Nanometer magnetic bead for removing endotoxin in protein sample as well as preparation method and application of nanometer magnetic bead

Abstract

The invention discloses a nano magnetic bead for removing endotoxin in a protein sample as well as a preparation method and application of the nano magnetic bead. The nano magnetic bead comprises an inner core, a middle layer and an outer layer, the inner core is a magnetic core, the middle layer is a metal oxide layer, and the outer layer is a boric acid ligand layer. The metal oxide layer is coordinated with a phosphate group in endotoxin, the boric acid ligand layer is combined with a cis-diphenol structure in the endotoxin, and the nano magnetic bead achieves an effect of removing the endotoxin through a dual mechanism. The nano magnetic bead for removing the endotoxin in the protein sample can reduce the content of the endotoxin in the protein to 0.5 EU / mL or below, meets the requirements of raw materials in the field of medicine, does not generate denaturation of the protein, and has the advantages of simplicity and rapidness in operation, low cost, high endotoxin removal rate, high protein recovery rate and the like.

Description

Technical Field

[0001] The invention relates to the technical field of protein purification, and in particular to a nanomagnetic bead for removing endotoxin in a protein sample, and a preparation method and application thereof. Background Art

[0002] Proteins are basic molecules that build life and play a core role in the biomedical field. Structural proteins such as collagen and elastin have excellent biological properties and are widely used in fields such as wound healing, tissue engineering, and regenerative medicine. However, when producing and applying these proteins, the problem of endotoxin contamination cannot be ignored. Endotoxins mainly come from the cell wall lysis of Gram-negative bacteria. Even if a trace amount of endotoxin enters the human body, it may cause a strong inflammatory response, leading to fever, diarrhea, shock, and even life-threatening. Therefore, it is crucial to ensure that the endotoxin content in protein products meets the medical application standards (0.5 EU / mL).

[0003] Traditional methods for removing endotoxins include high-temperature treatment at over 250°C for at least 30 minutes and inactivation using strong acids and alkalis. However, high temperatures can lead to denaturation of protein structure and loss of function, while strong acid and alkali conditions can destroy the three-dimensional structure of proteins and the chemical bonds of amino acids, thereby rendering the proteins 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 costly; ion exchange chromatography relies on the negative charge of endotoxins, which is not suitable for other negatively charged substances in the solution and high salt environments; 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 removal efficiency of proteins is not high; liquid phase separation uses the two-phase separation characteristics of detergents to separate endotoxins and targets, which is prone to residual detergents and complicated 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 adsorption on the surface of the adsorbent, which is easy to adsorb effective ingredients and has low efficiency.

[0005] There have been many studies in this field on the removal of endotoxins. For example, Chinese invention patent CN115386562A discloses a method for removing endotoxins from recombinant proteins using a highly efficient endotoxin-removing affinity filler FF. The inventors added a highly efficient endotoxin-removing affinity filler FF to the target protein solution after BlueSepHarose 6FastFlow affinity chromatography and lysine HyperD affinity chromatography, and then sterilized and filtered it with a 0.22 μm filter to obtain a purified protein solution; Chinese invention patent CN115298197A discloses a composition and a kit for removing lipopolysaccharide, which contains a polypeptide having the ability to bind to lipopolysaccharide or its salt substitute as an active ingredient, and the polypeptide or its salt substitute is separated 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, mixing Sushi123 magnetic beads with the sample to be tested and separating them using a magnetic frame. 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 purpose of the present invention is to provide a nanomagnetic bead for removing endotoxin from a protein sample and a preparation method and application thereof in view of the above technical problems. The nanomagnetic bead for removing endotoxin from a protein sample of the present invention can simply and quickly reduce the endotoxin content in the protein sample to the original drug requirement (0.5 EU / mL) in the pharmaceutical field without denaturing the protein, and has the advantages of simple operation, low cost, high endotoxin removal rate and high protein recovery rate.

[0007] The first aspect of the present invention provides a nanomagnetic bead for removing endotoxin in a protein sample, wherein 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 boric 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 size of the metal oxide layer is 1 to 500 nm.

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

[0014] The magnetic core is synthesized 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 is removed, washed, and dried to obtain the nanomagnetic beads for removing endotoxins in protein samples.

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

[0020] The third aspect of the present invention provides an application of nanomagnetic beads for removing endotoxins in protein samples, which is used to remove endotoxins in 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 in any one of the above items with a washing solution, mixing, performing magnetic separation, and removing the washing solution to obtain standby nanomagnetic beads;

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

[0024] Preferably, in the above method for removing endotoxin 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 endotoxin 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 inner 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 by 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, will not cause denaturation of protein components, and achieve a high protein recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2 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 standard curve of endotoxin concentration 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 in removing endotoxin 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 the present invention is not limited thereto. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0040] The invention provides a nanomagnetic bead for removing endotoxin in a protein sample. 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 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 in protein samples, comprising the following steps:

[0045] The magnetic core is synthesized 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 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 (DMF), 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 is removed, washed, and dried to obtain the nanomagnetic beads for removing endotoxins in protein samples.

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

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

[0052] The spare nanomagnetic beads are resuspended with the protein sample, incubated at 4°C to 70°C for 10 min to 360 min, and the supernatant is 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 Endotoxin 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 min. The uniformly mixed solution was transferred to a 100 mL reactor and reacted at 200 °C for 10 h, followed by slow cooling for 24 h. The supernatant was discarded using an external magnet, and the magnetic product was washed with ultrapure water, ethanol, and methanol, respectively, and dried in vacuum at 55 °C.

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

[0059] 200 mg of 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 make them uniformly dispersed. 2 mL of tetrabutyl titanate was added dropwise to the above solution, and mechanically stirred for reaction at room temperature for 30 min. The solution was transferred to a 100 mL reactor, reacted at 200 °C for 22 h, and cooled for 4 h. After separation using an external magnet, the supernatant was discarded, the product was washed with ultrapure water and ethanol, respectively, and vacuum dried 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, and the mixture was uniformly dispersed by ultrasonication for 30 min, and then reacted at 70°C for 8 h under nitrogen protection. After the reaction, the supernatant was discarded after separation using an external magnet, and the product was washed with ethanol and dried in vacuum at 55°C to obtain magnetic nanoparticles Fe3O4@TiO2-NH2 with amino groups on the surface.

[0062] (4) reacting the nanomagnetic beads with amino groups on the surface described in step (3) 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 with acetic acid and water in sequence, 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 value 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 DBA and 218 μL N, N-diisopropylethylamine were dissolved in DMF (20 mL), and then 500 mg Fe3O4@TiO2-NH2 was added and ultrasonically dispersed for 30 min, followed by reaction at 90°C for 8 h under nitrogen protection. After the reaction, the supernatant was discarded using an external magnet, washed with DMF and ethanol in turn, and dried in vacuum at 55°C to obtain Fe3O4@TiO2-DBA.

[0065] The preparation process of the nanomagnetic beads for removing endotoxin 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 prepared in Example 1 were characterized by nanomagnetic beads, and the specific steps were as follows:

[0069] (1) Scanning electron microscopy (SEM) characterization: The nanoparticles in each step of Example 1 were dispersed in ethanol respectively, and a small amount was dropped on a silicon wafer until it was completely dried. The dried sample was subjected to gold spraying 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-1 corresponds to the Fe-O bond; -1 and 690 cm -1 The absorption peaks at 1110 cm-1 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 They are related to the stretching vibration of -CH2 group and -NH2 respectively; 1380 cm -1 The small peak at comes from the BO bond of DBA. XPS characterization further shows that the Fe3O4@TiO2-DBA has been successfully synthesized. VSM shows that the synthesized nanomagnetic beads have superparamagnetism and Fe3O4@TiO2-DBA can be quickly separated 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 min, and recover the supernatant using an external magnet.

[0078] (3) The standard curve of endotoxin concentration was obtained by using the dynamic turbidity method of horseshoe crab reagent and following the method described in the kit instructions: lgT = 2.8791-0.2120 lgC (R 2 = 0.9962; T is the reaction time between the kinetic turbidity method horseshoe crab reagent and the sample, unit s; C is the endotoxin concentration in the sample, unit EU / mL). The results are as follows Figure 4 (a) shown.

[0079] (4) Using the kinetic turbidity method, determine the endotoxin concentration 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 removal ability with a removal rate of 39.4%, while the endotoxin removal rate of Fe3O4@TiO2-DBA is 99.8%, indicating that Fe3O4@TiO2-DBA has dual endotoxin removal capabilities.

[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 effect of removing endotoxin from 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 use an external magnet to remove the washing solution. Repeat this washing process 3-5 times.

[0084] (2) Add 1 mL of Ela sample containing endotoxin (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 method horseshoe crab reagent, determine the endotoxin content of the supernatant in (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 times dilution, that is, the endotoxin concentration was greater than 0.125 EU / mL; the endotoxin test result was negative after the supernatant was diluted 2 times after the treatment with nanomagnetic beads Fe3O4@TiO2-DBA, and the concentration was lower than 0.125 EU / mL. It can be seen that Fe3O4@TiO2-DBA can remove endotoxins exceeding 2500 EU / mL in Ela samples, and the endotoxin content of elastin samples after treatment meets the medical application standards.

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

[0088] The Fe3O4@TiO2-DBA prepared in Example 1 was tested for its effect of removing endotoxin from 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 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 horseshoe crab reagent to determine the endotoxin content in the supernatant of (2) according to the method described in the kit instructions.

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

[0093] 5. Nano-magnetic 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 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 horseshoe crab reagent to 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 is 101.70 EU / mL. The nanomagnetic beads Fe3O4@TiO2-DBA can remove 99.9% of the endotoxin in the R-Col sample. (2) The endotoxin concentration in the supernatant is 0.042 EU / mL, which meets the medical application standards.

[0099] 6. Protein recovery rate determination

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

[0101] (1) The BCA protein concentration determination 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 the removal of endotoxin 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, after removing endotoxin, the recovery rates of recombinant elastin (Ela), recombinant collagen (R-Col) and animal extracted collagen (Col-Ⅰ) were all greater than 92%, achieving a high protein recovery rate.

[0104] 7. Determination of denatured collagen content

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

[0106] (1) A 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 the endotoxin removal in step 4 of this test example was measured according to the method described in the instructions of the denatured collagen detection kit.

[0108] As shown in Table 1, the nanomagnetic beads will 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 it is easy to realize large-scale 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 in protein samples, and the reaction conditions are mild, will not cause protein denaturation, and achieve a high protein recovery rate.

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

Claims

1. A nanomagnetic bead for removing endotoxin from a protein sample, characterized in that: The nano magnetic 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 boric acid ligand layer.

2. The nanomagnetic beads for removing endotoxin from protein samples according to claim 1, characterized in that: 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.

3. The nanomagnetic beads for removing endotoxin 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.

4. The nanomagnetic beads for removing endotoxin from protein samples according to claim 1, characterized in that: The metal oxide layer includes at least one of titanium dioxide, copper oxide, zinc oxide, aluminum oxide and iron oxide.

5. The method for preparing nanomagnetic beads for removing endotoxin from protein samples according to any one of claims 1 to 4, characterized in that: The following steps are involved: The magnetic core is synthesized 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.

6. The method for preparing nanomagnetic beads for removing endotoxin from protein samples according to claim 5, 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, dispersed, reacted at 0°C to 100°C for 1 to 24 hours under the protection of inert gas, magnetically separated, the supernatant is removed, washed, and dried to obtain the endotoxin-double-removing nanomagnetic beads.

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

8. Use of the nanomagnetic beads for removing endotoxins from protein samples according to any one of claims 1 to 4, characterized in that: Used to remove endotoxins from protein samples.

9. 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 4 with a washing solution, mix well, perform magnetic separation, remove the washing solution, and obtain the nanomagnetic beads for use; The spare nanomagnetic beads are resuspended with the protein sample, incubated at 4°C to 70°C for 10 min to 360 min, and the supernatant is removed.

10. A method for removing endotoxins from a protein sample according to claim 9, characterized in that: The concentration of the standby nanomagnetic beads is 0.001 mg / mL to 100 mg / mL.

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