HBsAg aptamer screening method based on magnetic separation

A nucleic acid aptamer and screening method technology, applied in biochemical equipment and methods, DNA preparation, recombinant DNA technology, etc., can solve the problems of tediousness, easy loss in the separation process, and long separation operation time, so as to simplify the operation process, Ease of long-term storage and transportation, shortening of screening time

Inactive Publication Date: 2014-03-26
SOUTHEAST UNIV
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Benefits of technology

This patented method involves taking an enzyme called hbead liver sinus (HBS) DNA into cells that have been genetically alterated or engineered to produce certain substances found naturally within them such as cholesterols and other lipids. These modifications help make these organisms easier to detect through immunological methods like Western blotting techniques. They also allow for easy manipulation without losing their original structure during preparations and modification processes. Overall this new approach allows researcher scientists to quickly identify specific proteins associated with diseases from complex mixtures containing different types of biomolecules.

Problems solved by technology

The technical problem addressed in this patented text relates to developing better ways to detect tiny magnetite grapeptide (MBG), specifically iron oxide). This material plays crucial roles in various applications including medicine, biosensing, diagnoses, therapy, and medical imagings. Previous techniques were limited because they had poor selectivity and weak binding ability towards target compounds. A novel approach called nanoimprint technique was developed based on combining special materials together into larger structures without affecting their functions. These nanosized MNAs made up of multiple atoms show superior performance compared to existing ones but also offer greater flexibility when being combined into large structures.

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  • HBsAg aptamer screening method based on magnetic separation
  • HBsAg aptamer screening method based on magnetic separation
  • HBsAg aptamer screening method based on magnetic separation

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preparation example Construction

[0029] The Fe 3 o 4 The preparation methods of magnetic nanoparticles include co-precipitation method, solvothermal method, soft template method, microemulsion method, thermal decomposition method and other physical or chemical methods. These methods can all be used in the present invention Fe 3 o 4 The preparation of magnetic nanoparticles and the preparation methods listed are only examples of the present invention, and cannot limit the scope of the present invention in any way.

[0030] The Fe 3 o 4 SiO 2 The carboxylation modification of the surface of magnetic nanoparticles is to firstly convert Fe 3 o 4 SiO 2 The surface of the magnetic nanoparticles is aminated, and then the surface is carboxylated using succinic anhydride.

[0031]The preserved carboxylated Fe 3 o 4 SiO 2 The organic solvent of the magnetic nanoparticles is ethanol, isopropanol, glycerol, acetone, n-butanol, tetrahydrofuran, or N,N-dimethylformamide (DMF) and other organic solvents. The li...

Embodiment 1

[0043] Embodiment 1 Carboxylated Fe 3 o 4 SiO 2 Preparation of Magnetic Nanoparticles

[0044] Fe 3 o 4 Magnetic nanoparticles were prepared by soft template method, Fe 3 o 4 SiO 2 Magnetic nanoparticles using seed polymerization and classical Preparation method; the specific preparation process is carried out with reference to Example 1 of the invention application CN102568728A. The prepared particles have the characteristics of uniform size and good dispersibility; and then carry out carboxylation modification on the surface.

[0045] The specific experimental process is as follows.

[0046] (1), first adopt the soft template method to prepare Fe with a particle size of about 500nm 3 o 4 Magnetic nanoparticles, followed by seed polymerization and classical Coated a layer of silicon shell on its surface to prepare Fe with a particle size of about 550nm 3 o 4 SiO 2 magnetic nanoparticles. figure 1 gives Fe 3 o 4 SiO 2 Transmission electron microscope image...

Embodiment 2

[0049] Embodiment 2 Carboxylated Fe 3 o 4 SiO 2 Magnetic nanoparticles activated and coupled to HBsAg by EDC in one step

[0050] (1), 2-(N-morpholine)-ethanesulfonic acid monohydrate (MES) buffer solution of 25mmol / L pH5 was used to clean the carboxylated Fe of Example 1 under the action of an external magnetic field 3 o 4 SiO 2 Magnetic nanoparticles 2 times to carboxylate Fe 3 o 4 SiO 2 Magnetic nanoparticles were dispersed in 5 mL of MES buffer.

[0051] (2) Before use, prepare a 10 mg / mL EDC solution with a 25 mmol / L pH5 MES solution stored at 4°C, and take the carboxylated Fe dispersed in the MES 3 o 4 SiO 2 Add 100 μL of EDC solution to 1 mL of magnetic nanoparticles, and shake slowly at 37° C. for 30 min.

[0052] (3) Discard the supernatant under the action of a magnetic field, wash with 25mmol / L pH5 MES buffer for 3 times, wash away the EDC not involved in the activation reaction, and obtain activated carboxylated Fe 3 o 4 SiO 2 magnetic nanoparticles a...

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Abstract

A disclosed HBsAg aptamer screening method based on magnetic separation comprises the following steps: preparing Fe3O4 magnetic nanometer particles and performing carboxylation modification; activating carboxylation magnetic nanometer particles and coupling with HBsAg; screening aptamers capable of specifically identifying HBsAg by employing SELEX technology; cloning and sequencing the screened product; and analyzing and predicting the secondary structure of the aptamer. By fixing the screening target molecule HBsAg to the surfaces of Fe3O4 magnetic nanometer particles with surfaces decorated by carboxyl groups, the fixing amount is large; and also by utilizing the magnetism of Fe3O4 magnetic nanometer particle, the rapid magnetic separation is facilitated and the screening time is saved. The screened aptamer is capable of specifically identifying HBsAg and has high affinity, and has wide application prospect in the field of HBsAg detection diagnosis.

Description

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Claims

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Application Information

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Owner SOUTHEAST UNIV
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