CN105561914A - Nano magnetic bead for nucleic acid purification - Google Patents
Nano magnetic bead for nucleic acid purification Download PDFInfo
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Abstract
The invention provides a nano magnetic bead for nucleic acid purification. A preparation method of the nano magnetic bead includes the processes of preparation and surface modification of nano microspheres. The biologically-magnetic microspheres of certain sizes are obtained through an aqueous phase reaction, groups capable of adsorbing nucleic acid are introduced to the surfaces of the biologically-magnetic microspheres, through the character that silicon dioxide is electrified to adsorb nucleic acid, multiple complex samples can be efficiently purified, and therefore automation of nucleic acid purification is achieved and the workload of working staff is greatly reduced. The nano magnetic bead has the advantages that dispersibility is good, cost is low, particle uniformity is good, and the size of the magnetic bead is high in controllability, the nano magnetic bead can be effectively used for diagnostic reagent earlier purification and clinical nucleic acid detection, and has wide application prospects and social and economic benefits in the field of sequencing and the fields of biologically-magnetic microsphere targeted therapy and biological medicine development.
Description
Technical field
The present invention relates to material science, particularly a kind of nanometer magnetic bead for nucleic acid purification.
Background technology
In recent years, nanometer magnetic bead (abbreviation magnetic bead) is more and more subject to the favor of people, has now been widely used in multiple field such as fixing, nucleic acid purification of cell separation, enzyme.Magnetic bead refers to the composite macromolecule microsphere with certain magnetic and special construction magnetic inorganic particle and organic polymer specific binding formed by methods such as physics, chemistry or biology.Paramagnetic particle method is first by cell lysis, by free nucleic acid molecules specific adsorption out to magnetic-particle surface, makes the impurity such as protein stay in the solution simultaneously.Then under magnetic fields, magnetic-particle separates with liquid, can obtain pure nucleic acid again after reject liquid through wash-out.Extract the method for nucleic acid compared to other tradition, use magnetic bead to extract nucleic acid and there is incomparable unique advantage.Be mainly manifested in high efficiency, high specific, high stability that magnetic bead extracts nucleic acid.In addition, magnetic bead simple universal, can direct rapid extraction nucleic acid from the multiple sample such as blood, tissue, and with low cost, is convenient to extensive use.Use paramagnetic particle method to extract nucleic acid, step is simple, without the need to centrifugal, and effectively can remove the inhibiting factor of the subsequent reactions such as great majority interference PCR, as protein, polysaccharose substance, reduce false negative rate, improve purity and the precision of nucleic acid extraction, ensure the true and reliable of testing result.
Varied for the magnetic bead of nucleic acid extraction at present, what comparatively often adopt has metal alloy as iron, nickel etc., iron oxide, ferrite etc.They, by wrapping up different activity functional groups in magnetic bead surfaces, make it have the function of adsorbs nucleic acid.Magnetic bead for nucleic acid extraction is mainly divided into carboxyl magnetic microsphere, amino-magnetic microballoon, silicone dioxide magnetic microsphere etc.The principle of silicone dioxide magnetic microsphere adsorbs nucleic acid is identical with conventional solid silicon class medium, can under specific pH and salt ionic concentration effective adsorbs nucleic acid, the nucleic acid integrality obtained is good, and purity is high.
More than absolutely prove that magnetic bead will occupy more and more consequence in extraction nuclei aoid methods.But in the prior art, the market operation of nucleic acid purification reagent is complicated, and technique falls behind, and the magnetic bead that market is sold exists unstable, and surface group is modified not exclusively, and need organic solvent dispersion, particle is irregular, and adsorption rate amount is little, the shortcoming that automaticity is low.Therefore how to ensure that magnetic bead can give full play to performance, adsorbs nucleic acid becomes major issue urgently to be resolved hurrily effectively completely.
Summary of the invention
The technical problem to be solved in the present invention there is instability for existing magnetic bead, surface group is modified not exclusively, need organic solvent dispersion, particle is irregular, and adsorption rate amount is little, the shortcoming that automaticity is low, provide a kind of good dispersion, cost is low, and particle homogeneity is good, the nanometer magnetic bead for nucleic acid purification that magnetic bead size controllability is strong.
In order to solve the problems of the technologies described above, the technical scheme that one aspect of the present invention provides is:
For a nanometer magnetic bead for nucleic acid purification, described nanometer magnetic bead is prepared from by following steps:
Steps A) preparation of Nano microsphere:
Under argon shield, in the aqueous solution containing 13 ~ 17g FeSO47H2O and 8 ~ 12g ferric chloride hexahydrate, dripped 1ml0.4M ~ 0.6M sodium hydroxide solution at interval of 10 minutes; When reaction solution becomes gluey emulsion, dripped 1ml0.2M ~ 0.3M sodium hydroxide solution at interval of 20 minutes, shaking table continues to shake up, shaking table speed 100 revs/min, after magnetic to appear, continues to react 4 ~ 10 hours under argon shield; In gained solution, add sodium chloride subsequently, make sodium chloride final concentration be 0.5M ~ 1M; Room temperature leaves standstill and uses rinsed with deionized water to pH value of solution to be 7 after 18 ~ 36 hours; The sodium hydroxide solution re-using 0.2M ~ 0.3M is made into the suspension containing magnetic beads that magnetic bead percent by volume is 20% ~ 40%, to obtain final product;
Step B) prepare nanometer magnetic bead by finishing:
By steps A) gained suspension containing magnetic beads passes into argon gas and uniform stirring under 50 DEG C ~ 60 DEG C conditions; Add 60g nine water sodium metasilicate to be stirred to and to dissolve completely, every 3min drips 0.5ml glacial acetic acid, and shaking table continues to shake up, shaking table speed 100 revs/min, stirs 2h; When pH value is 6 ~ 8, uses 3 times to the 0.3M sodium chloride solution rinsing 5 times of described suspension containing magnetic beads volume, re-use 0.3M sodium chloride solution and be mixed with the suspension containing magnetic beads that magnetic bead percent by volume is 20% ~ 40%; In the suspension containing magnetic beads obtained, add 30gPEG-1750, shaking table continues to shake up, shaking table speed 100 revs/min, stirs 24h; Use 3 times to the sodium chloride solution rinsing 5 times of the 0.3M of described suspension containing magnetic beads volume; Re-use 0.3M sodium chloride solution and Sodium azide is mixed with the suspension containing magnetic beads that magnetic bead percent by volume is 40%-60%, obtain described nanometer magnetic bead.
A kind of nanometer magnetic bead for nucleic acid purification provided by the invention modifies to be prepared from aqueous phase reactions, and it is wide adaptability not only, can be used for the purifying of multiple nucleic acids purifying sample, and good dispersion, cost is low, and particle homogeneity is good, and magnetic bead size controllability is strong.
The nanometer magnetic bead that the present invention is used for nucleic acid purification obtains according to following formula I:
In steps A of the present invention) in, when the aqueous medium solution containing 13 ~ 17g FeSO47H2O and 8 ~ 12g ferric chloride hexahydrate becomes gluey emulsion, the interval time of titration should be changed, and 0.2M ~ 0.3M sodium hydroxide solution of 1ml is dripped at interval of 20min, until particle occurs, thus the Nano microsphere of different size can be obtained by different titers.
As preferably, steps A) solvent of the described aqueous solution containing FeSO47H2O and ferric chloride hexahydrate is deionized deoxygenated water, can effectively prevent ferrous ion oxidized like this.
As preferably, steps A) described be 15g containing the quality of FeSO47H2O in the aqueous solution of FeSO47H2O and ferric chloride hexahydrate, the quality of ferric chloride hexahydrate is 10g.
As preferably, steps A) magnetic bead percent by volume is 30% in the suspension containing magnetic beads that is made into of described use 0.2M ~ 0.3M sodium hydroxide solution.
In the present invention, inventor is by directly using deionized water dissolving FeSO47H2O and ferric chloride hexahydrate, under the prerequisite not changing ionic environment, for subsequent operation provides the reaction environment of liquid, and the Nano microsphere of different size can be obtained by the titer controlling alkaline solution.Meanwhile, the use of sodium chloride can increase the ion concentration in liquid, makes magnetic bead obtain good suspension and dispersiveness.Prevent growing and effectively removing the albumen in suspension containing magnetic beads of bacterium simultaneously.
At step B of the present invention) in, inventor equally under aqueous phase condition to steps A) Nano microsphere that obtains carries out finishing.First, react under 50 DEG C ~ 60 DEG C conditions, can fast reaction speed, shorten magnetic bead surfaces and modify the time.
As preferably, step B) described in pass into argon gas and uniform stirring carries out under 55 DEG C of conditions.
As preferably, step B) described in drip in acid solution process and by controlling the addition of described acid solution, the finishing particle of different modifying degree can be obtained.
As preferably, step B) in use 0.3MNaCl and Sodium azide to be mixed with 50% suspension containing magnetic beads, the ion concentration in liquid can be increased like this, make magnetic bead obtain good suspension and dispersiveness.Prevent growing and effectively removing the albumen in suspension containing magnetic beads of bacterium simultaneously.
Another aspect of the present invention there is provided a kind of Nucleic acid purification kits, and this kit comprises nanometer magnetic bead prepared by said process.
Beneficial effect of the present invention is:
The present invention obtains a certain size biomagnetism microballoon by aqueous phase reactions, introduce the group of energy adsorbs nucleic acid on its surface, utilize silica charging property to inhale the character of nucleic acid, energy efficiently purifying Various Complex sample, thus realize the automation of nucleic acid purification, greatly reduce the workload of staff.And nanometer magnetic bead of the present invention has good dispersion, cost is low, particle homogeneity is good, the feature that magnetic bead size controllability is strong, can be effective to diagnostic reagent purifying and clinical detection of nucleic acids in early stage, in order-checking field, biomagnetism microballoon targeted therapy, biological medicine development field have a wide range of applications and economic results in society.
Accompanying drawing explanation
Fig. 1 is the Electronic Speculum figure of nanometer magnetic bead prepared by the embodiment of the present invention 1;
Fig. 2 is the real-time fluorescence quantitative PCR testing result figure carrying out HBVDNA after the nanometer magnetic bead utilizing embodiment 1 to prepare carries out nucleic acid purification;
Fig. 3 is the real-time fluorescence quantitative PCR testing result comparison diagram carrying out HBVDNA after utilizing the present invention and commercially available nanometer magnetic bead to carry out nucleic acid purification respectively.
Detailed description of the invention
The invention discloses a kind of nanometer magnetic bead for nucleic acid purification, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.It needs to be noted, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention, and related personnel obviously can change content described herein on the basis not departing from content of the present invention, spirit and scope or suitably change and combination, realizes and applies the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has the implication that those skilled in the art understand usually.Meanwhile, in order to understand the present invention better, provide definition and the explanation of relational language below.
The term " magnetic bead " used in the present invention, refers to magnetic microsphere (abbreviation magnetic bead), has now been widely used in multiple field such as fixing, nucleic acid purification of cell separation, enzyme.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Experiment material and instrument
The chemical reagent used in following examples is commercially available AR.
Pcr amplification instrument used in following examples is the real-time fluorescence quantitative PCR instrument that Shanghai Hong Shi medical science and technology Co., Ltd produces.
Embodiment 1: for the preparation of the nanometer magnetic bead of nucleic acid purification
Steps A) preparation of Nano microsphere:
Under the protection of argon gas, in the aqueous solution containing 15g FeSO47H2O and 10g ferric chloride hexahydrate, dripped the sodium hydroxide solution of 1ml0.5M at interval of 10 minutes.When reaction solution becomes gluey emulsion, dripped the NaOH of the 0.25M of 1ml at interval of 20 minutes, shaking table continues to shake up, shaking table speed 100 revs/min, after magnetic to appear, reacts 4 ~ 10 hours under continuing argon shield.Subsequently according to magnetic bead volume, add sodium chloride, make its final concentration be 1M.Room temperature leaves standstill and uses rinsed with deionized water after 18 ~ 36 hours, to pH value of solution close to 7.The NaOH of 0.25M is used to be made into the suspension containing magnetic beads of 30%;
Step B) finishing of nanometer magnetic bead:
By steps A) under 55 DEG C of conditions, pass into argon gas and uniform stirring.Add 60 gram of nine water sodium metasilicate to be stirred to and to dissolve completely, every 3min drips the pure glacial acetic acid of 0.5ml, and shaking table continues to shake up, shaking table speed 100 revs/min, Keep agitation 2h.Until pH value 6 ~ 8 time, the NaCl rinsing of the 0.3M of use 3 times of suspension containing magnetic beads volumes 5 times, uses 0.3MNaCl to be mixed with 30% suspension containing magnetic beads.Add 30gPEG-1750, shaking table continues to shake up, shaking table speed 100 revs/min, Keep agitation 24h.The NaCl rinsing of the 0.3M of use 3 times of suspension containing magnetic beads volumes 5 times.0.3MNaCl and Sodium azide is used to be mixed with 50% suspension containing magnetic beads.
The form of the nanometer magnetic bead prepared with electron microscopy observation.Result as shown in Figure 1.As can be seen from the figure, magnetic bead is regular spherical, and mean size is about 400nm, has good dispersiveness.After magnetic bead surfaces is modified, define the finishing clad that thickness is about 20nm, the magnetic Nano microsphere of synthesis has good hydrophily, is that magnetic bead disperses in aqueous easily.
The real-time fluorescence quantitative PCR of embodiment 2:HBVDNA detects
The method identical with embodiment 1 is utilized to carry out the preparation of magnetic bead.
Lysate and the cleaning solution of magnetic bead is mixed with according to following component ratio preparation:
The bromophenol blue of 0.3N NaOH, 0.45M potassium chloride, 0.03%N-sodium lauroyl sarcosine, 5mMEDTA, 0.45MTris-HCL, 1% triton x-100,3mMDTT and 0.001wt%.
Be that 1:100 adds the natural sedimentation magnetic bead of 24 hours according to the volume ratio of magnetic bead and lysate, mixing.
Cleaning solution: the malachite green of 0.3M potassium chloride, 0.001wt%, with acetic acid, pH value is adjusted to 4.
Use above lysate and cleaning solution clear and definite and HBVDNA quantitative values is 5IU/ml, 10IU/ml, 20IU/ml, 2 × 10 through detecting to clinical diagnosis
2iU/ml, 2 × 10
3iU/ml, 2 × 10
4iU/ml, 2 × 10
5iU/ml, 2 × 10
6iU/ml, 2 × 10
7iU/ml, 2 × 10
8iU/ml, 2 × 10
9the Serum of Patients with Hepatitis B of IU/ml carries out the operation of following steps:
(1) packing of PCR lysate: the lysate being mixed with magnetic bead prepared is dispensed in special PCR pipe according to every pipe 100 μ l.
(2) application of sample: the serum getting 100 μ l joins above-mentioned point and is equipped with in the PCR pipe of the lysate being mixed with magnetic bead, blows and beats mixing gently 5 times with suction nozzle, room temperature leaves standstill 5 minutes.
(3) liquid is abandoned in suction: above-mentioned PCR pipe be placed on eight townhouse magnetic frames, leaves standstill 2 minutes, is sopped up by liquid with pipettor at magnetic bead offside, notes not sopping up magnetic bead.
(4) wash: take off PCR pipe from eight townhouse magnetic frames, and by the cleaning solution prepared often pipe add 200 μ l, reappose on eight townhouse magnetic frames, directly inhale with pipettor or negative pressure pump without the need to waiting time and abandon cleaning solution, note exhaustion raffinate.
(5) by the Taq DNA polymerase of 2.0 μ l (1U/ μ l) unit and 38.0 μ lPCR reactant liquors, (described reactant liquor is for comprising concentration 40nmol/ μ l upstream and downstream primer and 30nmol/ μ l probe; The Tris alkali of 20nmol/ μ l; 20mmol/ μ l magnesium chloride; The fluorescent PCR amplifing reagent mixed liquor of the malachite green indicator of dNTP and 0.001wt% of 50mmol/ μ l potassium chloride, 200umol/ μ l) mixing, join in the pcr amplification pipe that step (4) obtains, capping, gently magnetic bead is springed down, ensure that magnetic bead and PCR reactant liquor thoroughly mix.
Wherein forward primer sequence is HBVF5 '-TAGGAGGCTGTAGGCATAAATTGG-3 ', and reverse primer sequences is HBVR5 '-GCACAGCTTGGAGGCTTGT-3 ', and probe sequence is HBVP5 '-TCACCTCTGCCTAATC-3 '.
(6) centrifugal: centrifugal 45 seconds of horizontal centrifuge 3000RPM.
(7) PCR pipe obtained above is positioned in PCR instrument increases.
Amplification program: 37 DEG C, 2min; 95 DEG C, 5 minutes; 95 DEG C that carry out 45-50 circulation, 10 seconds and 61 DEG C, in 45 seconds, detect fluorescence signals at 61 DEG C.
As shown in the figure, wherein, it is 5IU/ml, 10IU/ml, 20IU/ml, 2 × 10 that dextrosinistral curve represents concentration according to this to experimental result
2iU/ml, 2 × 10
3iU/ml, 2 × 10
4iU/ml, 2 × 10
5iU/ml, 2 × 10
6iU/ml, 2 × 10
7iU/ml, 2 × 10
8iU/ml, 2 × 10
9the sample of IU/ml.Result shows, the present invention can detect that concentration range is in 5IU/ml ~ 2 × 10
9the HBVDNA sample of IU/ml, shows that the sensitivity of the inventive method can be low to moderate 5IU/ml simultaneously.
Comparative example 1
The nanometer magnetic bead using the nanometer magnetic bead of preparation in embodiment 1 and conventional method to make respectively, carries out the preparation of lysate, washing lotion according to the method in embodiment 2, clear and definite and be 10IU/ml, 2 × 10 through detecting HBVDNA quantitative values to clinical diagnosis
7the Serum of Patients with Hepatitis B of IU/ml carries out extraction and the amplification of viral nucleic acid.
As shown in Figure 3, use nanometer magnetic bead of the present invention to extract the viral nucleic acid of identical amount, its Ct value and efficiency are all higher than commercially available product to experimental result.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. for a nanometer magnetic bead for nucleic acid purification, it is characterized in that, described nanometer magnetic bead is prepared from by following steps:
Steps A) preparation of Nano microsphere:
Under argon shield, in the aqueous solution containing 13 ~ 17g FeSO47H2O and 8 ~ 12g ferric chloride hexahydrate, dripped 1ml0.4M ~ 0.6M sodium hydroxide solution at interval of 10 minutes; When reaction solution becomes gluey emulsion, dripped 1ml0.2M ~ 0.3M sodium hydroxide solution at interval of 20 minutes, shaking table continues to shake up, shaking table speed 100 revs/min, after magnetic to appear, continues to react 4 ~ 10 hours under argon shield; In gained solution, add sodium chloride subsequently, make sodium chloride final concentration be 0.5M ~ 1M; Room temperature leaves standstill and uses rinsed with deionized water to pH value of solution to be 7 after 18 ~ 36 hours; The sodium hydroxide solution re-using 0.2M ~ 0.3M is made into the suspension containing magnetic beads that magnetic bead percent by volume is 20% ~ 40%, to obtain final product;
Step B) prepare nanometer magnetic bead by finishing:
By steps A) gained suspension containing magnetic beads passes into argon gas and uniform stirring under 50 DEG C ~ 60 DEG C conditions; Add 60g nine water sodium metasilicate to be stirred to and to dissolve completely, every 3min drips 0.5ml glacial acetic acid, and shaking table continues to shake up, shaking table speed 100 revs/min, stirs 2h; When pH value is 6 ~ 8, uses 3 times to the 0.3M sodium chloride solution rinsing 5 times of described suspension containing magnetic beads volume, re-use 0.3M sodium chloride solution and be mixed with the suspension containing magnetic beads that magnetic bead percent by volume is 20% ~ 40%; In the suspension containing magnetic beads obtained, add 30gPEG-1750, shaking table continues to shake up, shaking table speed 100 revs/min, stirs 24h; Use 3 times to the sodium chloride solution rinsing 5 times of the 0.3M of described suspension containing magnetic beads volume; Re-use 0.3M sodium chloride solution and Sodium azide is mixed with the suspension containing magnetic beads that magnetic bead percent by volume is 40% ~ 60%, obtain described nanometer magnetic bead.
2. the nanometer magnetic bead for nucleic acid purification according to claim 1, is characterized in that, steps A) solvent of the described aqueous solution containing FeSO47H2O and ferric chloride hexahydrate is deionized deoxygenated water.
3. the nanometer magnetic bead for nucleic acid purification according to claim 1, it is characterized in that, steps A) described be 15g containing the quality of FeSO47H2O in the aqueous solution of FeSO47H2O and ferric chloride hexahydrate, the quality of ferric chloride hexahydrate is 10g.
4. the nanometer magnetic bead for nucleic acid purification according to claim 1, is characterized in that, steps A) magnetic bead percent by volume is 30% in the suspension containing magnetic beads that is made into of described use 0.2M ~ 0.3M sodium hydroxide solution.
5. the nanometer magnetic bead for nucleic acid purification according to claim 1, is characterized in that, step B) described in pass into argon gas and uniform stirring carries out under 55 DEG C of conditions.
6. the nanometer magnetic bead for nucleic acid purification according to claim 1, is characterized in that, step B) in re-use 0.3MNaCl and Sodium azide is mixed with the suspension containing magnetic beads that magnetic bead percent by volume is 50%.
7. a Nucleic acid purification kits, is characterized in that, described kit comprises the nanometer magnetic bead in claim 1 ~ 10 described in any one.
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Cited By (6)
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| CN110233020A (en) * | 2019-06-14 | 2019-09-13 | 迪佰(厦门)生物科技有限公司 | A kind of extracting method for the magnetic material of nucleic acid extraction, nucleic acid-extracting apparatus and nucleic acid |
| WO2020083372A1 (en) * | 2018-10-26 | 2020-04-30 | 南京金斯瑞生物科技有限公司 | Semi-continuous flow magnetic bead purification system |
| CN112007605A (en) * | 2019-05-30 | 2020-12-01 | 苏州海狸生物医学工程有限公司 | Hydroxyl nano magnetic bead for nucleic acid extraction and preparation method thereof |
| CN112604645A (en) * | 2020-12-10 | 2021-04-06 | 广东省测试分析研究所（中国广州分析测试中心） | Nano magnetic particle and preparation method and application thereof |
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| CN112725328B (en) * | 2020-12-28 | 2023-05-05 | 苏州白垩纪生物科技有限公司 | Large-scale manufacturing method of diagnostic magnetic beads and biological application thereof |
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