Nanomagnetic beads for removing endotoxins from protein samples, preparation method and application thereof

Abstract

The present invention discloses a nanomagnetic bead for removing endotoxins from protein samples, as well as a preparation method and application thereof. The nanomagnetic bead comprises an inner core, an intermediate layer, and an outer layer; the inner core is a magnetic core, the intermediate layer is a metal oxide layer, and the outer layer is a boronic acid ligand layer. The metal oxide layer coordinates with the phosphate group in the endotoxin, and the boronic acid ligand layer combines with the cis-diphenol structure in the endotoxin. The nanomagnetic beads of the present invention achieve an endotoxin-clearing effect through a dual mechanism. The nanomagnetic beads for removing endotoxins from protein samples of the present invention can reduce the endotoxin content in the protein to below 0.5 EU / mL, meet the raw material requirements in the pharmaceutical field, and the protein will not be denatured. It has the advantages of simple and rapid operation, low cost, high endotoxin removal rate, and high protein recovery rate.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular to nanomagnetic beads for removing endotoxins from protein samples, and a preparation method and application thereof. Background Art

[0002] Proteins are fundamental building blocks of life and play a central role in biomedicine. Structural proteins such as collagen and elastin possess excellent biological properties and are widely used in wound healing, tissue engineering, and regenerative medicine. However, endotoxin contamination is a significant concern in the production and application of these proteins. Endotoxins primarily originate from the cell wall lysis of Gram-negative bacteria. Even trace amounts of endotoxins entering the human body can trigger a strong inflammatory response, leading to fever, diarrhea, shock, and even life-threatening consequences. Therefore, ensuring that the endotoxin content in protein products meets the standard for pharmaceutical applications (0.5 EU / mL) is crucial.

[0003] Traditional endotoxin removal methods involve high-temperature treatment above 250°C for at least 30 minutes and inactivation using strong acids and bases. However, high temperatures can lead to protein denaturation and loss of function, while strong acids and bases can disrupt the protein's three-dimensional structure and the chemical bonds between amino acids, rendering it inactive. Therefore, these extreme conditions make it difficult to effectively remove endotoxins while maintaining protein function.

[0004] Common methods for removing endotoxins include affinity chromatography, ion exchange chromatography, molecular sieve chromatography, hydrophobic chromatography, liquid phase separation, ultrafiltration, and adsorption. Affinity chromatography removes endotoxins by binding to specific ligands, which is relatively expensive. Ion exchange chromatography relies on the negative charge of endotoxins, which is not suitable for the presence of other negatively charged substances in the solution or in a high-salt environment. Molecular sieve chromatography uses molecular size differences for separation, but the separation effect is limited due to the close molecular weight of endotoxins and proteins. Hydrophobic chromatography is based on the hydrophobicity of endotoxins and is suitable for high-salt conditions, but the protein removal efficiency is not high. Liquid phase separation uses the two-phase separation characteristics of detergents to separate endotoxins and targets, which is prone to detergent residues and complex operations. Ultrafiltration uses the molecular weight difference between endotoxins and targets for separation, which results in a large loss of protein activity. Adsorption removes endotoxins by adsorbing them on the surface of the adsorbent, but the adsorbent easily adsorbs the active ingredients and has low efficiency.

[0005] There have been many studies in this field regarding endotoxin removal. For example, Chinese invention patent CN115386562A discloses a method for removing endotoxins from recombinant proteins using a high-efficiency endotoxin-removing affinity filler FF. The inventors added the high-efficiency endotoxin-removing affinity filler FF to a target protein solution after BlueSepHarose 6FastFlow affinity chromatography and lysine HyperD affinity chromatography, and then sterilized and filtered it through a 0.22 μm filter to obtain a purified protein solution. Chinese invention patent CN115298197A discloses a composition and kit for removing lipopolysaccharide, which contains a polypeptide capable of binding to lipopolysaccharide or its salt substitute as an active ingredient, and separates the polypeptide or its salt substitute from the protein sample by centrifugation or using a column. Chinese invention patent CN115876545A removes endotoxins by recombinantly expressing the three Sushi regions at the N-terminus of Factor C and coupling them to polymer magnetic microspheres. The Sushi123 magnetic beads are then mixed with the test sample and separated using a magnetic rack. However, the above patents still have problems such as low sample recovery rate, complicated endotoxin removal steps, and high requirements for users. Summary of the Invention

[0006] The present invention aims to address the above-mentioned technical problems by providing nanomagnetic beads for removing endotoxins from protein samples, as well as a preparation method and application thereof. The nanomagnetic beads of the present invention can simply and quickly reduce the endotoxin content in protein samples to the pharmaceutical technical requirement of 0.5 EU / mL without denaturing the protein. These beads offer advantages such as simple operation, low cost, high endotoxin removal rate, and high protein recovery rate.

[0007] In a first aspect of the present invention, a nanomagnetic bead for removing endotoxins from a protein sample is provided. The nanomagnetic bead comprises an inner core, an intermediate layer and an outer layer; the inner core is a magnetic core, the intermediate layer is a metal oxide layer, and the outer layer is a boronic acid ligand layer.

[0008] The metal oxide layer coordinates with the phosphate group in the endotoxin, and the boric acid ligand layer combines with the cis-diphenol structure in the endotoxin. The nanomagnetic beads of the present invention achieve the effect of clearing endotoxin through a dual mechanism.

[0009] Preferably, the boronic acid ligand layer includes at least one of 3,3'-(6-chloro-1,3,5-triazine-2,4-diyl)bis(azadiyl)bis(3,1-phenylene)diboronic acid, 4-carboxyphenylboronic acid, 4-hydroxyphenylboronic acid and 2,4-difluoro-3-formylphenylboronic acid.

[0010] Preferably, the magnetic core includes at least one of ferroferric oxide, cobalt ferrite, manganese ferrite and zinc ferrite.

[0011] Preferably, the metal oxide layer includes at least one of titanium dioxide, copper oxide, zinc oxide, aluminum oxide and iron oxide.

[0012] Preferably, the pore diameter of the metal oxide layer is 1 to 500 nm.

[0013] A second aspect of the present invention provides a method for preparing nanomagnetic beads for removing endotoxins from protein samples, comprising the following steps:

[0014] synthesizing the magnetic core by a solvothermal method;

[0015] Using a solvothermal method to wrap the magnetic core with a layer of the metal oxide layer;

[0016] Modifying the metal oxide layer with a silane coupling agent to obtain magnetic nanoparticles with amino groups on the surface;

[0017] The boric acid ligand layer is coated on the surface of the magnetic nanoparticles with amino groups on the surface to obtain the endotoxin dual-clearing nanomagnetic beads.

[0018] Preferably, the method for coating the boronic acid ligand layer on the surface of the magnetic nanoparticles with amino groups on the surface is: the boronic acid ligand layer and N,N-diisopropylethylamine are dissolved in N,N-dimethylformamide, the magnetic nanoparticles with amino groups on the surface are added, dispersed, reacted at 0°C to 100°C for 1 to 24 hours under the protection of inert gas, magnetically separated, the supernatant removed, washed, and dried to obtain the nanomagnetic beads for removing endotoxins from protein samples.

[0019] Preferably, the silane coupling agent is 3-aminopropyltriethoxysilane.

[0020] A third aspect of the present invention provides an application of nanomagnetic beads for removing endotoxins from protein samples.

[0021] A fourth aspect of the present invention provides a method for removing endotoxins from a protein sample, comprising the following steps:

[0022] resuspending the nanomagnetic beads for removing endotoxin from a protein sample described above with a washing solution, mixing, performing magnetic separation, and removing the washing solution to obtain the nanomagnetic beads for use;

[0023] The prepared nanomagnetic beads were resuspended with the protein sample, incubated at 4°C to 70°C for 10 min to 360 min, and the supernatant was removed.

[0024] Preferably, in the above method for removing endotoxins from a protein sample, the concentration of the standby nanomagnetic beads is 0.001 mg / mL to 100 mg / mL.

[0025] Preferably, in the above method for removing endotoxins from protein samples, all solutions are prepared with pyrogen-free water, the pipette tips used are pyrogen-free, test tubes and other glass containers are subjected to high-temperature pyrogen removal treatment at 250°C, and all operations are performed in a clean bench.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The preparation process of the nanomagnetic beads for removing endotoxins from protein samples provided by the present invention is simple and convenient, and can be easily scaled up for production.

[0028] (2) The present invention obtains a nanomagnetic bead with dual endotoxin removal by wrapping a layer of porous (pore size 1 to 500 nm) metal oxide outside the magnetic core and fixing phenylboronic acid material on the surface of the metal oxide.

[0029] (3) The core of the nanomagnetic beads provided by the present invention is a magnetic core, which can be quickly separated from the solution within 30 seconds in the presence of an external magnetic field.

[0030] (4) The method for removing endotoxins from protein samples provided by the present invention is simple to operate. It only requires mixing the washed nanomagnetic beads with the sample, incubating the mixture, and then separating the mixture using an external magnetic field to remove the endotoxins from the sample.

[0031] (5) The nanomagnetic beads for removing endotoxins from protein samples provided by the present invention can efficiently remove endotoxins from protein samples, and the reaction conditions are mild, which will not cause denaturation of protein components and achieve a high protein recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flow chart for the preparation of nanomagnetic beads for removing endotoxins from protein samples;

[0033] Figure 2 For the high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy characterization of DBA;

[0034] Figure 3 For the characterization of nanomagnetic beads;

[0035] Figure 4 It is the endotoxin concentration standard curve and the double endotoxin clearance verification of Fe3O4@TiO2-DBA;

[0036] Figure 5 This is a picture showing the effect of nanomagnetic beads in removing endotoxin from elastin samples;

[0037] Figure 6 This is a picture showing the effect of nanomagnetic beads removing endotoxins from collagen samples;

[0038] Figure 7 The standard curve of protein concentration and protein recovery rate are shown in Figure 2. DETAILED DESCRIPTION

[0039] The present invention is further described below by way of examples, but is not intended to limit the present invention. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included within the scope of protection of the present invention.

[0040] The invention provides nanomagnetic beads for removing endotoxins in protein samples. The nanomagnetic beads comprise an inner core, an intermediate layer and an outer layer; the inner core is a magnetic core, the intermediate layer is a metal oxide layer, and the outer layer is a boric acid ligand layer.

[0041] In some examples, the boronic acid ligand layer includes at least one of 3,3'-(6-chloro-1,3,5-triazine-2,4-diyl)bis(azadiyl)bis(3,1-phenylene)diboronic acid (DBA), 4-carboxyphenylboronic acid, 4-hydroxyphenylboronic acid, and 2,4-difluoro-3-formylphenylboronic acid.

[0042] In some examples, the magnetic core includes at least one of ferroferric oxide, cobalt ferrite, manganese ferrite, and zinc ferrite.

[0043] In some examples, the metal oxide layer includes at least one of titanium dioxide, copper oxide, zinc oxide, aluminum oxide, and iron oxide.

[0044] The present invention provides a method for preparing nanomagnetic beads for removing endotoxins from protein samples, comprising the following steps:

[0045] synthesizing the magnetic core by a solvothermal method;

[0046] Using a solvothermal method to wrap the magnetic core with a layer of the metal oxide layer;

[0047] Modifying the metal oxide layer with a silane coupling agent to obtain magnetic nanoparticles with amino groups on the surface;

[0048] The boric acid ligand layer is coated on the surface of the magnetic nanoparticles with amino groups on the surface to obtain the nanomagnetic beads for removing endotoxins in protein samples.

[0049] In some examples, the method for coating the surface of the magnetic nanoparticles with amino groups on the surface with the boronic acid ligand layer is as follows: the boronic acid ligand layer and N,N-diisopropylethylamine are dissolved in N,N-dimethylformamide (DMF), the magnetic nanoparticles with amino groups on the surface are added and dispersed, and the reaction is carried out at 0°C to 100°C for 1 to 24 hours under the protection of inert gas, and magnetic separation is performed. The supernatant is removed, washed, and dried to obtain the nanomagnetic beads for removing endotoxins from protein samples.

[0050] The present invention provides a method for removing endotoxins from a protein sample, comprising the following steps:

[0051] resuspending the nanomagnetic beads for removing endotoxin from a protein sample described above with a washing solution, mixing, performing magnetic separation, and removing the washing solution to obtain the nanomagnetic beads for use;

[0052] The prepared nanomagnetic beads were resuspended with the protein sample, incubated at 4°C to 70°C for 10 min to 360 min, and the supernatant was removed.

[0053] In some examples, in the method for removing endotoxins from a protein sample, the concentration of the standby nanomagnetic beads is 0.001 mg / mL to 100 mg / mL.

[0054] Unless otherwise specified, all reagents used in the examples can be obtained commercially.

[0055] Example 1 Preparation of Nanomagnetic Beads for Removing Endotoxins from Protein Samples

[0056] (1) Synthesis of Fe3O4 magnetic core of nanomagnetic beads by solvothermal method

[0057] 3.24 g of ferric chloride hexahydrate ( ) and 7.24 g of anhydrous sodium acetate were added to ethylene glycol (60 mL) and mechanically stirred for 30 minutes. The homogeneously mixed solution was transferred to a 100 mL reactor and reacted at 200°C for 10 hours, followed by slow cooling for 24 hours. The supernatant was discarded using an external magnet, and the magnetic product was washed with ultrapure water, ethanol, and methanol, respectively, and dried in vacuo at 55°C.

[0058] (2) The Fe3O4 magnetic core is wrapped with a layer of porous TiO2 by a solvent thermal method.

[0059] 200 mg of the Fe3O4 magnetic cores obtained in step (1) were added to a mixture of N,N-dimethylformamide (20 mL) and isopropanol (60 mL) and ultrasonicated for 30 min to uniformly disperse them. 2 mL of tetrabutyl titanate was added dropwise to the above solution and mechanically stirred at room temperature for 30 min. The solution was transferred to a 100 mL reactor and reacted at 200°C for 22 h, followed by cooling for 4 h. After separation using an external magnet, the supernatant was discarded, the product was washed with ultrapure water and ethanol, respectively, and dried in vacuo at 55°C.

[0060] (3) Modifying the surface of the porous TiO2 coating described in step (2) with 3-aminopropyltriethoxysilane

[0061] 300 mg of Fe3O4@TiO2 obtained in step (2) and 0.9 mL of 3-aminopropyltriethoxysilane were added to 30 mL of toluene. The mixture was uniformly dispersed by ultrasonication for 30 min and then reacted at 70°C under nitrogen for 8 h. After the reaction, the supernatant was discarded after separation using an external magnet. The product was washed with ethanol and dried in vacuo at 55°C to obtain Fe3O4@TiO2-NH2 magnetic nanoparticles with amino groups on the surface.

[0062] (4) The nanomagnetic beads with amino groups on the surface described in step (3) are reacted with DBA to obtain the nanomagnetic beads for removing endotoxins from the protein sample.

[0063] 1.39 g of 3-aminophenylboronic acid monohydrate and 0.92 g of sodium acetate were dissolved in acetic acid-water (10 mL, 1:1, v / v), and then a solution of 0.84 g of cyanuric chloride in acetic acid (20 mL) was added. After reacting in an ice-water bath for 3 h, the resulting precipitate was collected by centrifugation, washed sequentially with acetic acid and water, and dried in vacuo at 80°C to obtain DBA. The product was characterized by autoflex speed high-resolution mass spectrometry (HRMS) and AVANCE 400 NMR spectrometer ( 1 H NMR) characterization of DBA, the results are as follows Figure 2 HRMS (MALDI-TOF, m / z) calculated values ​​of C 15 H 14 B2ClN5O4[M+H] + 386.10, measured value 386.008. 1 H NMR (400 MHz, DMSO-d6) δ is as follows: 10.142 (s, 2H), 8.031 (s, 4H), 7.779 (s, 4H), 7.500 (d, 2H), 7.276 (t, 2H).

[0064] 500 mg of DBA and 218 μL of N,N-diisopropylethylamine were dissolved in 20 mL of DMF. Then, 500 mg of Fe₃O₄@TiO₂-NH₂ was added and ultrasonically dispersed for 30 minutes. The mixture was then reacted at 90°C for 8 hours under nitrogen. After the reaction, the supernatant was discarded using an external magnet, washed sequentially with DMF and ethanol, and dried in vacuo at 55°C to obtain Fe₃O₄@TiO₂-DBA.

[0065] The preparation process of the nanomagnetic beads for removing endotoxins from protein samples of the present invention is as follows: Figure 1 shown.

[0066] Test example

[0067] 1. Characterization of Nanomagnetic Beads for Removing Endotoxins from Protein Samples

[0068] The Fe3O4, Fe3O4@TiO2, Fe3O4@TiO2-NH2, and Fe3O4@TiO2-DBA nanoparticles prepared in Example 1 were characterized respectively. The specific steps are as follows:

[0069] (1) Scanning electron microscopy (SEM) characterization: The nanoparticles in each step of Example 1 were dispersed in ethanol, and a small amount was dropped on a silicon wafer until completely dry. The dried sample was sprayed with gold and characterized using a Sigma 360 scanning electron microscope.

[0070] (2) Fourier transform infrared spectrometer (FTIR) characterization: The nanoparticles in each step of Example 1 were mixed and ground with potassium bromide at a mass ratio of 1:200. The ground powders were pressed into tablets and characterized using a TENSOR 27 infrared spectrometer.

[0071] (3) X-ray photoelectron spectroscopy (XPS) characterization: The full spectrum and B element fine spectrum of the nanomagnetic beads Fe3O4@TiO2-DBA were characterized using a Shimadzu Axis Supra X-ray photoelectron spectrometer.

[0072] (4) Vibrating sample magnetometer (VSM) characterization: The nanoparticles in each step of Example 1 were characterized using an EV9 vibrating sample magnetometer.

[0073] like Figure 3 As shown in the figure, the Fe3O4@TiO2, Fe3O4@TiO2-NH2 and Fe3O4@TiO2-DBA all have porous structures; FTIR characterizes the successful synthesis of each step of Fe3O4@TiO2-DBA, among which 580 cm -1 The absorption peak at 480 cm corresponds to the Fe-O bond; -1 and 690 cm -1 The absorption peaks at 1110 cm in the FT-IR curve of Fe3O4@pTiO2-NH2 are Ti-O and Ti-O-Ti stretching vibration peaks respectively. -1 and 1030 cm -1 Corresponding to the stretching vibration of Si-O-Si bond, 2930 cm -1 and 3435 cm -1 are related to the stretching vibration of -CH2 group and -NH2 respectively; 1380 cm -1 The small peak at π / 4 is derived from the BO bond of DBA. XPS characterization further demonstrates the successful synthesis of the Fe₃O₄@TiO₂-DBA. VSM reveals that the synthesized nanoparticles possess superparamagnetic properties, enabling rapid separation of the Fe₃O₄@TiO₂-DBA from the sample.

[0074] 2. Double endotoxin removal effect of nanomagnetic beads

[0075] The Fe3O4, Fe3O4@TiO2, and Fe3O4@TiO2-DBA prepared in Example 1 were tested for their dual endotoxin removal effects. The specific steps are as follows:

[0076] (1) Weigh 5 mg of the nanomagnetic beads Fe3O4, Fe3O4@TiO2, and Fe3O4@TiO2-DBA in Example 1 respectively into a pyrogen-free test tube, add 1 mL of pyrogen-free water to resuspend the nanomagnetic beads, shake to mix, and then use an external magnet to remove the washing solution. Repeat this washing process 3-5 times.

[0077] (2) Add 1 mL of endotoxin solution (25 EU / mL) to resuspend the nanomagnetic beads, incubate with shaking at 25°C for 60 minutes, and recover the supernatant using an external magnet.

[0078] (3) Using the kinetic turbidimetric method, the standard curve of endotoxin concentration was obtained according to the method described in the kit instructions: lgT = 2.8791-0.2120 lgC (R 2 = 0.9962; T is the reaction time between the lysate and the sample by kinetic turbidimetric method, in seconds; C is the endotoxin concentration in the sample, in EU / mL). Figure 4 (a) shown.

[0079] (4) Using the kinetic turbidimetric method, determine the endotoxin concentrations of the endotoxin solution and supernatant in (2) according to the method described in the kit instructions, and calculate the endotoxin removal rate.

[0080] The experimental results are as follows Figure 4 As shown in (b), it can be seen that Fe3O4@TiO2 has a certain endotoxin clearance ability with a clearance rate of 39.4%, while the endotoxin clearance rate of Fe3O4@TiO2-DBA is 99.8%, indicating that Fe3O4@TiO2-DBA has dual endotoxin clearance ability.

[0081] 3. Nanomagnetic beads to remove endotoxins from recombinant elastin (Ela) samples

[0082] The Fe3O4@TiO2-DBA prepared in Example 1 was tested for its endotoxin removal effect on Ela samples. The specific steps are as follows:

[0083] (1) Weigh 5 mg of Fe3O4@TiO2-DBA nanoparticles into a pyrogen-free test tube, add 1 mL of pyrogen-free resuspended magnetic nanoparticles, shake to mix, and then use an external magnet to remove the washing solution. Repeat this washing process 3-5 times.

[0084] (2) Add 1 mL of endotoxin-containing Ela sample (0.6 mg / mL), resuspend the magnetic nanoparticles with the sample, incubate with shaking at 25°C for 60 min, and use an external magnet to recover the Ela supernatant after removing endotoxin.

[0085] (3) Using the gel-based limulus amebocyte lysate, determine the endotoxin content in the supernatant of step (2) according to the method described in the kit instructions.

[0086] The experimental results are as follows Figure 5 As shown, the sensitivity of the gel-based limulus amebocyte lysate is 0.125 EU / mL, and the 0.6 mg / mL Ela sample is diluted 2×10 4 The endotoxin test result was positive after 2-fold dilution, indicating an endotoxin concentration greater than 0.125 EU / mL. The endotoxin test result of the supernatant treated with Fe3O4@TiO2-DBA nanoparticles was negative after 2-fold dilution, indicating an endotoxin concentration less than 0.125 EU / mL. This indicates that Fe3O4@TiO2-DBA can remove endotoxins exceeding 2500 EU / mL in the Ela sample, and the endotoxin content of the treated elastin sample meets the standards for medical applications.

[0087] 4. Nanomagnetic beads to remove endotoxins from animal-extracted collagen (Col-Ⅰ) samples

[0088] The Fe3O4@TiO2-DBA prepared in Example 1 was tested for its endotoxin removal effect on Col-Ⅰ samples. The specific steps are as follows:

[0089] (1) Weigh 5 mg of Fe3O4@TiO2-DBA nanoparticles into a pyrogen-free test tube, add 1 mL of pyrogen-free water to resuspend the magnetic nanoparticles, shake to mix, and then use an external magnet to remove the washing solution. Repeat this washing process 3-5 times.

[0090] (2) Add 1 mL of endotoxin-containing Col-Ⅰ sample (1 mg / mL), resuspend the magnetic nanoparticles with the sample, incubate with shaking at 25°C for 60 min, and use an external magnet to recover the Col-Ⅰ supernatant after removing endotoxin.

[0091] (3) Use the kinetic turbidimetric method with Limulus amebocyte lysate and determine the endotoxin content in the supernatant of (2) according to the method described in the kit instructions.

[0092] like Figure 6 As shown in the figure, the endotoxin concentration in the Col-Ⅰ sample was 3.00 EU / mL, and the nanomagnetic beads Fe3O4@TiO2-DBA were able to remove 98.99% of the endotoxin in the Col-Ⅰ sample. The endotoxin concentration in the supernatant (2) was 0.031 EU / mL, which met the medical application standards.

[0093] 5. Nanomagnetic beads to remove endotoxins from recombinant collagen (R-Col) samples

[0094] The Fe3O4@TiO2-DBA prepared in Example 1 was tested for its endotoxin removal effect on the R-Col sample. The specific steps are as follows:

[0095] (1) Weigh 5 mg of Fe3O4@TiO2-DBA nanoparticles into a pyrogen-free test tube, add 1 mL of pyrogen-free water to resuspend the nanoparticles, shake to mix, and use an external magnet to remove the washing solution. Repeat this washing process 3-5 times.

[0096] (2) Add 1 mL of endotoxin-containing R-Col sample (0.1 mg / mL), resuspend the nanomagnetic beads with the sample, incubate with shaking at 25°C for 60 min, and use an external magnet to recover the R-Col supernatant after removing endotoxin.

[0097] (3) Use the kinetic turbidimetric method with Limulus amebocyte lysate and determine the endotoxin content in the supernatant of (2) according to the method described in the kit instructions.

[0098] like Figure 6 As shown, the endotoxin concentration in the R-Col sample was 101.70 EU / mL. The nanomagnetic beads Fe3O4@TiO2-DBA were able to remove 99.9% of the endotoxin in the R-Col sample. The endotoxin concentration in the supernatant (2) was 0.042 EU / mL, which met the medical application standards.

[0099] 6. Protein recovery determination

[0100] The protein recovery rate in steps 2, 3, and 4 of this test example was determined as follows:

[0101] (1) The BCA protein concentration assay kit was used and the protein concentration standard curve was obtained according to the method described in the kit instructions: y = 0.8944x + 0.1465 (R 2 = 0.9987);

[0102] (2) The protein concentration of the solution before and after endotoxin removal in steps 2, 3, and 4 of this test example was determined according to the method described in the instructions of the BCA protein concentration detection kit, and the protein recovery rate was calculated.

[0103] like Figure 7 As shown in the results, after the removal of endotoxin, the recovery rates of recombinant elastin (Ela), recombinant collagen (R-Col) and animal extracted collagen (Col-Ⅰ) were all greater than 92%, achieving high protein recovery rates.

[0104] 7. Determination of denatured collagen content

[0105] The content of denatured collagen in the solution before and after the endotoxin removal in step 4 of this test example was determined in the following specific steps:

[0106] (1) Denatured collagen detection kit (Gansu Tianji Biotechnology Co., Ltd.) was used, and the standard curve of denatured collagen concentration was obtained according to the method described in the kit instructions: y = -2595.4 lgC + 40692, R 2 =0.9922; C is the concentration of denatured collagen, in ng / mL;

[0107] (2) The denatured collagen concentration of the solution before and after endotoxin removal in step 4 of this test example was determined according to the method described in the instructions of the denatured collagen detection kit.

[0108] As shown in Table 1, the nanomagnetic beads do not cause protein denaturation after removing endotoxin from protein samples.

[0109] Table 1 Denatured collagen content before and after endotoxin removal

[0110]

[0111] The above experimental results show that the preparation process of the nanomagnetic beads with dual endotoxin removal provided by the present invention is simple and convenient, and can be easily scaled up for production; the endotoxin removal method provided by the present invention is easy to operate and has rapid separation; the nanomagnetic beads with dual endotoxin removal provided by the present invention can efficiently remove endotoxins from protein samples, and the reaction conditions are mild, without causing protein denaturation, thereby achieving a high protein recovery rate.

[0112] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, and the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A nanomagnetic bead for removing endotoxins from protein samples, characterized in that: The nanomagnetic beads include an inner core, an intermediate layer, and an outer layer; the inner core is a magnetic core, the intermediate layer is a metal oxide layer, and the outer layer is a boronic acid ligand layer; the boronic acid ligand layer includes 3,3'-(6-chloro-1,3,5-triazine-2,4-diyl)bis(azadiyl)bis(3,1-phenylene)diboronic acid; the metal oxide layer is porous titanium dioxide with a diameter of 1 to 500 nm; the metal oxide layer is modified with a silane coupling agent to obtain magnetic nanoparticles with amino groups on the surface; and the boronic acid ligand layer is coated on the surface of the magnetic nanoparticles with amino groups on the surface.

2. The nanomagnetic beads for removing endotoxins from protein samples according to claim 1, characterized in that The magnetic core includes at least one of ferroferric oxide, cobalt ferrite, manganese ferrite and zinc ferrite.

3. The method for preparing nanomagnetic beads for removing endotoxins from protein samples according to any one of claims 1 to 2, characterized in that: The following steps are involved: synthesizing the magnetic core by a solvothermal method; Using a solvothermal method to wrap the magnetic core with a layer of the metal oxide layer; Modifying the metal oxide layer with a silane coupling agent to obtain magnetic nanoparticles with amino groups on the surface; The boric acid ligand layer is coated on the surface of the magnetic nanoparticles with amino groups on the surface to obtain the nanomagnetic beads for removing endotoxins in protein samples.

4. The method for preparing nanomagnetic beads for removing endotoxins from protein samples according to claim 3, characterized in that: The method for coating the boronic acid ligand layer on the surface of the magnetic nanoparticles with amino groups on the surface is as follows: the boronic acid ligand layer and N,N-diisopropylethylamine are dissolved in N,N-dimethylformamide, the magnetic nanoparticles with amino groups on the surface are added and dispersed, and the reaction is carried out at 0°C to 100°C for 1 to 24 hours under the protection of inert gas, and magnetic separation is performed. The supernatant is removed, and the solution is washed and dried to obtain the endotoxin-double-removing nanomagnetic beads.

5. The method for preparing nanomagnetic beads for removing endotoxins from protein samples according to claim 4, characterized in that: The silane coupling agent is 3-aminopropyltriethoxysilane.

6. Use of the nanomagnetic beads according to any one of claims 1 to 2 for removing endotoxins from protein samples, characterized in that: Used to remove endotoxins from protein samples.

7. A method for removing endotoxins from a protein sample, characterized in that: The following steps are involved: Resuspend the nanomagnetic beads for removing endotoxin from a protein sample according to any one of claims 1 to 2 with a washing solution, mix well, perform magnetic separation, remove the washing solution, and obtain the nanomagnetic beads for use; The prepared nanomagnetic beads were resuspended with the protein sample, incubated at 4°C to 70°C for 10 min to 360 min, and the supernatant was removed.

8. The method for removing endotoxins from a protein sample according to claim 7, wherein: The concentration of the standby nanomagnetic beads is 0.001 mg / mL to 100 mg / mL.

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