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Rapid detection method and kit for mercury poisoning based on magnetic separation and quantum dot marking

A kit and rapid technology, applied in measurement devices, analytical materials, material excitation analysis, etc., can solve the problems of complex pretreatment, unsuitable for rapid detection and screening, etc., and achieve the effects of high photochemical stability, low cost and high efficiency

Inactive Publication Date: 2014-04-02
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Urinary mercury detection methods mainly include cold atomic absorption spectrophotometry (AAS) and atomic fluorescence spectrometry (AFS). Although these methods are mature and the results are highly reliable, the samples require complex pretreatment, which is not suitable for rapid detection and Screening of large numbers of samples

Method used

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  • Rapid detection method and kit for mercury poisoning based on magnetic separation and quantum dot marking
  • Rapid detection method and kit for mercury poisoning based on magnetic separation and quantum dot marking
  • Rapid detection method and kit for mercury poisoning based on magnetic separation and quantum dot marking

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Effect test

Embodiment 1

[0046] 1) Synthesis of Hg immune antigen: Hg (National Center for Analysis and Testing of Nonferrous Metals and Electronic Materials, GSB04-1729-2004), bifunctional chelating agent [(R)-2-thiocyano-3-(4-aminophenyl) )Propyl]-(SS)cyclohexane-1,2 diethylenetriaminepentaacetic acid (p-SCN-Bn-CHX-A"-DTPA) (Macroyclics, USA), hemocyanin (KLH) (Sigma, United States, H7017), triethylamine (TEA) in accordance with the ratio of Hg:p-SCN-Bn-CHX-A"-DTPA:KLH:TEA=9:12:200:13(W / W / W / W), The reaction was shaken at 25°C for 22h; the reaction product was washed with PBS buffer, and the unreacted small molecules were filtered through an ultrafiltration tube (30KD); the obtained Hg immune antigen was diluted with PBS buffer.

[0047] 2) Hg detection antigen synthesis: Hg, p-SCN-Bn-CHX-A"-DTPA, bovine serum albumin (BSA) (Sigma, USA, A1933) and TEA are combined according to Hg: p-SCN-Bn-CHX- A"-DTPA:BSA:TEA=9:12:200:13 (W / W / W / W) ratio of synthetic detection antigen. The identification of immune ant...

Embodiment 2

[0049] Preparation and purification of anti-Hg monoclonal antibody (HgMAb):

[0050] The 1.0 mg / ml Hg immune antigen and an equal volume of Freund's complete adjuvant (FCA) are thoroughly mixed by the syringe double push method to form a white water-in-oil emulsion, that is, an antigen emulsifier. Multipoint immunization was used to immunize 6-week-old female Balb / C mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 211), immunized once every 2 weeks, and immunized 5 times in total. In the second, third, and fourth immunizations, mix the Hg immune antigen with an equal volume of Freund’s incomplete adjuvant (FIA) and boost the immunization at the same dose. For the fifth immunization, only use the Hg immune antigen without adjuvant. Agent. The titer and specificity of mouse serum after mouse immunization are as follows Figure 3-4 Shown. The spleen cells were prepared from the immunized mice with the highest antiserum titer, and the spleen cells were combine...

Embodiment 3

[0052] Anti-Hg immunomagnetic beads (HgMAb-Fe 3 O 4 ) Preparation: Because nano magnetic beads ( M-280Tosylactivated, lifetechnologies, USA, 14203) has a low background value, and the antibody is covalently attached to the surface of the beads, which is an excellent choice for immunoprecipitation of protein complexes. The magnetic aggregation of the beads is gentle and fast and the incubation time is very short. Therefore, the choice of nano magnetic beads M-280Tosylactivated, its particle size is 280nm. The synthesis steps of anti-Hg immunomagnetic beads are as follows:

[0053] (1) Take 165 μL of nano magnetic beads, magnetically separate for 1 min, and remove the supernatant;

[0054] (2) Add 100μg of anti-Hg monoclonal antibody and 0.1M pH7.4 phosphate buffer to make the volume become 150μL, vortex and shake;

[0055] (3) Add 100μL 3M(NH 4 ) 2 SO 4 、Vortex and shake 0.1M pH7.4 phosphate buffer;

[0056] (4) Shaking at 37℃ for 12-18h;

[0057] (5) Magnetic separation for 2 min, ...

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Abstract

The invention provides a rapid detection method for mercury in a liquid biological sample based on magnetic separation and quantum dots (QDs) marking. The method comprises the steps: (1) preparing mercury-resistant monoclonal antibodies; (2) coupling the mercury-resistant monoclonal antibodies with magnetic nanobeads through covalent bonds to prepare mercury-resistant immunomagnetic nanobeadd; and (3) adding a difunctional chelating agent, bovine serum albumin (BSA) and triethylamine (TEA) into the liquid biological sample to incubate, then, adding the mercury-resistant immunomagnetic nanobeads to sufficiently mix, next, carrying out magnetic separation, and adding biotinylated BSA antibodies and streptavidinylated QDs into a sediment, and carrying out fluorescence detection by using a fluorescence microplate, wherein the sediment is obtained through magnetic separation. The invention also provides a rapid detection kit for mercury in a liquid biological sample.

Description

Technical field [0001] The invention belongs to the technical field of biology and new medicine. Specifically, the present invention provides a method for rapid detection of mercury content in urine based on magnetic separation and quantum dot labeling technology, and a kit for rapid detection of mercury content in urine. Background technique [0002] Due to its special physical and chemical properties, mercury (Hg) exhibits four distinctive characteristics of persistence, mobility, high toxicity, and bioaccumulation, which are extremely harmful to the human body. However, despite the strong toxicity of mercury, due to the limitations of the current technology level, mercury is still widely used in various products and processes, including pressure gauges, thermometers, electrical switches, dental fillings, batteries, and PVC made by calcium carbide. . At present, man-made mercury pollution sources have been widely exposed. Humans mainly ingest methylmercury through diet (espec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/533G01N21/64
CPCG01N33/54326G01N33/542
Inventor 黄沛力孙湖泊王萌萌王吉龙
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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