PH-sensitive oligopeptide magnetic bead as well as preparation method and application thereof
By loading iron tetroxide nanoparticles on dendritic silicon ball templates and modifying pH-sensitive oligopeptides, pH-sensitive oligopeptide magnetic beads were prepared, which solved the complex problems of the existing magnetic bead preparation process, and achieved simplified process and efficient nucleic acid capture and release effects.
Patent Information
- Application Number
- CN202311205015.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-19
- Publication Date
- 2025-03-21
AI Technical Summary
The existing magnetic bead preparation process is complex and requires the coating process of the holes on the surface of the magnetic bead, which has high requirements.
The dendritic silicon sphere template was used to load iron tetraoxide nanoparticles, and the surface of the magnetic beads was modified by maleimide-modified pH-sensitive oligopeptide magnetic beads, which simplified the process flow.
The preparation of magnetic beads is achieved, and the coating process of the magnetic beads is avoided. The magnetic beads can effectively adsorb nucleic acids under low pH conditions and release nucleic acids under high pH conditions.
Smart Images
Figure CN119673601A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of magnetic composite nanomaterials, and in particular to pH-sensitive oligopeptide magnetic beads and a preparation method and application thereof. Background Art
[0002] Traditional cfDNA extraction methods include phenol-chloroform method, Trizol method, and adsorption column method. They use toxic phenol and chloroform reagents and adopt precipitation and centrifugation processes. The extraction process requires a large amount of samples, and the sample processing process is cumbersome and complicated. The recovery efficiency of cfDNA is low, and it is difficult to achieve high-throughput and automated operations. At present, the mainstream extraction method of cfDNA is the magnetic bead method. The principle of magnetic bead method for extracting nucleic acids is: using magnetic beads as carriers, the magnetic beads can adsorb nucleic acids under high salt and low pH, and separate from nucleic acids under low salt and high pH, and then move the magnetic beads or transfer liquids to achieve the entire extraction and purification process of nucleic acids.
[0003] Magnetic beads are a type of nano- to micron-sized magnetic biomaterial, usually composed of internal magnetic inorganic materials and surface organic materials. According to the relative positions of inorganic and organic materials, magnetic beads can be divided into core-shell, sandwich and diffuse structures, among which the core-shell type is the more commonly used structure. The organic material on the surface of the magnetic beads itself will introduce some basic groups, such as -OH, -COOH, -NH2, -SH, etc. The basic magnetic beads containing these groups can be used for nucleic acid extraction, cell separation and protein purification. In addition, the basic magnetic beads can be further coupled to oligonucleotides and proteins, and the modified magnetic beads can be used in immunoassays, immobilized enzymes, drug delivery, etc.
[0004] Magnetic beads are widely used in early cancer screening due to their simple separation method, fast separation speed, and easy automation. Currently, most early cancer screening technologies are based on deep gene sequencing of tumor free DNA (cfDNA) shed by tumor cells into the blood. The first step in using cfDNA for testing is to extract and purify cfDNA in human peripheral blood. The quality of the extracted product directly affects the accuracy and reliability of subsequent test results. However, the content of cfDNA is very low and is easily degraded. Only magnetic beads with uniform particle size and strong adsorption can ensure the stability and accuracy of the diagnostic process.
[0005] Most of the magnetic beads used for nucleic acid extraction in the prior art are prepared by the swelling method, which uses polymer microspheres with a porous structure as a template and deposits magnetic iron oxide nanoparticles inside. In this method, the polymer microspheres have a porous structure inside, and the inner wall of the pores is modified with a compound precursor that can bind to magnetic materials, and then hydrolyze, nucleate, and crystallize under alkaline and heating conditions to form magnetic beads. The magnetic beads obtained by this method have the advantages of uniform particle size and high magnetic content, but the seed swelling method has many steps and a complicated process, and the pores on the surface of the magnetic beads formed need to be fully filled and closed in the subsequent coating process, which has high requirements for the coating process. Summary of the invention
[0006] In view of this, in order to solve the above problems, the present invention provides a pH-sensitive oligopeptide magnetic bead and a preparation method and application thereof, aiming to solve the technical problem of complex magnetic bead preparation process in the prior art.
[0007] In order to achieve the above object, the present invention provides a method for preparing pH sensitive oligopeptide magnetic beads, comprising the following steps:
[0008] S1. Preparing an iron precursor mixture: mixing iron acetylacetonate, ethanol and a dendritic silicon sphere template to obtain the iron precursor mixture;
[0009] S2. Preparation of ferroferric oxide / dendritic silicon sphere magnetic composite: transferring the iron precursor mixture to a non-metallic material reactor for thermal reaction to obtain the ferroferric oxide / dendritic silicon sphere magnetic composite, wherein the ferroferric oxide is loaded inside the dendritic silicon sphere template;
[0010] S3. Preparation of thiolated ferroferric oxide / dendritic silicon ball magnetic composite: Dispersing the ferroferric oxide / dendritic silicon ball magnetic composite in ethanol, adding ammonia and 3-mercaptopropyltrimethoxysilane, and reacting with mechanical stirring at room temperature to obtain the thiolated ferroferric oxide / dendritic silicon ball magnetic composite;
[0011] S4. Preparation of pH-sensitive oligopeptide magnetic beads: mechanically stirring the thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex and the maleimide-modified pH-sensitive oligopeptide in a HEPES buffer at pH = 7.4, wherein the pH-sensitive oligopeptide is modified on the surface of the dendritic silica sphere template to obtain the pH-sensitive oligopeptide magnetic beads.
[0012] Preferably, in S1, the mass ratio of ferric acetylacetonate to dendritic silicon sphere template is (0.1-1.0): (0.01-1.0); the stirring time is 15-30 min; and the volume of ethanol added is 5-150 mL / g based on the mass of ferric acetylacetonate.
[0013] Preferably, in S2, the solvent thermal reaction temperature is 160-200° C., and the reaction time is 5-10 h.
[0014] Preferably, in S3, based on the mass of the ferroferric oxide / dendritic silicon sphere magnetic composite, the added volume of ethanol is 1-10 mL / mg, the added volume of ammonia water is 2-10 mL / g, and the added volume of 3-mercaptopropyltrimethoxysilane is 0.1-20 mL / g.
[0015] Preferably, in S4, based on the mass of the thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex, the mass of the pH sensitive oligopeptide is 0.01-1 g / g, and the volume of the HEPES buffer is 0.1-5 mL / mg.
[0016] Preferably, the preparation method of the dendritic silicon spheres comprises: dissolving triethanolamine in deionized water and stirring to form a triethanolamine solution;
[0017] Add hexadecyltrimethylammonium bromide, sodium salicylate and ethyl orthosilicate to the triethanol solution in sequence, and after the reaction is completed, cool to room temperature and collect precipitate A by centrifugation;
[0018] The precipitate A is dispersed in a mixed solution of hydrochloric acid and ethanol to react. After the reaction is completed, the precipitate B is collected by centrifugation to obtain the dendritic silicon sphere.
[0019] Preferably, the mass volume concentration of the triethanolamine solution is 0.1-10 mg / mL.
[0020] Preferably, in the mixed solution of hydrochloric acid and ethanol, the volume ratio of hydrochloric acid to ethanol is (1-5):(25-100).
[0021] Preferably, the entire process of preparing the dendritic silicon spheres is carried out under stirring conditions, and the reaction temperature is 60-80°C.
[0022] Preferably, the mass ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate is (0.1-10):(1-10):(1-10).
[0023] To achieve another purpose, the present invention also provides the above-mentioned pH-sensitive oligopeptide magnetic beads, including: ferrosoferric oxide nanoparticles; a dendritic silica sphere template, in which the ferrosoferric oxide nanoparticles are loaded; and a pH-sensitive oligopeptide, in which the pH-sensitive oligopeptide is modified on the surface of the dendritic silica sphere template.
[0024] Preferably, after the pH-sensitive oligopeptide magnetic beads are dispersed in ethanol, the particle size thereof is 350 nm to 450 nm.
[0025] The pH-sensitive oligopeptide magnetic beads prepared based on the above technical solution can be used for nucleic acid capture or nucleic acid release, wherein the nucleic acid includes DNA ladder, cfDNA in plasma or urine.
[0026] Preferably, the method for nucleic acid capture comprises the following steps:
[0027] (1) dispersing the pH-sensitive oligopeptide magnetic beads prepared by the above technical solution into deionized water to obtain a magnetic bead suspension;
[0028] (2) The nucleic acid, the acidic buffer and the magnetic bead suspension are mixed and incubated at room temperature under shaking conditions until the adsorption of the nucleic acid by the magnetic beads reaches equilibrium, and then the product in the mixed solution obtained by the incubation is collected by a magnet. The collected product is then washed with an acidic buffer and ethanol respectively to remove protein and salt, thereby obtaining a magnetic bead-nucleic acid complex and completing the capture of the nucleic acid.
[0029] Preferably, the method for releasing nucleic acid comprises the following steps:
[0030] The magnetic bead-nucleic acid complex is dispersed in an alkaline buffer and eluted at room temperature under shaking conditions until the desorption of the nucleic acid by the magnetic beads reaches equilibrium, so that the nucleic acid is separated from the magnetic beads, that is, the release of the nucleic acid is completed, and then the magnetic beads in the eluted mixed solution are collected with a magnet.
[0031] Preferably, the concentration of the magnetic bead suspension is 10-50 mg / mL.
[0032] Preferably, the acidic buffer comprises: 5-6 mol / L guanidine thiocyanate, 0.1-1 mmol / L polyoxyethylene ether Brij58, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, and 40%-60% (v / v) isopropanol.
[0033] Preferably, the alkaline buffer comprises: 50 mM TE buffer, pH=10.0.
[0034] Preferably, the amount of the alkaline buffer is at least enough to submerge the magnetic bead-nucleic acid complex.
[0035] The principle of nucleic acid extraction is that under acidic conditions, the magnetic beads are positively charged and adsorb nucleic acids, while under alkaline conditions, the magnetic beads are negatively charged and release nucleic acids.
[0036] Beneficial technical effects obtained by the present invention:
[0037] 1. The pH-sensitive oligopeptide magnetic beads provided by the technical solution of the present invention have a simple preparation method. Different from the swelling method in the prior art, there is no need to coat the pores on the surface of the magnetic beads. Only the surface of the template needs to be modified to obtain the pH-sensitive oligopeptide magnetic beads.
[0038] 2. By using the pH-sensitive oligopeptide magnetic beads provided by the technical solution of the present invention, it is only necessary to load the iron-containing compound into the dendritic silica sphere template and then perform surface modification to obtain the pH-sensitive oligopeptide magnetic beads, so that the composite particles are sensitive to the pH of the system, can effectively adsorb nucleic acids under low pH conditions, and release nucleic acids under high pH conditions. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 This is a transmission electron microscope image of the dendritic silicon sphere provided in Example 1 of the present invention.
[0040] Figure 2 This is a transmission electron microscope image of the ferroferric oxide / dendritic silicon sphere magnetic composite provided in Example 1 of the present invention.
[0041] Figure 3 This is a transmission electron microscopy image of the pH-sensitive oligopeptide magnetic beads provided in Example 1 of the present invention.
[0042] Figure 4a and Figure 4b They are respectively the transmission electron microscope picture and the magnified picture provided for comparative example 1 of the present invention. DETAILED DESCRIPTION
[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.
[0044] The present invention provides a method for preparing pH-sensitive oligopeptide magnetic beads, comprising the following steps:
[0045] (1) preparing an iron precursor mixture: mixing iron acetylacetonate, ethanol and a dendritic silicon sphere template to obtain the iron precursor mixture;
[0046] (2) Preparing a ferroferric oxide / dendritic silicon sphere magnetic composite: transferring the iron precursor mixture to a non-metallic material reactor for thermal reaction to obtain the ferroferric oxide / dendritic silicon sphere magnetic composite, wherein the ferroferric oxide is loaded inside the dendritic silicon sphere template;
[0047] (3) magnetically separating the ferroferric oxide / dendritic silicon sphere magnetic composite and cleaning it;
[0048] (4) preparing a thiolated ferroferric oxide / dendritic silicon sphere magnetic composite: dispersing the ferroferric oxide / dendritic silicon sphere magnetic composite in ethanol, adding ammonia water and 3-mercaptopropyltrimethoxysilane, and reacting by mechanical stirring at room temperature to obtain the thiolated ferroferric oxide / dendritic silicon sphere magnetic composite;
[0049] (5) Magnetic separation of the thiolated ferroferric oxide / dendritic silicon sphere magnetic complex and cleaning thereof.
[0050] As a preferred embodiment, in step (1), the mass ratio of ferric acetylacetonate to dendritic silica sphere template is (0.1-1.0): (0.01-1.0); the stirring time is 15-30 min; and the volume of ethanol added is 5-150 mL / g based on the mass of ferric acetylacetonate.
[0051] As a preferred solution, the solvent thermal reaction temperature in step (2) is 160-200° C., and the reaction time is 5-10 h.
[0052] As a preferred embodiment, in step (4), based on the mass of the ferroferric oxide / dendritic silicon sphere magnetic composite, the added volume of ethanol is 1 to 10 mL / mg, the added volume of ammonia water is 2 to 10 mL / g, and the added volume of 3-mercaptopropyltrimethoxysilane is 0.1 to 20 mL / g.
[0053] As a preferred solution, in step (6), based on the mass of the thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex, the mass of the pH sensitive oligopeptide is 0.01-1 g / g, and the volume of the HEPES buffer is 0.1-5 mL / mg.
[0054] As a preferred solution, after the pH-sensitive oligopeptide magnetic beads are dispersed in ethanol, the particle size thereof is 350 nm to 450 nm.
[0055] In some specific embodiments, the method for preparing the ferroferric oxide / dendritic silicon sphere magnetic composite comprises:
[0056] After mixing ferric acetylacetonate, ethanol and dendritic silicon sphere template, a solvent thermal reaction is carried out, and the product is collected by magnetic separation and washed with ethanol for several times to obtain a ferrosoferric oxide / dendritic silicon sphere magnetic composite.
[0057] In some specific embodiments, the preparation method of the ferroferric oxide / dendritic silicon sphere magnetic composite comprises: dispersing 50-100 mg of the ferroferric oxide / dendritic silicon sphere magnetic composite in 10-50 mL of ethanol, ultrasonically mixing, adding 0.1-1.0 mL of ammonia water and 0.01-1 mL of 3-mercaptopropyltrimethoxysilane, reacting at room temperature by mechanical stirring for 30-60 min, collecting the product by magnetic separation, and washing it with ethanol several times to obtain the thiolated ferroferric oxide / dendritic silicon sphere magnetic composite.
[0058] In some specific embodiments, the preparation method of pH sensitive oligopeptide magnetic beads comprises: in HEPES buffer, stirring and reacting the thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex with maleimide-modified pH sensitive oligopeptide to obtain pH sensitive oligopeptide magnetic beads.
[0059] Furthermore, the preparation method of pH sensitive oligopeptide magnetic beads specifically includes: in 10-50 mL HEPES buffer (pH=7.4, 10 mM), 10-100 mg of thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex and 1-10 mg of pH sensitive oligopeptide are mechanically stirred and reacted for 30-60 minutes to obtain pH sensitive oligopeptide magnetic beads.
[0060] Further preferably, the preparation method of dendritic silicon spheres comprises the following steps: dissolving triethanolamine in deionized water, stirring and mixing, then adding hexadecyltrimethylammonium bromide and sodium salicylate, and continuing stirring; then adding ethyl orthosilicate, heating and stirring to react; cooling the product to room temperature, collecting the precipitate by centrifugation, and washing with ethanol several times; dispersing the product in concentrated hydrochloric acid and ethanol, and after reflux reaction, collecting the precipitate by centrifugation to obtain dendritic silicon spheres.
[0061] In some specific embodiments, the method for preparing dendritic silicon spheres comprises the following steps:
[0062] 1) Dissolve 0.01-1.0 g triethanolamine in 10-100 mL deionized water and stir magnetically at 60-80 °C for 15-30 min;
[0063] 2) Add 0.1-1.0 g of hexadecyltrimethylammonium bromide and 0.1-1.0 g of sodium salicylate, and stir magnetically at 60-80° C. for 15-30 min;
[0064] 3) Add 1-10 mL of ethyl orthosilicate and stir magnetically at 60-80°C for 1-10 hours;
[0065] 4) The product was cooled to room temperature, the precipitate was collected by centrifugation, and washed several times with ethanol;
[0066] 5) The product is dispersed in 1-5 mL of concentrated hydrochloric acid and 25-100 mL of ethanol and refluxed at 60-80°C for 12-24 h;
[0067] 6) Collect the precipitate by centrifugation to obtain dendritic silicon spheres.
[0068] The pH-sensitive oligopeptide magnetic beads prepared by the above method are used in nucleic acid capture or nucleic acid release, and the nucleic acid is DNA ladder, cfDNA in plasma or urine.
[0069] In some specific embodiments, the steps of nucleic acid adsorption are as follows:
[0070] (1) dispersing pH-sensitive oligopeptide magnetic beads in deionized water to prepare a suspension of thiol-functionalized magnetic beads with a concentration of 10 to 50 mg / mL;
[0071] (2) Mix the nucleic acid, acidic buffer and magnetic bead suspension, incubate at room temperature under shaking conditions until the adsorption of the magnetic beads to the nucleic acid reaches equilibrium, then use a magnet to collect the product in the mixed solution obtained by incubation, and then wash the collected product with acidic buffer and ethanol respectively to remove protein and salt, thereby obtaining a magnetic bead-nucleic acid complex and completing the capture of the nucleic acid.
[0072] In some specific embodiments, the steps of nucleic acid release are as follows: dispersing the magnetic bead-nucleic acid complex in an alkaline buffer, eluting at room temperature under shaking conditions until the desorption of nucleic acid by the magnetic beads reaches equilibrium, separating the nucleic acid from the magnetic beads, i.e., completing the release of the nucleic acid, and then collecting the magnetic beads in the eluted mixed solution with a magnet; the amount of the alkaline buffer used is at least enough to submerge the magnetic bead-nucleic acid complex.
[0073] In some specific embodiments, the concentration of the magnetic bead suspension is 10-50 mg / mL.
[0074] In some specific embodiments, the acidic buffer comprises 5-6 mol / L guanidine thiocyanate, 0.1-1 mmol / L polyoxyethylene ether Brij58, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, and 40%-60% (v / v) isopropanol.
[0075] In some specific embodiments, the alkaline buffer comprises: 50 mM TE buffer, pH=10.0.
[0076] In some specific embodiments, the amount of the alkaline buffer is at least enough to submerge the magnetic bead-nucleic acid complex.
[0077] It should be noted that, unless otherwise specified, the raw materials and reagents involved in the present invention are all commercially available, and the source of pH-sensitive oligopeptides is Shanghai Hongpeptide Biotechnology Co., Ltd.
[0078] The technical solution of the present invention is described in detail below through specific embodiments.
[0079] Example 1
[0080] The preparation method of hydroxy magnetic beads provided in this embodiment specifically includes:
[0081] 1. Provide dendritic silicon spheres, the preparation method comprising:
[0082] 1) 0.68 mg of triethanolamine was dissolved in 25 mL of deionized water and stirred at 80 °C for 15 min;
[0083] 2) Add 380 mg of hexadecyltrimethylammonium bromide and 218 mg of sodium salicylate, and stir magnetically at 80°C for 15-30 min;
[0084] 3) Add 4 mL of ethyl orthosilicate and stir under magnetic stirring at 80°C for 3 h;
[0085] 4) The product was cooled to room temperature, the precipitate was collected by centrifugation, and washed several times with ethanol;
[0086] 5) The product was dispersed in 3 mL of concentrated hydrochloric acid (concentration: 37%) and 50 mL of ethanol and heated at 60°C for 24 h;
[0087] 6) Collect the precipitate by centrifugation to obtain dendritic silicon spheres.
[0088] 2. Provide a ferroferric oxide / dendritic silicon sphere magnetic composite, the preparation method comprising:
[0089] 0.1 g of ferric acetylacetonate, 15 mL of ethanol and 0.05 g of dendritic silicon sphere template were mixed evenly by ultrasonication for 30 min, and the mixed solution was transferred to a non-metallic reaction kettle and thermally reacted at 180°C for 6 h. When the temperature dropped to room temperature, the product was collected by magnetic separation and washed several times with ethanol to obtain a ferrosoferric oxide / dendritic silicon sphere magnetic composite.
[0090] 3. Provide pH-sensitive oligopeptide magnetic beads, the preparation method of which comprises:
[0091] 100 mg of ferroferric oxide / dendritic silica sphere magnetic complex was dispersed in 10 mL of ethanol, 250 μL of ammonia water and 56 μL of 3-mercaptopropyltrimethoxysilane were added, and the mixture was stirred at room temperature for 12 h. The product was collected by magnetic separation and washed several times with ethanol to obtain the thiolated ferroferric oxide / dendritic silica sphere magnetic complex. 20 mg of thiolated ferroferric oxide / dendritic silica sphere magnetic complex was taken, 1 mL of pH-sensitive oligopeptide (1 mg / mL), 2 mL of HEPES 7.4 (50 mM), and 7 mL of deionized water were added, and the mixture was stirred mechanically at 300 rpm for 30 min to obtain pH-sensitive oligopeptide magnetic beads.
[0092] See also Figure 1, is a transmission electron microscope picture of the dendritic silicon spheres prepared in the above step 1. It can be seen from the figure that the dendritic silicon spheres obtained by the technical solution of the present invention have uniform particle sizes, a large number of pores on the surface of the silicon spheres, and regular shapes.
[0093] See also Figure 2 , is a scanning electron microscope image of the ferroferric oxide / dendritic silicon ball magnetic composite prepared in the above step 2. It can be seen from the figure that in the ferroferric oxide / dendritic silicon ball magnetic composite prepared in this embodiment, the ferroferric oxide particles are loaded inside the dendritic silicon ball, and the pores of the dendritic silicon ball are almost completely filled, forming a microsphere shape with a dense structure, and the size of the particles has obvious uniformity, which can provide a uniform size of the core structure for the preparation of mercapto magnetic beads. In particular, it can be clearly observed that the pores on the surface of the composite have been loaded and filled with ferroferric oxide, indicating that the technical solution of this embodiment can successfully load ferroferric oxide into the interior of the dendritic silicon ball. Obviously, by obtaining a dendritic mesoporous silicon with a uniform particle size distribution, the particle size of the obtained composite is also uniform.
[0094] See also Figure 3 , Transmission electron microscopy image of pH-sensitive oligopeptide magnetic beads. It can be seen from the figure that the obtained magnetic beads have uniform particle size and uniform distribution.
[0095] The pH-sensitive oligopeptide magnetic beads prepared by the technical solution of the present invention can reversibly capture and release nucleic acids in an acidic / alkaline environment, thereby enabling nucleic acid extraction from DNA ladders, plasma or cfDNA in urine.
[0096] Example 2
[0097] The pH-sensitive oligopeptide magnetic beads prepared based on the technical solution of Example 1 above are applied to the extraction of cfDNA from human plasma, specifically including DNA capture and DNA release, and the specific steps include:
[0098] (1) Take 2 mL of plasma and transfer it to a 10 / 15 mL centrifuge tube. Add 2.5 mL of acidic buffer (5 mol / L guanidine thiocyanate, 0.5 mmol / L polyoxyethylene ether Brij58, 50 mmol / L pH = 5.0 NaAc-HAc buffer, 40% (v / v) isopropanol, and the rest is water) and 30 μL of magnetic bead suspension (40 mg / mL), mix well, and vortex mix at room temperature for 10 min; then centrifuge at 1000 rpm for 1-2 min; then place on a magnetic rack for 5 min or until the solution is clear, and remove the clear solution by aspiration.
[0099] (2) Remove the centrifuge tube from the magnetic rack, add 1 mL of 80% ethanol, shake for 30 seconds, and centrifuge. Place the centrifuge tube on the magnetic rack for 2 minutes or until the solution is clear; use a 200 μL pipette tip to remove the clear solution as thoroughly as possible, and repeat once; open the centrifuge tube cap and air-dry the magnetic beads for 2 to 5 minutes.
[0100] (3) Remove the centrifuge tube from the magnetic rack, add 15 μL of alkaline buffer, and mix by pipetting back and forth; shake for 5 minutes and then centrifuge briefly, then place the centrifuge tube on the magnetic rack for 2 minutes or until the liquid is clear, and then use a pipette to draw the clarified liquid into a new 1.5 mL centrifuge tube to obtain cfDNA. Repeat the extraction 3 times. The cfDNA quantitative results are as follows:
[0101] cfDNA mass in 2mL plasma (ng) 1 16.65 2 19.54 3 18.76
[0102] Comparative Example 1
[0103] This comparative example provides the effect of preparing the ferroferric oxide / dendritic silicon sphere magnetic composite by a step-by-step method, which specifically includes:
[0104] 1. Mix 0.1g of ferric acetylacetonate and 15mL of ethanol by ultrasonic for 30min, transfer the mixture to a non-metallic reactor, and heat react at 180℃ for 6h. When the temperature drops to room temperature, collect the product by magnetic separation, and wash it with ethanol several times to obtain ferrosoferric oxide.
[0105] 2. 60 mg of ferrosoferric oxide and 0.05 g of dendritic silica sphere template were evenly dispersed in 30 mL of deionized water, mechanically stirred at room temperature for 12 h, magnetically separated and collected using a magnetic stand, washed several times with ethanol, and the product was dispersed in ethanol.
[0106] See also Figure 4a and Figure 4b , which are the transmission electron microscope pictures and magnified pictures of the product obtained in the above step 2. From the pictures, the structure of ferroferric oxide of about 10 nm can be seen, and the structure of dendritic mesoporous silicon is not seen, indicating that the comparative example cannot obtain the ferroferric oxide / dendritic silicon ball magnetic composite.
[0107] The technical solution based on Comparative Example 1 cannot produce a magnetic composite.
[0108] Obviously, by adopting the technical scheme of the present invention, the iron-containing compound is loaded into the dendritic silica sphere template and then the surface is modified by the pH-sensitive oligopeptide to obtain the pH-sensitive oligopeptide magnetic beads, so that the composite particles of the entire magnetic beads are sensitive to the pH of the system, and can effectively adsorb nucleic acids under low pH conditions and release nucleic acids under high pH conditions.
[0109] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or capable of achieving the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.
Claims
1. A method for preparing pH-sensitive oligopeptide magnetic beads, characterized in that: The following steps are involved: S1. Preparing an iron precursor mixture: mixing iron acetylacetonate, ethanol and a dendritic silicon sphere template to obtain the iron precursor mixture; S2. Preparation of ferroferric oxide / dendritic silicon sphere magnetic composite: transferring the iron precursor mixture to a non-metallic material reactor for thermal reaction to obtain the ferroferric oxide / dendritic silicon sphere magnetic composite, wherein the ferroferric oxide is loaded inside the dendritic silicon sphere template; S3. Preparation of thiolated ferroferric oxide / dendritic silicon ball magnetic composite: Dispersing the ferroferric oxide / dendritic silicon ball magnetic composite in ethanol, adding ammonia and 3-mercaptopropyltrimethoxysilane, and reacting with mechanical stirring at room temperature to obtain the thiolated ferroferric oxide / dendritic silicon ball magnetic composite; S4. Preparation of pH-sensitive oligopeptide magnetic beads: mechanically stirring the thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex and the maleimide-modified pH-sensitive oligopeptide in a HEPES buffer at pH = 7.4, wherein the pH-sensitive oligopeptide is modified on the surface of the dendritic silica sphere template to obtain the pH-sensitive oligopeptide magnetic beads.
2. The method for preparing pH sensitive oligopeptide magnetic beads according to claim 1, characterized in that: In S1, the mass ratio of ferric acetylacetonate to the dendritic silica sphere template is (0.1-1.0): (0.01-1.0); the stirring time is 15-30 min; the volume of ethanol added is 5-150 mL / g based on the mass of ferric acetylacetonate; In S2, the solvent thermal reaction temperature is 160-200°C, and the reaction time is 5-10h; In S3, based on the mass of the ferroferric oxide / dendritic silicon sphere magnetic composite, the added volume of ethanol is 1 to 10 mL / mg, the added volume of ammonia water is 2 to 10 mL / g, and the added volume of 3-mercaptopropyltrimethoxysilane is 0.1 to 20 mL / g; In S4, based on the mass of the thiolated ferrosoferric oxide / dendritic silica sphere magnetic complex, the mass of the pH sensitive oligopeptide is 0.01-1 g / g, and the volume of the HEPES buffer is 0.1-5 mL / mg.
3. The method for preparing pH sensitive oligopeptide magnetic beads according to claim 1, characterized in that: The preparation method of the dendritic silicon sphere comprises: dissolving triethanolamine in deionized water and stirring to form a triethanolamine solution; Add hexadecyltrimethylammonium bromide, sodium salicylate and ethyl orthosilicate to the triethanolamine solution in sequence, and after the reaction is completed, cool to room temperature and collect precipitate A by centrifugation; The precipitate A is dispersed in a mixed solution of hydrochloric acid and ethanol to react. After the reaction is completed, the precipitate B is collected by centrifugation to obtain the dendritic silicon sphere.
4. The method for preparing pH sensitive oligopeptide magnetic beads according to claim 3, characterized in that: The mass volume concentration of triethanolamine solution is 0.1-10 mg / mL; And / or, in the mixed solution of hydrochloric acid and ethanol, the volume ratio of hydrochloric acid to ethanol is (1-5): (25-100); the concentration of hydrochloric acid is 36%-38%; And / or, the entire process of preparing the dendritic silicon spheres is carried out under stirring conditions, and the reaction temperature is 60-80° C.; And / or, the mass ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate is (0.1-10):(1-10):(1-10).
5. A pH-sensitive oligopeptide magnetic bead prepared by the preparation method according to any one of claims 1 to 4, comprising: Fe3O4 nanoparticles; A dendritic silicon sphere template, wherein ferrosoferric oxide nanoparticles are loaded in the dendritic silicon sphere template; The pH-sensitive oligopeptide is modified on the surface of the dendritic silica sphere template.
6. The pH sensitive oligopeptide magnetic beads according to claim 5, characterized in that After the pH sensitive oligopeptide magnetic beads are dispersed in ethanol, the particle size thereof is 350nm-450nm.
7. The use of the pH-sensitive oligopeptide magnetic beads as claimed in claim 5 or 6 in nucleic acid capture or nucleic acid release can achieve reversible capture and release of nucleic acids in an acidic / alkaline environment; wherein, The nucleic acid includes cfDNA in DNA ladder, plasma or urine.
8. The use according to claim 7, wherein the method for nucleic acid capture comprises the following steps: (1) dispersing the pH-sensitive oligopeptide magnetic beads into deionized water to obtain a magnetic bead suspension; (2) The nucleic acid, the acidic buffer and the magnetic bead suspension are mixed and incubated at room temperature under shaking conditions until the adsorption of the nucleic acid by the magnetic beads reaches equilibrium, and then the product in the mixed solution obtained by the incubation is collected by a magnet. The collected product is then washed with an acidic buffer and ethanol respectively to remove protein and salt, thereby obtaining a magnetic bead-nucleic acid complex and completing the capture of the nucleic acid.
9. The use according to claim 8, wherein the method for releasing nucleic acid comprises the following steps: The magnetic bead-nucleic acid complex is dispersed in an alkaline buffer and eluted at room temperature under shaking conditions until the desorption of the nucleic acid by the magnetic beads reaches equilibrium, so that the nucleic acid is separated from the magnetic beads, that is, the release of the nucleic acid is completed, and then the magnetic beads in the eluted mixed solution are collected with a magnet.
10. The use according to claim 9, characterized in that The concentration of the magnetic bead suspension is 10 to 50 mg / mL; The acidic buffer comprises: 5-6 mol / L guanidine isothiocyanate, 0.1-1 mmol / L polyoxyethylene ether Brij58, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, and 40%-60% (V / V) isopropanol; The alkaline buffer comprises: 50 mM TE buffer, pH=10.0; The amount of the alkaline buffer is at least enough to submerge the magnetic bead-nucleic acid complex.