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Monodispersed nano magnetic bead used for rapid sample pretreatment and preparation method thereof

A nano-magnetic bead, monodisperse technology, applied in the field of nano-biomedical materials, can solve the problems of poor dispersion ability of polydisperse magnetic beads, non-uniform size and shape, reduced use efficiency of magnetic beads, etc., and achieves low cost and sedimentation speed. Slow, stable and reliable results

Active Publication Date: 2014-12-17
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the commonly used magnetic bead nucleic acid extraction technology currently uses magnetic beads, most of which are polydisperse magnetic beads. Due to the non-uniform size and shape, the dispersion ability of polydisperse magnetic beads in a liquid environment is poor. This disadvantage Prolong their settling time and have insufficient interaction with the solution, resulting in a reduction in the use efficiency of magnetic beads
[0006] Aiming at many problems such as the high requirements for sample pretreatment in the current on-site detection of infectious disease pathogens, the limited application of the traditional polydisperse magnetic bead nucleic acid extraction method, and the fact that most of the commercially available magnetic Technology, molecular biology technology and biomedical technology combine high-tech products to provide monodisperse nano-magnetic beads that can be applied to immunomagnetic separation and nucleic acid extraction

Method used

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  • Monodispersed nano magnetic bead used for rapid sample pretreatment and preparation method thereof
  • Monodispersed nano magnetic bead used for rapid sample pretreatment and preparation method thereof
  • Monodispersed nano magnetic bead used for rapid sample pretreatment and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1, preparation of monodisperse nano magnetic beads.

[0021] (1) Monodisperse iron ferric oxide nanospheres were prepared by solvothermal method. The specific preparation process was as follows: Weighed 2.0 g of sodium acetate and 0.25 g of ferric chloride hexahydrate, respectively, and added them to 50 ml of ethylene glycol solution. After magnetic stirring for 1 hour, the system was transferred to a reaction kettle, and reacted at 100° C. for 10 hours. After the reaction was completed, the resulting black solution was centrifuged, and washed 3 times with deionized water and ethanol successively to obtain monodisperse ferric oxide nanospheres (Fe 3 o 4 ), the resulting product was dried in an oven at 60°C for use; the monodisperse ferric oxide nanosphere scanning electron microscope characterization results were as follows figure 1 As shown in a, it can be seen from the figure that the prepared magnetic nanomaterial is a nanosphere structure with a rough sur...

Embodiment 2

[0024] Example 2, applying the monodisperse nano-magnetic beads prepared in Example 1 to immunomagnetic separation of pathogenic microorganisms, comprising the following steps:

[0025] (1) Prepare simulated pathogen sample solution and complex medium dilution.

[0026] Simulated pathogen sample solution: add 20ul enterovirus EV71 (10MOI) to 20ml PBS buffer, mix well;

[0027] Complex medium diluent: Take a normal human feces sample (solid like the size of a pea) in a biological safety cabinet, dissolve it in 20ml of PBS buffer, and shake vigorously with a vortex shaker to mix evenly;

[0028] (2) Coating Fe with antibody 3 o 4 SiO 2 -AEAPS monodisperse nano-magnetic beads: take 40 μl (100 μg / ml) monoclonal antibody of EV71 structural protein VP1 and 0.1 g monodisperse nano-magnetic beads and incubate overnight at room temperature. In order to eliminate the non-specific adsorption of EV71 on the surface of magnetic beads, the Incubate the incubated magnetic beads with BSA (b...

Embodiment 3

[0030] Example 3, the results of real-time fluorescent quantitative PCR for the application of monodisperse nano-magnetic beads to the immunomagnetic separation of pathogenic microorganisms are as follows: figure 2 As shown, the nano-magnetic beads can specifically bind to the target antigen after being modified by a specific antibody, and the antigen-antibody immune complex obtained after enrichment and separation can be detected by fluorescent quantitative PCR. In order to avoid errors as much as possible, we made three parallel samples and a negative control. It can be seen from the figure that curve 4 is a negative control with a CT value of 26.62, and curves 1, 2, and 3 are three parallel samples with a CT value of They were 14.21, 14.64, and 15.02, respectively, indicating that the antigen concentration captured by immunomagnetic separation was relatively high, and the separation effect was ideal.

[0031] Example 3, the monodisperse nano-magnetic beads prepared in Exam...

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Abstract

The invention discloses monodispersed nano magnetic beads for rapid sample pretreatment and a preparation method thereof. The nano magnetic material has chemical structure of Fe3O4@SiO2-AEAPS, is a core-shell multifunctional magnetic nano material with abilities of targeting transport, monodispersity and specific combination, and can quickly and efficiently enrich pathogenic microorganisms in field diagnosis of infectious diseases. Furthermore, the monodispersed nano magnetic bead can be used for nucleic acid extraction of the obtained pathogen. The material provided by the invention is a high-tech product combining nanotechnology, molecular biological technology and biological medicine.

Description

technical field [0001] The invention belongs to the field of nano biomedical materials, and relates to a monodisperse nano magnetic bead used for rapid sample pretreatment and a preparation method thereof. Background technique [0002] Usually the medium of infectious disease pathogens is a mixed system such as soil, blood, urine, etc., and it needs to go through tedious pretreatment steps such as continuous centrifugation, extraction, dilution, etc. before the collected samples can be used for subsequent biological detection. This process takes a long time. , has greatly restricted the development of rapid pathogen detection technology, making the pre-treatment of samples the key to determine the detection efficiency and identification results among many rapid biological detection methods. Immunomagnetic separation technology, which has emerged in recent years, uses magnetic materials modified by antibodies or gene probes to identify targets, and under the action of an exte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/10B01J20/28B01J20/30H01F1/10C12N15/10
Inventor 郝荣章宋宏彬李杨刘雪林赵荣涛卢晓董世彪邱少富王勇李鹏贾雷立王立贵谢靖吴志豪
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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