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Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria

A nanosphere, dual-function technology, applied in nanotechnology, nanotechnology, nanotechnology for sensing, etc., can solve the problems of inability to meet the urgent needs of rapid on-site detection of microorganisms, time-consuming, and complicated steps.

Inactive Publication Date: 2015-07-01
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional microbial culture method is time-consuming, cumbersome, requires a variety of media and reagents, and cannot meet the urgent needs of rapid on-site microbial detection in some emergencies in today's society

Method used

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  • Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of bifunctional nanosphere

[0032] In the present invention, the bifunctional nanosphere is improved by obtained by the method. Specifically, it is obtained by hydrolyzing ethyl orthosilicate in ethanol solution under the catalysis of a certain concentration of ammonia water. By adjusting the relative concentrations of ammonia, tetraethyl orthosilicate, ethanol, water, magnetic nanoparticles, and quantum dots, the properties and sizes of the bifunctional nanospheres can be changed. A typical synthesis process is as follows:

[0033] Mix 24.75mL water, 16.25mL ethanol and 9.0mL ammonia water thoroughly, then add 20μL magnetic iron oxide nanoparticles and 20μL cadmium telluride quantum dots, then quickly add the mixed solution of 4.5mL tetraethyl orthosilicate and 45.5mL ethanol, and stir the reaction After 2 hours, nanospheres are formed. Through magnetic separation and ethanol washing three times, and vacuum drying overnight, bifuncti...

Embodiment 2

[0034] Embodiment 2: the construction of immune bifunctional silicon-based nanosphere probe

[0035] Firstly, the surface of the bifunctional composite nanospheres prepared in Example 1 was modified. Specifically, 0.5 g of nanospheres were dissolved in 50 mL of pyridine, 0.2 g of succinic anhydride and 0.02 g of 4-dimethylaminopyridine were added, stirred overnight at room temperature, and magnetically separated , washed twice with methanol and water, respectively.

[0036] Then enterohemorrhagic Escherichia coli (E.coli O157:H7) antibody is covalently linked to bifunctional nanospheres, and the operation steps are as follows:

[0037] Take 500μg of nanospheres in a 1.5mL centrifuge tube, add 1mL of washing buffer, mix well and wash thoroughly, magnetically separate and wash twice, resuspend in 250μL of 2-(N-morpholino)ethanesulfonic acid, and then add 500μg1 -Ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 750 μg N-hydroxysuccinimide, activated at 37°C for 15 minutes, then wa...

Embodiment 3

[0038] Embodiment 3: the construction of immune quantum dot probe

[0039] Take 500 μL of quantum dots in a 1.5 mL centrifuge tube, add 1 mL of washing buffer, mix well and wash thoroughly, centrifuge and wash twice, resuspend in 250 μL 2-(N-morpholino)ethanesulfonic acid, and then add 500 μg1 -Ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 750 μg N-hydroxysuccinimide, activated at 37°C for 15 minutes, then washed twice with MES, resuspended and added 50 μg of The antibody was reacted at room temperature for 2 hours, and the antibody was coupled to the surface of the nanosphere to obtain the immune nanosphere. Wash the coupled magnetic beads twice with PBS, add 50 μL of phosphate buffered saline (pH=7.4) containing 1% bovine serum albumin, and store in a 4°C refrigerator until use.

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Abstract

The invention provides a difunctional composite nanosphere and a method for rapidly detecting food-borne pathogenic bacteria. The difunctional composite nanosphere is characterized in that silicon dioxide is adopted and quantum dots and magnetic nanoparticles are embedded simultaneously to construct the composite nanosphere having the optical property and the superparamagetism. The corresponding quantum dot and the nanosphere are connected with a monoclonal antibody capable of specifically recognizing food-borne pathogenic bacteria, and an immune quantum dot probe capable of carrying out antigen-antibody reaction with antigens on the surface of the bacteria and an immune composite nanosphere probe are acquired. The composite nanosphere with the composite structure can be used as a carrier for immunologically recognizing and separating the pathogenic bacteria and also can be used as a signal enhancer element of the immune quantum dot probe, secondary amplification of a detection signal is realized, and target microorganisms of a sample to be detected are acquired by adopting an optical detection method. According to the method, the detection time less than or equal to 2h can be greatly shortened, the sensitivity (102cfu / mL) is improved, and the method is suitable for on-site rapid detection of foods and environment samples and can be popularized and applied in grass roots.

Description

technical field [0001] The invention relates to a method for rapidly detecting food-borne pathogenic bacteria based on bifunctional immune composite nanospheres, more precisely, the present invention relates to bifunctional composite nanospheres and a method for rapidly detecting foodborne pathogenic bacteria, belonging to microbial detection It can be used in medical diagnosis, food safety, environmental monitoring, etc. Background technique [0002] Bacterial pathogens seriously endanger human health. There are many kinds of foodborne pathogenic microorganisms, and the lack of sensitive, convenient and specific rapid detection technology is one of the main reasons why food safety cannot be effectively guaranteed. Therefore, it is an urgent need for food safety and national security to develop fast, sensitive and reliable detection methods and on-site and portable detection instruments for pathogenic bacteria. [0003] The traditional microbial selection culture method is...

Claims

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Application Information

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IPC IPC(8): G01N33/577B82Y15/00B82Y40/00B01J13/02
CPCG01N33/56911B82Y15/00B82Y40/00G01N33/54326G01N33/54346
Inventor 葛玉卿金庆辉毛红菊赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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