Visualized immunoassay method based on nanogold growth

An immune analysis and nano-gold technology, applied in the field of analytical chemistry and nanotechnology, can solve the problems of high price, single color development, poor quantitative effect, etc., and achieve the effect of fast reaction speed, good color development effect and easy promotion

Inactive Publication Date: 2016-03-30
FUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of complex operation and high price of existing instruments, single color development and poor quantitative effect of traditional visualized enzyme-linked immunosorbent assay, the present invention proposes a fast, sensitive and multi-color visual detection and analysis method

Method used

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  • Visualized immunoassay method based on nanogold growth
  • Visualized immunoassay method based on nanogold growth
  • Visualized immunoassay method based on nanogold growth

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Experimental program
Comparison scheme
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Embodiment 1

[0038] Embodiment 1: the synthesis of nano gold nucleus

[0039] First, add 5mL0.20M CTAB into a 15mL glass bottle, then add 0.25mL0.01MHAuCl 4 and 4.75mL of water, stir vigorously to mix. Then add 0.6mL of freshly made frozen 0.01M NaBH to this solution 4 , the final product is a brown-yellow gold nano-nucleus solution. Quickly mix for 2 minutes, and stand at room temperature for more than 10 minutes for later use.

Embodiment 2

[0040]Embodiment 2: the establishment of working curve

[0041] Firstly, in the carbonate buffer system of pH 9.6, the primary antibody was added to the microwell plate and left overnight at 4°C. After washing three times with PBST, 5 wt.% BSA was added to each well as a blocking agent. PSA pre-dispersed in serum was added to the microplate at concentrations ranging from 10 -3 To 200pg / mL, the serum-only solvent was used as a blank control, and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μL of alkaline phosphatase-labeled secondary antibody was added to each well, incubated for 30 min, and washed three times. 50 μL (20 mM) ascorbic acid superphosphate was added to each well, incubated at 37°C for 1 hour, then 20 μL was taken out and mixed with gold rod growth solution, and 10 μL of gold nucleus solution was added. These mixed solutions need to be incubated at room temperature for 1 hour, and then the absorption spectrum of the grown gold rods is m...

Embodiment 3

[0042] Embodiment 3: detection of simulated sample to be tested by standard addition method

[0043] The whole process adopts the standard addition method to detect the simulated samples to be tested, that is, artificially add PSA samples of known concentration in normal human serum as positive samples, and normal human serum as negative samples, and then use our method to detect and verify the reliability of the method sex. First, in a carbonate buffer system with pH 9.6, 100 μL of the primary antibody was added to a 96-well microplate and left overnight at 4°C. It was subsequently washed three times with PBST, and 5 wt.% BSA was added to each well as a blocking agent. Add 100 μL of 500-fold diluted positive sample serum (containing PSA10pg / mL), negative sample serum and blank control (Tris-HCl buffer) to each well, and incubate at 37°C for 1 hour. After washing three times with PBST, 100 μL of alkaline phosphatase-labeled secondary antibody was added to each well, incubate...

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Abstract

The invention discloses a visualized immunoassay method based on nanogold growth, which comprises the following steps: (1) adding antigens that have different concentrations and are capable of combining with specificity of an elisa plate into the elisa plate that is coated with a target acquisition antibody; (2) adding an enzyme labeled detection antibody into the elisa plate; (3) adding ascorbic acid ultraphosphate ester into the elisa plate, so that the ascorbic acid ultraphosphate ester reacts with labeled enzyme to generate ascorbic acid; (4) adding 20muL of the obtained reaction liquid to 220muL of gold rod growth liquid and then adding 10muL of gold core liquid, and after reaction, using a microplate reader for measuring absorption spectrum, and using a digital camera for recording the liquid color; (5) processing data and drawing a work curve; and (6) detecting and simulating a sample to be detected according to the above experimental steps. The method has the advantages that the operation is simple, the applicability is wide, the reaction is fast, various color changes can be generated, and the method can realize visualization quantitative determination for a target object.

Description

technical field [0001] The invention relates to a visual immunoassay method based on nano-gold growth, which belongs to the field of analytical chemistry and nanotechnology. Background technique [0002] Colorimetric sensors have been widely used in scientific research and industry due to their simplicity and practicability. The sensor can be detected with UV instruments and can even be inspected with the naked eye, making it ideal for applications such as point-of-care testing and environmental food detection. An ideal colorimetric sensor should at least satisfy the following aspects: high sensitivity, wide range of applicable analytes, and naked-eye detection. In this invention, we demonstrate a colorimetric sensor based on the combination of traditional ELISA and enzyme-induced growth of gold rods, which can almost meet all the conditions mentioned above. [0003] ELISA is a widely used immunoassay method that utilizes the biological properties of enzymes and the specif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/543
Inventor 郭隆华李言言马小明林振宇邱彬陈国南
Owner FUZHOU UNIV
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