CN113005117A

CN113005117A - Magnetic bead drying protection solution, drying magnetic bead and preparation method thereof

Info

Publication number: CN113005117A
Application number: CN202110222434.6A
Authority: CN
Inventors: 张瑜; 蔡亦梅; 贾欣月; 李洁昆; 蒋鹏翀; 高静; 任鲁风
Original assignee: Beijing Integrated Biosystems Co ltd
Current assignee: Beijing Integrated Biosystems Co ltd; Beijing Linke Biotechnology Co ltd
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2021-06-22
Anticipated expiration: 2041-02-26
Also published as: CN113005117B

Abstract

The invention relates to the technical field of biochemical reagents, in particular to a magnetic bead drying protective solution, a drying magnetic bead and a preparation method thereof. The magnetic bead drying protective solution comprises, by mass, 5-15% of cellobiose, 0.005-0.02% of tween 20, 0.05-0.2% of sucrose ester and the balance of water; the sucrose ester had an HLB value of 15. The magnetic bead drying protection solution provided by the invention can protect the performance of dried hydroxyl magnetic beads to the maximum extent, and the dried hydroxyl magnetic beads can be stored and transported at room temperature, have long storage life and are beneficial to being applied to a microfluidic chip.

Description

Magnetic bead drying protection solution, drying magnetic bead and preparation method thereof

Technical Field

The invention relates to the technical field of biochemical reagents, in particular to a magnetic bead drying protective solution, a drying magnetic bead and a preparation method thereof.

Background

The molecular biology technology of nucleic acid is one of the common research means for pathogenic microorganism detection, species identification, species origin, diversity evaluation and its genetic relationship, system evolution, etc. Whether high-quality nucleic acid can be extracted or not is the key in molecular biology experiments, and the sensitivity and specificity of the extraction method are directly related to the success or failure of subsequent experiments. Therefore, nucleic acid extraction is one of the most critical methods in molecular biology.

With the wide application of molecular biology, new requirements are provided for nucleic acid extraction technology, and the method is efficient, convenient, environment-friendly, high-throughput and automatic, and becomes the mainstream direction for the development of nucleic acid extraction technology. From the 1 st generation chemical methods such as phenol-chloroform method, Trizol method, to the 2 nd generation adsorption column method, and the 3 rd generation magnetic bead method developed in recent years, the extraction technique and efficiency are continuously improved and optimized.

The surface of the hydroxyl magnetic bead is modified with a large number of silanol groups (hydroxyl groups), and the silanol groups can be specifically combined with nucleic acid in a solution under the conditions of high salt and low pH value through hydrophobic interaction, hydrogen bond interaction, electrostatic interaction and the like, and are not combined with other impurities (such as protein), so that the method can be used for quickly separating the nucleic acid from a biological sample, is safe and simple to operate, and is very suitable for automatic and high-flux extraction of the nucleic acid.

However, almost all the magnetic beads on the market are transported and used in a storage solution, and the performance of the magnetic beads is greatly damaged by drying, but along with the increasing popularization of a magnetic bead purification method and the research, development and progress of a microfluidic device, how to preset dried magnetic beads becomes very important, because the miniaturization of the device and a chip can reduce the volume of the chip by reducing the magnetic beads stored in a reagent bin independently, and the dried magnetic beads can be preset at any position of the chip, so that the complexity of liquid flowing in the microfluidic process can be greatly reduced, a developer is provided with a larger space to research, develop and optimize, and more convenient use and control are provided.

Chinese patent application CN109725148A discloses an immunomagnetic bead preservation solution, which contains buffer solution, emotional protein, glycerol, water-soluble sugar, artificially synthesized high molecular polymer, protein stabilizer, reducing substance, amino acid, integration agent, serum, surfactant and preservative. The immunomagnetic bead preservation solution provided by the invention enables immunomagnetic beads to maintain higher activity within one year under the preservation condition of 4 ℃, and provides guarantee for the application of immunomagnetic beads in practical detection.

The Chinese patent application CN109725141A discloses a streptavidin coupled magnetic bead (SA-magnetic bead) freeze-dried working solution, an SA-magnetic bead freeze-dried product and a preparation method of the freeze-dried product, wherein the freeze-dried working solution is suitable for an alkaline phosphatase luminescent system and comprises a protein protective agent, and the protein protective agent is casein sodium. The freeze-drying working solution also comprises a buffer solution, wherein the buffer solution is a TBS buffer solution, the TBS buffer solution comprises 10-50mM Tris-HC1, 0.9% NaCl and has the pH value of 7.4, and the freeze-drying working solution comprises sodium caseinate and the TBS buffer solution, so that the freeze-drying working solution is more suitable for SA-magnetic bead freeze-dried products of an alkaline phosphatase reaction system: the mixed solution of SA-magnetic beads and freeze-drying working solution is spotted into a chip in a micro-droplet form and then freeze-dried in situ to obtain a freeze-dried product, the freeze-dried product can keep good biotin-binding activity after redissolving, and after the sandwich reaction is carried out on the freeze-dried product and biotinylated antibody, antigen and alkaline phosphate alcohol labeled antibody, the influence on the activity of an enzyme catalysis substrate is small, and the luminous value is high and stable.

The Chinese patent application CN108008125A discloses a freeze-drying working solution suitable for immunomagnetic beads, wherein the freeze-drying working solution is a combination of a freeze-drying buffer solution, a freeze-drying protective agent and a preservative, and the mass ratio of the freeze-drying buffer solution to the freeze-drying protective agent to the preservative is 1-10:1-500: 1; the freeze-drying buffer solution is one or a combination of more of a tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution, a phosphate buffer solution, a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution and a boric acid shed sand buffer solution; the freeze-drying protective agent comprises a saccharide substance and a polymer, wherein the mass ratio of the saccharide substance to the polymer is 0.1-20: 1.

The above protective solution is freeze-drying protective solution, needs freeze drying and is used for preserving immunomagnetic beads, and hydroxyl magnetic beads can cause magnetic bead agglomeration under the conditions of freezing, drying and centrifugal operation, are not easy to resuspend and disperse, and influence the chemical activity of functional groups on the surfaces of the magnetic beads.

Therefore, the development of a drying protective solution suitable for magnetic hydroxyl beads is an important problem to be solved urgently.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the drying protection solution for the hydroxyl magnetic beads, so that the performance of the dried hydroxyl magnetic beads is protected to the maximum extent, the dried hydroxyl magnetic beads can be stored and transported at room temperature, the storage life is long, and the hydroxyl magnetic beads can be stored for 50 days at 52 ℃.

The above purpose of the invention is realized by the following technical scheme:

a magnetic bead drying protective solution comprises cellobiose, Tween 20, sucrose ester and water;

preferably, the components comprise, by mass, 5-15% of cellobiose, 0.005-0.02% of tween 20, 0.05-0.2% of sucrose ester and the balance of water; the sucrose ester had an HLB value of 15.

Preferably, the components comprise, by mass, 8-12% of cellobiose, 0.008-0.01% of tween 20, 0.08-0.15% of sucrose ester and the balance of water.

Preferably, the component further comprises dextran; the average weight average molecular weight of dextran was 2000-9000.

Preferably, the mass percent of the glucan is 2-5%.

The second purpose of the invention is to provide a preparation method of the magnetic bead drying protection solution, which is characterized by comprising the following steps:

(1) mixing Tween and water uniformly to obtain a Tween aqueous solution; preferably, the concentration of the tween aqueous solution is 8-12%; preferably 10%;

(2) then, uniformly mixing sucrose ester and water to obtain a sucrose aqueous solution; preferably, the concentration of the sucrose aqueous solution is 8-12%; preferably 10%;

(3) and (3) finally, adding the sucrose aqueous solution into the step (1), adding the rest magnetic bead drying protection solution components for several times or once, and uniformly mixing at 45-55 ℃.

The invention also aims to provide a dried magnetic bead, which comprises a magnetic bead and a magnetic bead drying protective agent, wherein the magnetic bead drying protective agent is formed by drying a magnetic bead drying protective solution; the magnetic bead drying protective solution is the magnetic bead drying protective solution optionally selected;

preferably, the mass-to-volume ratio of the magnetic beads to the magnetic bead drying protective solution is 20-30: 1 mg/mL.

Preferably, the dried magnetic beads are dried silicon hydroxyl magnetic beads.

The invention also aims to provide a preparation method of the dried magnetic beads, which comprises the following steps: and dispersing the magnetic beads in the magnetic bead drying protection solution, and airing at room temperature or drying in an oven at 45-60 ℃ for 30-50min to obtain the magnetic bead drying protection solution.

Preferably, the magnetic beads are silicon hydroxyl magnetic beads;

preferably, the mass-to-volume ratio of the magnetic beads to the magnetic bead drying protective solution is 20-30: 1 mg/mL;

preferably, the magnetic beads are subjected to ultrasonic treatment for 5-15min under 500-1200W in advance, and supernatant is removed through adsorption;

preferably, the airing is airing for 10-15 h.

The invention also aims to provide the application of the magnetic bead drying protective solution or the drying magnetic beads in a microfluidic chip.

The invention has the beneficial effects that:

(1) the invention discloses a magnetic bead drying protective solution which comprises cellobiose, tween, sucrose ester and water; the magnetic beads dried by the magnetic bead drying protective solution can ensure the performance of the dried hydroxyl magnetic beads and can be stored and transported at room temperature;

(2) the invention further provides the optimal range proportion of each component of the magnetic bead drying protective solution, the performance of the dried hydroxyl magnetic beads can be further obviously improved, and the protection period can be prolonged, and researches prove that the dried hydroxyl magnetic beads can be stored for 50 days at the room temperature of 52 ℃; the storage period can be calculated by theory to be more than 12 months at normal temperature and more than 3 years at the refrigerated temperature of 4 ℃.

(3) The magnetic bead drying protective solution is convenient to use, and can be used by directly adding lysis solution and a sample.

(4) The magnetic bead drying protection liquid dried hydroxyl magnetic bead has wide application range, can be widely used in the aspects of magnetic bead method purification and microfluidics, particularly in the aspect of microfluidic chips, can reduce the volume of a chip by reducing the magnetic beads stored in a reagent bin independently, and can be preset at any position of the chip after drying, so that the complexity of liquid flowing in the microfluidic process can be greatly reduced, a developer can have more space for research and development and optimization, and more convenient use and control are provided.

(5) The invention discovers that cellobiose can play the best role in protection compared with other substances such as sucrose, polyethylene glycol, lactose and the like by adding cellobiose into the magnetic bead drying protection solution, and simultaneously, glucan can play a good role in synergy, and the molecular weight of glucan is unexpectedly found to have a relatively obvious effect on the protection of dried magnetic beads, and the purification quality of the dried magnetic beads can be influenced when the molecular weight is too small.

(6) Reducing disaccharide exists in the drying protective agent, the effect of combining water can be replaced in the drying process, hydroxyl magnetic beads are well protected, the antioxidation effect is realized on the magnetic beads, and the tween can better disperse the magnetic beads and is favorable for the redissolution of the magnetic beads.

Drawings

FIG. 1 is a diagram showing the effect of dried magnetic beads in example 1;

FIG. 2 is a graph of the amplification of triple RT-qPCR target 1 using dried magnetic beads purified product stored at 52 ℃ for 50 days; the abscissa is the cycle number, and the ordinate is the fluorescence value variation; the terminal of the curve is sequentially provided with a hydroxyl magnetic bead curve graph and dried hydroxyl magnetic bead curves prepared by the protection solutions of the embodiment 2, the embodiment 1 and the embodiment 3 from top to bottom;

FIG. 3 is a graph of the triple RT-qPCR target 2 amplification curve for a dried magnetic bead purified product stored at 52 ℃ for 50 days; the abscissa is the cycle number, and the ordinate is the fluorescence value variation; the terminal of the curve is a hydroxyl magnetic bead curve graph, and the dried hydroxyl magnetic bead curves prepared by the protection solutions of the embodiment 5, the embodiment 6 and the embodiment 4 are sequentially arranged from top to bottom.

FIG. 4 is a graph of the amplification of triple RT-qPCR target 3 using dried magnetic bead purified products stored at 52 ℃ for 50 days; the abscissa is the cycle number, and the ordinate is the fluorescence value variation; the terminal of the curve is sequentially provided with a hydroxyl magnetic bead curve graph and an anhydration hydroxyl magnetic bead curve graph prepared by the protection solution of the embodiment 2 and the embodiment 1 from top to bottom.

FIG. 5 is a triple RT-qPCR target 1 amplification curve diagram of a dried magnetic bead purified product stored at the same room temperature for 11 days; the abscissa is the cycle number, and the ordinate is the fluorescence value variation; the curve end is a graph of dried hydroxyl magnetic beads prepared by the protective solution of each of example 2, example 1, example 3, comparative example 1, comparative example 2 and comparative example 3 from top to bottom.

Detailed Description

The invention may be further understood by reference to the following examples. However, it is to be understood that these examples do not limit the present invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.

Some experimental material sources in the following examples:

polyethylene glycol is from sigma, cat # 1546605;

the silicon hydroxyl magnetic beads are from Shanghai Aladdin Biotechnology Co., Ltd, the product number S8036-A300nm-1 EA.

Example 1

The magnetic bead drying protective solution comprises the following components: 10% cellobiose, 0.01% tween 20 and 0.1% sucrose ester, the balance being water, wherein the sucrose ester has an HLB value of 15.

Example 2

The magnetic bead drying protective solution comprises the following components: 15% cellobiose, 0.02% tween 20 and 0.2% sucrose ester, the balance being water. Wherein the sucrose ester has an HLB value of 15.

Example 3

The magnetic bead drying protective solution comprises the following components: 5% cellobiose, 0.005% tween 20 and 0.05% sucrose ester, the balance being water. Wherein the sucrose ester has an HLB value of 15.

Example 4

The magnetic bead drying protective solution comprises the following components: 12% cellobiose, 0.01% tween 20 and 0.1% sucrose ester, the balance being water. Wherein the sucrose ester has an HLB value of 15.

Example 5

The magnetic bead drying protective solution comprises the following components: 10% cellobiose, 0.01% tween 20, 0.1% sucrose ester, 2% dextran Mw 9000, the balance water. Wherein the sucrose ester has an HLB value of 15. Wherein dextran Mw 9000 is purchased from Shanghai assist saint Biotech, Inc. under the designation 61205ES 25.

Example 6

The magnetic bead drying protective solution comprises the following components: 5% cellobiose, 0.01% tween 20, 0.1% sucrose ester, 5% dextran Mw 2000, the balance water. Wherein the sucrose ester has an HLB value of 15. Wherein dextran Mw 2000 is available from Shanghai assist saint Biotech, Inc. under the designation 61202ES 25.

Comparative example 1

The difference between this comparative example and example 1 is that cellobiose was replaced with trehalose; the specific magnetic bead drying protective solution comprises the following components: 10% trehalose, 0.01% tween 20 and 0.1% sucrose ester, the balance being water. Wherein the sucrose ester has an HLB value of 15.

Comparative example 2

The difference between the comparative example and the example 1 is that cellobiose is replaced by sucrose, and the specific magnetic bead drying protective solution comprises the following components: 10% of sucrose, 0.01% of Tween 20, 0.1% of sucrose ester SE-15 and the balance of water. Wherein the sucrose ester has an HLB value of 15.

Comparative example 3

The difference between the comparative example and the example 1 is that cellobiose is replaced by polyethylene glycol, and the specific magnetic bead drying protection solution comprises the following components: 10% of polyethylene glycol, 0.01% of Tween 20, 0.1% of sucrose ester and the balance of water. Wherein the sucrose ester has an HLB value of 15.

Wherein the polyethylene glycol

Comparative example 4

The difference between the comparative example and the example 1 is that the proportions of tween and sucrose ester are different, and the specific magnetic bead drying protection solution comprises the following components: 10% cellobiose, 0.1% tween 20 and 0.01% sucrose ester, the balance being water. Wherein the sucrose ester has an HLB value of 15.

Comparative example 5

The difference between this comparative example and example 6 is that the dextran is dextran Mw 15000; the specific magnetic bead drying protective solution comprises the following components: 5% of cellobiose, 0.01% of Tween 20, 0.1% of sucrose ester, 5% of glucan and the balance of water. Wherein the sucrose ester has an HLB value of 15. Dextran Mw 15000 was purchased from san Francisco Biotech, Inc. in Shanghai, under the designation 61207ES 25.

Comparative example 6

The comparative example is different from example 6 in that 5% of cellobiose was removed and the mass percentage of glucan was adjusted to 10%, and the rest was the same as example 6. The specific magnetic bead drying protective solution comprises the following components: 0.01% of Tween 20, 0.1% of sucrose ester, 10% of glucan and the balance of water. Wherein the sucrose ester has an HLB value of 15.

Comparative example 7

The mass percentages of the components of the comparative example are not in the optimal proportioning range, and the specific magnetic bead drying protective solution comprises 3% of cellobiose, 0.05% of tween 20, 0.01% of sucrose ester, 12% of glucan Mw 9000 and the balance of water. Wherein the sucrose ester has an HLB value of 15. Wherein dextran Mw 9000 is purchased from Shanghai assist saint Biotech, Inc. under the designation 61205ES 25.

Preparation of magnetic bead drying protective solution for examples 1-6 and comparative examples 1-7:

(1) firstly, diluting Tween 20 and water to 10% concentration according to the mass volume ratio of 1:10 g/ml;

(2) dissolving sucrose ester and water according to the mass volume ratio of 1:10g/ml to obtain 10% sucrose ester solution;

(3) adding 10% sucrose ester solution and the rest raw materials of examples 1-6 and comparative examples 1-7 into (1), adding 90% of water, mixing, standing at 50 deg.C for dissolving completely, adding the rest water, and mixing

Example 7 preparation of dried magnetic beads for examples 1-6 and comparative examples 1-7:

(1) ultrasonic cleaning is carried out on 500 mu L of silicon hydroxyl magnetic beads for 10min under 800W, so as to play a role in ultrasonic dispersion;

(2) completely adsorbing with magnet until the solution is clear, and removing the supernatant;

(3) adding the dried protective solution into silicon hydroxyl magnetic beads, and uniformly mixing the dried protective solution with shaking to obtain a mixed solution, wherein the mass-to-volume ratio of the silicon hydroxyl magnetic beads to the dried protective solution is 25:1 g/ml;

(4) and adding the 30 mu L mixed solution into a 1.5mL EP tube, and airing at room temperature for 12 hours in a clean environment or drying in an oven at 50 ℃ for 30-40 min.

Example 8 extraction procedure for pseudoviral nucleic acids:

the components of the reagent system used in this example are shown in Table 1

TABLE 1 Components of the reagent systems used in the examples

(1) Putting the hydroxyl drying magnetic beads into an EP tube, adding 80 mu L of lysine reagent, and carrying out vortex oscillation until the magnetic beads are uniformly mixed;

(2) then adding 20 mu L of pseudovirus; mixing the solution by vortex oscillation, and standing for 40 s; adsorbing the solution by using a magnetic frame until the solution is clarified, sucking out supernatant and discarding;

(3) adding 100 mu L of Ethanol wash into an EP tube, shaking and uniformly mixing, adsorbing magnetic beads by using a magnet, and removing supernatant; keeping the magnet to adsorb, adding 100 μ L desalting solution along the inner wall of the tube, standing for 3s, and discarding the supernatant (without scattering magnetic beads);

adding 100 mu L of Elution into an EP tube, shaking and uniformly mixing, carrying out water bath at 60 ℃ for 1min, and shaking and uniformly mixing again; using a magnet to adsorb the magnetic beads, and sucking out the supernatant to obtain the product.

Effect example 1

The nucleic acid samples were measured for A260, A260/A280, A260/A230 using an ultraviolet spectrophotometer Nanodrop 2000 and the recovery rates were calculated. The concentration and purity results of the pseudoviral nucleic acid extracted from the dried magnetic beads of this example are shown in tables 2, 3 and 4 below.

TABLE 2 concentration of RNA extracted from dry magnetic beads/ug for different storage times in the examples

TABLE 3 RNA purity A260/A280 values from dry magnetic beads for different storage times in the examples

TABLE 4 purity A260/A230 value of RNA extracted from dry magnetic beads in different storage times in each example

Effect example 2 RT-qPCR triple fluorescent amplification verification

The test method comprises the following steps: preparing reagents for PCR experiments according to the requirements of Table 5, uniformly mixing, taking 25 mu L as one part, and subpackaging into eight rows of tubes; then 25 μ L template or sample was added; the eight rows of tubes were then placed in a real-time fluorescent quantitative PCR instrument ABI 7500 and the setup procedure was as shown in table 6 below.

Set concentration of 5X 10²Copy number, and the fluorescence curves of the dried hydroxyl magnetic beads prepared by the un-dried hydroxyl magnetic beads and the protective solutions of examples 1 to 6 (see results in FIGS. 2 to 4) for the purified pseudovirus products after being stored at 52 ℃ for 50 days, and the fluorescence curves of the dried hydroxyl magnetic beads prepared by the protective solutions of examples 1 to 3 and comparative examples 1 to 3 (see results in FIG. 5) for the purified pseudovirus products after being stored at the same room temperature for 11 days. It can be seen from FIGS. 2 to 4 that the purification yield of examples 1 to 6 was still high after storage at 52 ℃ for 50 days, and from FIG. 5 that the yield was remarkably decreased after the comparative example showed a delay in the curve after storage at the same room temperature for 11 days.

TABLE 5 detailed description of PCR reagent preparation

TABLE 6 real-time fluorescent quantitative PCR instrument parameters

Claims

1. A magnetic bead drying protective solution comprises cellobiose, Tween 20, sucrose ester and water.

2. The magnetic bead drying protection solution as claimed in claim 1, wherein the components include, by mass, 5-15% cellobiose, 0.005-0.02% tween 20 and 0.05-0.2% sucrose ester, and the balance being water; the sucrose ester had an HLB value of 15.

3. The magnetic bead drying protection solution as claimed in claim 2, wherein the components include, by mass, 8-12% cellobiose, 0.008-0.01% tween 20, 0.08-0.15% sucrose ester, and the balance water.

4. The magnetic bead drying protection solution according to any one of claims 1 to 3, wherein the component further comprises dextran; the weight average molecular weight of dextran was 2000-9000.

5. The magnetic bead drying protection solution as claimed in claim 4, wherein the dextran is 2-5% by mass.

6. The preparation method of the magnetic bead drying protection solution according to any one of claims 1 to 5, characterized by comprising the following steps:

(1) mixing Tween and water uniformly to obtain a Tween aqueous solution; preferably, the concentration of the tween aqueous solution is 8-12%;

(2) then, uniformly mixing sucrose ester and water to obtain a sucrose aqueous solution; preferably, the concentration of the sucrose aqueous solution is 8-12%;

(3) finally, adding the sucrose aqueous solution in the step (1), adding the rest magnetic bead drying protective solution components for times or once,

mixing at 45-55 deg.C.

7. The drying magnetic bead is characterized by comprising a magnetic bead and a magnetic bead drying protective agent, wherein the magnetic bead drying protective agent is formed by airing a magnetic bead drying protective solution; the magnetic bead drying protective solution is the magnetic bead drying protective solution of any one of claims 1 to 5; preferably, the mass-to-volume ratio of the magnetic beads to the magnetic bead drying protective solution is 20-30: 1 mg/mL.

8. A preparation method of dried magnetic beads is characterized by comprising the following steps: dispersing magnetic beads in the magnetic bead drying protection solution of any one of claims 1 to 5, and airing at room temperature or drying in an oven at 45 to 60 ℃ for 30 to 50 min.

9. The method for preparing the dried magnetic bead as claimed in claim 8, wherein the magnetic bead is pre-sonicated at 500-1200W for 5-15min, and the supernatant is removed by adsorption; preferably, the airing time is 10-15 h.

10. Use of the magnetic bead drying protection solution according to any one of claims 1 to 5 or the drying magnetic bead according to claim 7 in a microfluidic chip.