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Preparation method of magnetic microspheres for trace nucleic acid extraction and purification

A technology of magnetic microspheres and a production method, which is applied in the fields of nanotechnology and biomedicine, can solve the problems that the efficiency of virus nucleic acid extraction affects the positive detection rate, the production process of magnetic microspheres cannot be satisfied, and the virus content is low, so as to improve the unit rate. Dispersion, reduce agglomeration, accelerate the effect of production

Pending Publication Date: 2020-06-26
上海领骏生物科技有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0003] With the rapid development of molecular biology technology, it is necessary to detect trace amounts of nucleic acid in many fields, such as forensic detection, genetically modified detection, free nucleic acid detection in liquid biopsy, virus detection, etc., especially in clinical samples. Low, the height of viral nucleic acid extraction efficiency will directly affect the positive detection rate of experiment, therefore put forward higher requirement to magnetic microsphere, for this, the manufacturing process of magnetic microsphere in the prior art can't satisfy

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  • Preparation method of magnetic microspheres for trace nucleic acid extraction and purification
  • Preparation method of magnetic microspheres for trace nucleic acid extraction and purification

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Embodiment 1

[0036] A method for manufacturing magnetic microspheres for extraction and purification of trace nucleic acids in this embodiment includes the following manufacturing steps:

[0037] S1. Preparation and screening of Fe3O4 magnetic microspheres: Fe3O4 was synthesized by ultrasonic-assisted aqueous co-precipitation method of iron salt in alkaline environment, and the surface of Fe3O4 magnetic microspheres was modified with citrate to make the particles dispersed and reduce agglomeration;

[0038] Specifically include:

[0039] S1.1. Weigh 32.5g FeCl3 and 12.7g FeCl2, dissolve them in 100ml ultrapure water, mix them evenly, and transfer them to a three-necked bottle;

[0040] S1.2. After the above mixed solution is stirred evenly, pass nitrogen gas to remove oxygen for 30 minutes, add alkali solution to the mixed solution to adjust the pH value to 10-12, turn on the ultrasonic wave and stir vigorously for 0.5 hours at a speed of 700r / min, add 1.47 Add 1 g of trisodium citrate so...

Embodiment 2

[0048] A method for manufacturing magnetic microspheres for extraction and purification of trace nucleic acids in this embodiment includes the following manufacturing steps:

[0049] S1. Preparation and screening of Fe3O4 magnetic microspheres: Fe3O4 was synthesized by ultrasonic-assisted aqueous co-precipitation method of iron salt in alkaline environment, and the surface of Fe3O4 magnetic microspheres was modified with citrate to make the particles dispersed and reduce agglomeration;

[0050] Specifically include:

[0051] S1.1. Weigh 270.3g FeCl3 6H2O and 139g FeSO4 7H2O in 1L ultrapure water, mix evenly, and transfer to a three-neck bottle;

[0052] S1.2. After the above mixed solution is stirred evenly, pass nitrogen gas to remove oxygen for 30 minutes, add ammonia solution to the mixed solution to adjust the pH value to 10-12, turn on the ultrasonic wave and stir vigorously for 0.5 hours at a speed of 700r / min, add 14.7 Add 1 g of trisodium citrate solution to the syste...

Embodiment 3

[0060] Apply the magnetic microspheres obtained in the above-mentioned embodiment 1 to the extraction of human serum free DNA, the steps are as follows:

[0061] 1. Cracking

[0062] Take 200 μl of serum sample and add it to EP tube, add 200 μl of cell lysate and 10 μl of proteinase K, mix well, put the EP tube in a constant temperature water tank at 70°C and incubate for 10-30 minutes;

[0063] 2. Combine

[0064] Take out the EP tube, add 10 μl of oscillating and mixed magnetic microspheres, add an equal volume of isopropanol, mix upside down, combine for 5 minutes, place the EP tube on a magnetic stand for magnetic separation, and discard the waste liquid;

[0065] 3. Washing

[0066] Add 500 μl of washing solution, mix up and down 5-10 times, then magnetically separate, suck up the residual liquid from the tube cap and bottom of the tube; repeat the washing once;

[0067] Open the lid and dry at room temperature for 10 minutes;

[0068] 4. Elution

[0069] Add 50 μl o...

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Abstract

The invention discloses a preparation method of magnetic microspheres for trace nucleic acid extraction and purification. The preparation method comprises the following preparation steps: S1, preparation and screening of Fe3O4 magnetic microspheres: synthesizing Fe3O4 by adopting an ultrasonic-assisted water-phase coprecipitation method, and modifying the surfaces of the Fe3O4 magnetic microspheres with citrate radicals, so that particles are in a dispersed state, and agglomeration is reduced; s2, coating and modifying the surfaces of the Fe3O4 magnetic microspheres: coating and modifying thesurfaces of the Fe3O4 magnetic microspheres with SiO2. According to the invention, Fe3O4 magnetic microspheres are prepared by adopting an ultrasonic-assisted water-phase coprecipitation method; citric acid radicals are used for modifying the surfaces of the Fe3O4 magnetic microspheres; the Fe3O4 / SiO2 submicron microspheres with strong magnetic responsiveness are prepared by adding the Fe3O4 magnetic microspheres into the dispersion liquid to enable the particles to be in a dispersed state, reducing agglomeration and then coating and modifying the surfaces of the Fe3O4 magnetic microspheres with SiO2, and the magnetic microspheres have excellent biocompatibility and hydrophilicity and very good chemical stability and colloid stability.

Description

technical field [0001] The embodiments of the present invention relate to the fields of nanotechnology and biomedicine, and in particular to a method for manufacturing magnetic microspheres for extraction and purification of trace nucleic acids. Background technique [0002] The use of magnetic microspheres for nucleic acid extraction is one of the important fields of application in the field of biomedicine. Any scientific or medical research related to nucleic acid is inseparable from the extraction of nucleic acid, and the efficiency and effect of nucleic acid extraction directly affect subsequent research or medical treatment. diagnosis. In particular, magnetic microspheres are used for DNA detection, which is the basis for in vitro diagnosis and treatment of diseases, and has the characteristics of high selectivity, high sensitivity, speed and convenience. Magnetic microspheres have broad application prospects in simplifying detection procedures, improving the accuracy ...

Claims

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Application Information

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IPC IPC(8): B01J20/30B01J20/28B01J20/06C12N15/10
CPCB01J20/28021B01J20/28009B01J20/06B01J20/28011C12N15/1013
Inventor 肖理慧王庆专孙凤娟
Owner 上海领骏生物科技有限公司
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