Method for measuring alpha fetoprotein by using bare nanogold as simulative peroxidase

A technology for peroxidase and alpha-fetoprotein, which is applied in analytical chemistry and nanometer fields, can solve the problems of expensive peroxidase, easy inactivation, complicated labeling process, etc., and achieves simple and fast preparation process, simple process and high activity stable effect

Inactive Publication Date: 2015-04-08
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the clinical detection of alpha-fetoprotein mostly adopts the enzyme-linked immunoassay method based on peroxidase, because peroxidase is expensive, easy to inactivate, and the labeling process is complicated

Method used

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  • Method for measuring alpha fetoprotein by using bare nanogold as simulative peroxidase
  • Method for measuring alpha fetoprotein by using bare nanogold as simulative peroxidase
  • Method for measuring alpha fetoprotein by using bare nanogold as simulative peroxidase

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The preparation of bare nano-gold: at first, 500 μ L concentration is that 0.1g / L chloroauric acid aqueous solution is diluted with the water of 39.5 milliliters, and adding 1.0 milliliter concentration is the sodium borohydride aqueous solution of 0.1 g / L under vigorous stirring (addition time is controlled at Within 5 minutes), the color of the reaction solution changed from light yellow to wine red, and the dark place continued to stir rapidly for 1 hour to obtain bare gold nanoparticles. Bare nano gold solution is wine red, and the maximum absorption wavelength is 516nm (see figure 1 ). All glassware used in the above process was soaked in aqua regia, washed thoroughly with double distilled water, and dried.

Embodiment 2

[0034] Naked nano-gold labeled antibody: Take 22 μg of mouse anti-human alpha-fetoprotein antibody and add 1.0 mL of the naked nano-gold solution prepared in Example 1, shake and react for 30 min, centrifuge at high speed for 1 h, remove the supernatant, and wash the precipitate with 1% BSA in PBS for two After three times, the volume was re-diluted to 1.0 mL to obtain the naked nano-gold-labeled antibody. The maximum absorption wavelength of bare gold nanoparticles is red-shifted to 522nm (see figure 2 ).

Embodiment 3

[0036] Determination of alpha-fetoprotein: coat alpha-fetoprotein antibody on the bottom of 96-well ELISA plate and block overnight at 4°C. Wash 3 times with PBS / T solution. Add 100 μL of alpha-fetoprotein standard substance with a concentration of 100 ng / mL, incubate at 37°C for 2 hours, wash with PBS / T solution 5 times, add naked nano-gold-labeled antibody (5-200 μL) prepared in Example 2, and incubate at 37°C for 2 hours . Wash 5 times with PBS / T solution. Add 2.4mmol / L 3,3',5,5'-tetramethylbenzidine hydrochloride and 6.0mol / L H 2 o 2 50 μL each, reacted in the dark at 45°C for 25 min, then added 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the reaction, and measured the absorbance at 450 nm with a microplate reader. Such as image 3 As shown, the absorbance reached the maximum when the amount of naked gold nanogold-labeled antibody was 50 μL.

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Abstract

The invention discloses a method for measuring alpha fetoprotein by using bare nanogold as simulative peroxidase. The method comprises the following steps: marking an alpha fetoprotein antibody by using the bare nanogold; catalyzing hydrogen peroxide and 3,3',5,5'-tetramethyl benzidine dihydrochloride developing system by a double sandwich antibody method and according to the simulative peroxidase characteristic of the bare nanogold; and detecting the alpha fetoprotein according to the absorbance characteristic of a developing product, wherein the absorbance of the developing product and the concentration of the alpha fetoprotein form a linear relationship within the range of 5 to 100 ng / mL; and the detection limit is 1.4 ng / mL.

Description

technical field [0001] The invention relates to an alpha-fetoprotein assay method using bare nano-gold as a simulated peroxidase, belonging to the fields of analytical chemistry and nanotechnology. Background technique [0002] Liver cancer has always been considered as one of the most common malignant tumors in the world, with a very low cure rate. Early detection and surgical resection are currently the most effective means of treating liver cancer. Alpha-fetoprotein is a typical tumor marker, which mainly exists in the serum of liver cancer cells, yolk sac cells and other malignant tumor patients. Under normal circumstances, alpha-fetoprotein exists in the liver and yolk sac in the early stage of fetal development, and it drops to normal levels within a few months to one year after birth. Therefore, except for pregnant women and newborns, only a small amount of alpha-fetoprotein can be detected in the human body, and the content of alpha-fetoprotein in normal human seru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33G01N33/68G01N33/574G01N33/532
Inventor 陈伟林新华刘爱林王胜邓豪华
Owner FUJIAN MEDICAL UNIV
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