, specifically relating to a magnetic composite material MagG@PDA@Au@Aptamer modified with nucleic acid aptamers and coated with polydopamine, its preparation, and its use for efficient and highly selective capture of recombinant proteins. Background Technology
[0002] Recombinant proteins are proteins obtained using recombinant DNA or recombinant RNA technology. They play a crucial role in biomedicine, bioengineering, proteomics, and other fields, and are widely used in basic life science research, including medical research, pharmaceutical research, laboratory medicine, and epidemiology. Therefore, the demand for recombinant protein expression and purification is increasing. Currently, immobilized metal ion affinity chromatography (IMAC) is one of the most common protein purification methods. Its purification principle utilizes the chelation between histidine residues exposed on the protein surface and metal ions immobilized on the matrix surface to achieve selective separation of recombinant proteins. However, this method is cumbersome, has limited enrichment capacity, and lacks strong selectivity and specificity. Therefore, it is necessary to develop a simple, efficient, selective, and specific method for recombinant protein purification.
[0003] Magnetic materials have been widely used in protein separation due to their excellent biocompatibility, ease of modification, and controllable magnetic properties. Graphene oxide not only has a large specific surface area but also contains a large number of oxygen-containing groups on its surface, making it easy to chemically modify and hydrophilically modify. Previous reports have described a complex and cumbersome preparation process involving functionalizing magnetic nanoparticles Fe3O4 and graphene oxide (GO) separately and then chemically linking them together, followed by further modifications. This process limits the widespread application and practicality of such materials. In addition, polydopamine is a biocompatible polymer with adhesive properties, self-assembly, and biocompatibility. Nucleic acid aptamers are single-stranded oligonucleotides (ssDNA or ssRNA) that can form specialized three-dimensional structures, exhibiting high affinity and specificity for target substances, good chemical stability, and ease of modification. Immobilizing nucleic acid aptamers as affinity ligands in magnetic graphene oxide composites can reduce non-specific adsorption and improve the selective enrichment of target proteins. However, the current immobilization capacity of nucleic acid aptamers needs improvement, and the preparation process is cumbersome. Summary of the Invention
[0004] This invention addresses the shortcomings of existing methods by preparing a simple, easy-to-operate, highly efficient, and selective magnetic graphene oxide composite material modified with nucleic acid aptamers and coated with polydopamine, which is then used for the purification and separation of recombinant proteins. Polydopamine (PDA) is coated onto the surface of the magnetic graphene oxide composite matrix through oxidative polymerization, and gold nanoparticles are generated in situ, improving the surface hydrophilicity of the composite material and the binding sites of the nucleic acid aptamers. Finally, nucleic acid aptamers are bonded to the material surface via a reaction between Au and thiol groups, increasing the immobilization capacity of nucleic acid aptamers.
[0005] The technical approach adopted in this invention is as follows:
[0006] This invention discloses a magnetic composite material modified with nucleic acid aptamers and coated with polydopamine, which has a magnetic graphene oxide core and a polydopamine shell, with alloy nanoparticles and nucleic acid aptamers bonded to the shell surface; the bonding loading of the nucleic acid aptamers is 1-45 nmol / mg.
[0007] In the above technical solution, the binding loading of the nucleic acid aptamer is further 45 nmol / mg.
[0008] In the above technical solution, the preparation method of the magnetic composite material further includes: synthesizing magnetic graphene oxide MagG in one step by a solvothermal method; dispersing MagG in an alkaline dopamine solution; preparing polydopamine-coated magnetic composite material MagG@PDA through the oxidative polymerization of dopamine; then, using the chemical bond between amino groups and the noble metal Au, bonding Au nanoparticles to the surface of MagG@PDA to obtain MagG@PDA@Au; and connecting a nucleic acid aptamer with a thiol group modified at the 5' end to the material surface to synthesize a nucleic acid aptamer-modified polydopamine-coated magnetic graphene oxide composite material MagG@PDA@Au@Aptamer.
[0009] In the above technical solution, the magnetic graphene oxide (MagG) is composed of iron oxide particles dispersed on graphene, the graphene sheet diameter is 500nm-4μm, and the iron oxide magnetic core particle diameter is 100-300nm.
[0010] In the above technical solution, the one-step solvothermal synthesis of magnetic graphene oxide (MagG) specifically involves:
[0011] 0.08-0.32g FeCl3·6H2O was used as the iron source, 0.015-0.06g sodium citrate was used as the stabilizer, and 0.015-0.06g graphene GO was used as the matrix for magnetic nucleus growth. 15-60mL ethylene glycol was added to dissolve the mixture, and it was sonicated for 1-4h in an ice bath at 0-4℃ to form a homogeneous suspension. Then 0.35-1.4g sodium acetate was added, and the mixture was mechanically stirred for 0.2-1h. The mixture was transferred to a reaction vessel and reacted at 160-220℃ for 5-10h. The prepared product was washed with water and ethanol 2-4 times each, and then vacuum dried to obtain the magnetic graphene oxide composite matrix material MagG.
[0012] In the above technical solution, the preparation method of the magnetic composite material further includes the following steps:
[0013] The first step involves the one-step synthesis of MagG using a solvothermal method.
[0014] The second step is to synthesize the polydopamine-coated magnetic composite material MagG@PDA: Weigh 15-60 mg of the MagG material prepared in the first step and ultrasonically disperse it in a mixture of 30-120 mL of ethanol, 15-60 mL of 5-20 mM Tris-HCl buffer solution and 20-90 mL of water. Add 60-240 mg of dopamine and mechanically stir at 25-60 °C for 6-12 h. Separate the product into solid and liquid components using a magnet, collect the solid product, and wash it with water and ethanol 2-4 times each. Then, vacuum dry it to obtain the polydopamine-coated magnetic composite material MagG@PDA.
[0015] The third step involves in-situ modification of nano-Au particles to prepare MagG@PDA@Au: 6-24 mg of the MagG@PDA material prepared in the second step is dispersed in 75-300 mL of deionized water, and 120-480 μL of 75-300 mM chloroauric acid is added. The reaction mixture is heated to 50-120 °C and maintained for 0.2-1 h. Then, 0.75-3 mL of 0.5-2.0 M sodium citrate is rapidly added, and the reaction continues for 0.5-2 h. The mixture is then washed 2-4 times with water and ethanol respectively, and then vacuum dried.
[0016] Step 4: Synthesis of the nucleic acid aptamer-modified magnetic graphene oxide composite material MagG@PDA@Au@Aptamer: Take 30-120 μL of 50-200 μM thiol-modified nucleic acid aptamer, add 30-120 μL of 50-200 μM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution and 60-240 μL of 50-200 mM Tris-HCl buffer solution, and incubate with shaking at room temperature for 0.5-2 h; after the reaction is complete, add the above solution to 40-160 μg of MagG@PDA@Au material, and incubate with shaking at 4-37℃ for 6-18 h, collect the material, wash with PBS, and then add 40-160 μL of 0.5-2 mM mercaptohexanol (MCH) for blocking; collect the material with a magnet, wash with PBS, and obtain MagG@PDA@Au@Aptamer.
[0017] In the above technical solution, the nucleic acid aptamer is further defined as an aptamer targeting the recombinant histidine tag protein (5'-(SH)-GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-3').
[0018] The present invention also discloses the application of the aforementioned nucleic acid aptamer-modified polydopamine-coated magnetic composite material as an affinity enrichment material.
[0019] In the above technical solution, the magnetic composite material is further added directly to the bacterial lysate of Escherichia coli in the application to efficiently and selectively capture the target protein.
[0020] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:
[0021] This invention employs a solvothermal method for one-step synthesis of graphene oxide composite materials. The preparation method is simple, avoids complex modification steps, and improves the material's practicality. Using the graphene oxide composite material as a substrate for nucleic acid aptamers not only increases the loading capacity of nucleic acid aptamers but also simplifies the operation. The magnetic response characteristics of the magnetic core enable controllable separation by an external magnetic field during the selective enrichment of histidine-tagged recombinant proteins. Coating the magnetic graphene oxide with polydopamine improves the material's hydrophilicity, and the abundant amino groups in polydopamine enhance the bonding capacity of Au nanoparticles, thereby increasing the nucleic acid aptamer loading capacity. The nucleic acid aptamer bonding capacity on the surface of this material is higher than that of most similar materials. Nucleic acid aptamer modification enables affinity enrichment of target proteins with high selectivity and specificity. This material has good practical value and application prospects in the field of protein purification. Attached Figure Description
[0022] Figure 1. Schematic diagram of the synthesis of the nucleic acid aptamer modified magnetic composite material MagG@PDA@Au@Aptamer.
[0023] Figure 2. Schematic diagram of the process for isolating and purifying recombinant histidine-tagged proteins from bacterial lysates using MagG@PDA@Au@Aptamer.
[0024] Figure 3. Transmission electron micrographs of different stages in the material preparation process. (a) GO, (b) MagG, (c) MagG@PDA, (d) MagG@PDA@Au.
[0025] Figure 4. Infrared characterization of MagG@PDA@Au@Aptamer material.
[0026] Figure 5. SDS-PAGE analysis of His-tagged RXRα protein isolated and purified from E. coli cell lysate by MagG@PDA@Au@Aptamer. Lane M: Standard protein marker; Lane L: Cell lysate; Lane S: Cell lysate supernatant after material treatment; Lane 1: Washing buffer after incubation with cell lysate; Lanes 2 and 3: Eluent obtained by elution twice with 0.5 mM imidazole after incubation with cell lysate. Detailed Implementation
[0027] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.
[0028] Example 1: Preparation of magnetic composite material modified with nucleic acid aptamers.
[0029] The first step involved a one-step solvothermal synthesis of the magnetic graphene oxide composite matrix material MagG: 0.16 g of FeCl3·6H2O was weighed as the iron source, 0.03 g of sodium citrate as the stabilizer, and 0.03 g of graphene GO as the matrix for magnetic nucleus growth. These were dissolved in 30 mL of ethylene glycol and sonicated in an ice bath for 3 hours to form a homogeneous suspension. Then, 0.7 g of sodium acetate was added, and the mixture was mechanically stirred for 30 minutes. The mixture was then transferred to a reaction vessel and reacted at 190 °C for 6 hours. The prepared product was washed three times each with water and ethanol, and then vacuum dried to obtain the magnetic graphene oxide composite matrix material MagG.
[0030] The second step is to synthesize the polydopamine-coated magnetic composite material MagG@PDA: 30 mg of the MagG material prepared in the first step is weighed and ultrasonically dispersed in a mixture of 60 mL ethanol, 30 mL of 10 mM Tris-HCl buffer and 45 mL of water. 120 mg of dopamine is added, and the mixture is mechanically stirred at room temperature for 8 h. The product is then separated into solid and liquid phases using a magnet. The solid product is collected and washed three times each with water and ethanol, and then vacuum dried to obtain the polydopamine-coated magnetic composite material MagG@PDA.
[0031] The third step involves in-situ modification of Au nanoparticles to prepare MagG@PDA@Au: 12 mg of the MagG@PDA material prepared in the second step was dispersed in 150 mL of deionized water, and 240 μL of a 158 mM chloroauric acid solution was added. The reaction mixture was heated to 85 °C and maintained for 30 min. Then, 1.5 mL of 1 M sodium citrate was rapidly added, and the reaction continued for 1 h. The mixture was then washed three times each with water and ethanol, and dried under vacuum.
[0032] Step 4: Synthesis of the nucleic acid aptamer-modified magnetic graphene oxide composite material MagG@PDA@Au@Aptamer: 60 μL of 100 μM thiol-modified nucleic acid aptamer was added to 60 μL of 100 μM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution and 120 μL of 100 mM Tris-HCl buffer solution. The mixture was incubated at room temperature with shaking for 1 h. After the reaction was complete, the above solution was added to 80 μg of MagG@PDA@Au material and incubated at 4 °C with shaking for 12 h. The material was collected, washed with PBS, and then blocked with 110 μL of 1 mM mercaptohexanol (MCH). The material was collected using a magnet, washed with PBS, and MagG@PDA@@Au@Aptamer was obtained. A schematic diagram of the synthesis is shown in Figure 1.
[0033] This application is for sheet-like graphene, as shown in Figure 3a. After one-step synthesis of MagG, it can be seen from Figure 3b that Fe3O4 particles are uniformly dispersed on the sheet-like graphene oxide. After coating with polydopamine, it can be seen from Figure 3c that a thin shell appears on the surface of Fe3O4, and the opacity increases. Figure 3d shows that Au nanoparticles are also uniformly distributed on the surface of the material.
[0034] Figure 4 shows the infrared characterization of the MagG@PDA@Au@Aptamer material; 1100-1400 cm⁻¹ -1 Stretching vibration peaks of CO and CN in dopamine; 1060 cm⁻¹ -1 The stretching vibration peak of the phosphate group in the nucleoside of the nucleic acid aptamer; 1650 cm⁻¹ -1 The stretching and bending vibration peaks of PO in nucleosides within nucleic acid aptamers.
[0035] The detection of nucleic acid aptamer immobilization capacity of the prepared polydopamine-coated magnetic composite material MagG@PDA@Au is shown in the table below.
[0036] MagG@PDA@Au Results: Nucleic acid aptamer immobilization load nmol / mg 45; Relative standard deviation (RSD, %) 0.39; Number of replicates (n) 3 surface
[0037] Example 2: Selective separation of His-tagged RXRα protein using nucleic acid aptamer-modified magnetic composite material (using the nucleic acid aptamer-modified magnetic composite material prepared in Example 1 as the enrichment material).
[0038] (1) Expression of recombinant histidine retinol X receptor (His-tagged RXRα protein): Escherichia coli BL21(DE3) capable of expressing His-tagged RXRα protein was inoculated into 800 mL LB liquid medium (containing 50 μg / mL kanamycin) and incubated with shaking at 220 rpm and 37 °C until OD600 = 0.5-0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was incubated at 180 rpm and 20 °C for 12 h to induce protein expression. The induced bacterial culture was collected and added to pre-weighed centrifuge tubes. The tubes were centrifuged at 4 °C and 6500 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was collected. The wet weight of the bacterial pellet was weighed and stored at -80 °C.
[0039] (2) Preparation of His-tagged RXRα protein lysis buffer: After thawing the bacterial cells, wash them once with PBS (pH 7.4), then resuspend them in buffer (50 mM K2HPO4, 150 mM NaCl, pH 7.4), and add the protease inhibitor PMSF to a final concentration of 1.0 mM. Sonicate the buffer in an ice-water bath for 15 min, then centrifuge at 12000 rpm and 4°C for 15 min to obtain the crude supernatant containing His-tagged RXRα from *E. coli*.
[0040] (3) Isolation and enrichment of His-tagged RXRα protein using the nucleic acid aptamer-modified magnetic composite material MagG@PDA@Au@Aptamer: Take 5 mg of MagG@PDA@Au@Aptamer prepared in Example 1, wash once with 100 μL of loading buffer (50 mM K2HPO4, 150 mM NaCl, 10 mM imidazole, pH 7.4), add 200 μL of the above bacterial lysis buffer, and incubate with shaking at 4 °C for 2 h. Separate the material using a magnet and collect the supernatant. Wash the material once with 100 μL of loading buffer, then wash twice with 50 μL of washing buffer (50 mM K2HPO4, 150 mM NaCl, 20 mM imidazole, pH 7.4). Finally, elute twice with 50 μL of elution buffer (50 mM K2HPO4, 150 mM NaCl, 0.5 M imidazole, pH 7.4) at 4 °C. The purified His-tagged RXRα protein was analyzed using SDS-PAGE. The specific purification results are shown in Figure 5. As can be seen from lanes L and S, the band of the target recombinant protein in the supernatant after material treatment was significantly weakened compared to the total lysis buffer, indicating that most of the target protein bound to the material. The elution band (lane 2) shows that the target recombinant protein was enriched and purified, demonstrating high material selectivity.
[0041] For anyone skilled in the art, many possible variations and modifications can be made to the technical solutions of this invention, or equivalent embodiments can be modified based on the disclosed technical content, without departing from the scope of the technical solutions of this invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this invention without departing from the content of the technical solutions of this invention should still fall within the protection scope of the technical solutions of this invention.
Claims
1. A magnetic composite material coated with polydopamine and modified with nucleic acid aptamers, characterized in that, The core is made of magnetic graphene oxide, coated with a polydopamine shell, and the shell surface is bonded with alloy nanoparticles and nucleic acid aptamers; the bonding loading of the nucleic acid aptamers is 1-45 nmol / mg.
2. The magnetic composite material coated with polydopamine modified with nucleic acid aptamers according to claim 1, characterized in that, The binding loading of the nucleic acid aptamer is 45 nmol / mg.
3. The magnetic composite material coated with polydopamine modified with nucleic acid aptamers according to claim 1, characterized in that, The method for preparing the magnetic composite material includes: synthesizing magnetic graphene oxide MagG in one step via a solvothermal method; dispersing MagG in an alkaline dopamine solution to prepare a polydopamine-coated magnetic composite material MagG@PDA; bonding Au nanoparticles to the surface of MagG@PDA to obtain MagG@PDA@Au; and connecting a nucleic acid aptamer with a thiol-modified 5-terminus to the material surface to synthesize a nucleic acid aptamer-modified polydopamine-coated magnetic graphene oxide composite material MagG@PDA@Au@Aptamer.
4. The nucleic acid aptamer-modified polydopamine-coated magnetic graphene oxide composite material according to claim 3, characterized in that: The magnetic graphene oxide (MagG) consists of graphene with iron oxide particles dispersed on it. The graphene sheet diameter is 500 nm-4 μm, and the iron oxide magnetic core particle diameter is 100-300 nm.
5. The nucleic acid aptamer-modified polydopamine-coated magnetic graphene oxide composite material according to claim 3, characterized in that, The one-step solvothermal synthesis of magnetic graphene oxide (MagG) specifically involves: using 0.08-0.32 g FeCl3·6H2O as an iron source, 0.015-0.06 g sodium citrate as a stabilizer, and 0.015-0.06 g graphene GO as a matrix for magnetic nucleus growth. 15-60 mL of ethylene glycol is added for dissolution, and the mixture is sonicated for 1-4 h in an ice bath at 0-4°C to form a homogeneous suspension. Then, 0.35-1.4 g sodium acetate is added, and the mixture is mechanically stirred for 0.2-1 h. The mixture is then transferred to a reaction vessel and reacted at 160-220°C for 5-10 h. The prepared product is washed 2-4 times each with water and ethanol, and then vacuum dried to obtain the magnetic graphene oxide composite matrix material MagG.
6. The nucleic acid aptamer-modified polydopamine-coated magnetic graphene oxide composite material according to claim 5, characterized in that, The preparation method of the magnetic composite material includes the following steps: First, a one-step synthesis of MagG using a solvothermal method; Second, synthesis of polydopamine-coated magnetic composite material MagG@PDA: Weigh 15-60 mg of the MagG material prepared in the first step and ultrasonically disperse it in a mixture of 30-120 mL ethanol, 15-60 mL of 5-20 mM Tris-HCl buffer, and 20-90 mL of water. Add 60-240 mg of dopamine and mechanically stir at 25-60 °C for 6-12 h to prepare the product. Solid-liquid separation was performed using a magnet, and the solid product was collected. It was then washed 2-4 times each with water and ethanol, and vacuum dried to obtain the polydopamine-coated magnetic composite material MagG@PDA. The third step involved in-situ modification of MagG@PDA@Au nanoparticles: 6-24 mg of the MagG@PDA material prepared in the second step was dispersed in 75-300 mL of deionized water, and 120-480 μL of 75-300 mM chloroauric acid was added. The reaction mixture was heated to 50-120 °C and maintained for 0.2-1 h. Rapidly add 0.75-3 mL of 0.5-2.0 M sodium citrate, and continue the reaction for 0.5-2 h. Then wash with water and ethanol 2-4 times each, and dry under vacuum. Fourth step: Synthesize the nucleic acid aptamer-modified magnetic graphene oxide composite material MagG@PDA@Au@Aptamer: Take 30-120 μL of 50-200 μM thiol-modified nucleic acid aptamer, add 30-120 μL of 50-200 μM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution and 60-2... 40 μL of 50-200 mM Tris-HCl buffer solution was added and incubated with shaking at room temperature for 0.5-2 h. After the reaction was complete, the above solution was added to 40-160 μg of MagG@PDA@Au material and incubated with shaking at 4-37 °C for 6-18 h. The material was collected, washed with PBS, and then blocked with 40-160 μL of 0.5-2 mM mercaptohexanol (MCH). The material was collected by magnet and washed with PBS to obtain MagG@PDA@Au@Aptamer.
7. The magnetic composite material coated with polydopamine modified with nucleic acid aptamers according to claim 1, characterized in that: The nucleic acid aptamer is an aptamer for recombinant histidine-tagged proteins (5'-(SH)-GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-3').
8. The application of the nucleic acid aptamer-modified polydopamine-coated magnetic composite material as described in claim 1 as an affinity enrichment material.
9. The application according to claim 8, characterized in that: The magnetic composite material is directly added to the bacterial lysate of Escherichia coli for efficient and selective capture of target proteins.