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Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof

An immunosensor and electrochemical technology, which is applied in the field of analytical chemistry and chemical sensors, can solve the problems of difficult to meet clinical rapid detection, long detection time, false negatives, etc., and achieve good electron transfer ability, convenient detection process, and good electrical conductivity Effect

Inactive Publication Date: 2013-02-06
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods all have the disadvantages of cumbersome operation, low detection sensitivity, and incapable of accurate quantitative detection.
As the most common method for clinical detection of Tg-IgM at present, enzyme-linked immunoassay not only has problems such as low sensitivity and specificity, but also requires a large amount of samples and a long detection time, which is difficult to meet the needs of rapid clinical detection
As another common clinical detection method, rapid colloidal gold immunochromatography can only be qualitative or semi-quantitative, and is prone to false negative and false positive results
There is no method for quantitative and real-time detection of Tg-IgM at home and abroad

Method used

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  • Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof
  • Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof
  • Electrochemical immunosensor for detecting toxoplasma gondii IgM antibody and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Fabrication of Immunosensors Based on Graphene, Polythionine, and Gold Nanoparticles

[0035] (1) Electrode pretreatment: the glassy carbon electrode is respectively treated with 1.0 and 3.0 μm Al on the suede 2 o 3 The slurry is polished to a mirror surface, then ultrasonicated in double distilled water and absolute ethanol for 5 minutes, and left to dry;

[0036](2) Preparation of Nafion-graphene mixed solution and gold nanoparticles: add 2mg / mL graphene solution into 1.0mL0.1% Nafion solution, and ultrasonicate for 30min to prepare a black homogeneous Nafion-graphene mixed solution ;Weigh 100mL of 0.01wt% chloroauric acid solution in a flask and heat to 80°C; quickly add 2.5mL of freshly prepared 1% trisodium citrate solution; heat and stir until the color of the solution turns deep purple; store after natural cooling In a brown volumetric flask (the average diameter of the prepared gold nanoparticles is 16nm);

[0037] (3) Preparation of graphene-polythionine-gol...

Embodiment 2

[0041] Determination of Tg-IgM by electrochemical immunosensor and drawing the working curve

[0042] (1) Preparation of Au-Fe 3 o 4 -HRP-anti-IgM composite detection probe: 1mg Au-Fe 3 o 4 Add 300 μL of HRP-anti-IgM solution, place in a constant temperature shaker at 37°C, and shake at 180 rpm for 20 minutes; apply an external magnetic field to magnetically separate the coupling reaction solution; wash the coupled particles with PBS repeatedly for 3 times; add 0.25% calf Serum (BSA) blocks the non-specific sites on the coupled particles; Au-Fe is obtained by rewashing and magnetic separation 3 o 4 -HRP-anti-IgM composite detection probe; the composite detection probe was added to PBS buffer, and stored at 4°C for later use;

[0043] (2) A series of Tg-IgM standard solutions of different concentrations were drip-coated on the surface of BSA / captured antigen / AuNPs / PTH / Nf-GS / glassy carbon electrode, and incubated at 37°C for 25min;

[0044] (3) Drop 5 μL of composite detec...

Embodiment 3

[0049] Determination of Tg-IgM content in serum samples of pregnant women

[0050] According to the working curve of Tg-IgM detection established in Example 2, the method of the present invention is used to measure the Tg-IgM content in the serum of pregnant women, and the standard addition method is used to measure the recovery rate.

[0051] (1) Pretreatment of serum samples of pregnant women: collect blood samples from clinically healthy pregnant women (from the Laboratory Department of the First People's Hospital of Jiulongpo District, Chongqing City), centrifuge at 3000rpm for 10min, and draw serum samples;

[0052] (2) Add 2.0, 4.0, 6.0, 8.0 AU / mL Tg-IgM standard substances in sequence to the above serum samples;

[0053] (3) Carry out the method identical with embodiment 1 and embodiment 2 to modify electrode and measure Tg-IgM, measure 5 times to each spiked sample, obtain the actual concentration of Tg-IgM in the sample according to working curve.

[0054] As shown i...

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Abstract

The invention belongs to the technical field of analytical chemistry and chemical sensors and discloses an electrochemical immunosensor for detecting a toxoplasma gondii IgM (Immunoglobulin m) antibody (Tg-IgM) of a gravida and a preparation method of the electrochemical immunosensor. The immunosensor is prepared by sequentially modifying graphene, polythionine, gold nanoparticles and capture antigen to the surface of a glassy carbon electrode. An enzyme-functionalized nano-composite detection probe with an electrical signal amplifying function is prepared by assembling enzyme and a second antibody with high proportions on an Au-Fe3O4 surface. According to the sandwich immunoassay principle, the concentration of Tg-IgM is determined by using an electrochemical signal generated by catalysis of enzyme to a substrate. According to the electrochemical immunosensor, the specificity of immunoreaction is combined with the sensitivity of electrochemical detection; the transmission of electronics is promoted by using the graphene, the polythionine, the gold nanoparticles, Au-Fe3O4 and other material; and the sensitivity of the detection is improved. The electrochemical immunosensor has the advantages of simplicity and convenience for operation, favorable regeneration performance and detection cost reduction. The electrochemical immunosensor prepared on the basis can be also used for detecting other immunological markers and has favorable application prospect in medical diagnosis.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry and chemical sensors, and relates to a preparation method of an electrochemical immunosensor for detecting toxoplasma gondii IgM antibody, specifically based on the reaction between antigen and antibody, for detecting toxoplasma gondii IgM antibody (Tg- IgM) immunosensor and preparation method thereof. Background technique [0002] With the change of modern eating habits and the increase of domestic pets, the infection rate of toxoplasmosis is increasing year by year. Toxoplasma infection during pregnancy can cause miscarriage, premature delivery, teratogenicity or stillbirth in pregnant women, and can cause neurological damage or visual impairment in newborns. Toxoplasma-specific IgM antibody (Tg-IgM) can appear in the serum of pregnant women 14 days after Toxoplasma infection, and can be used as an effective indicator for recent infection and early diagnosis of Toxoplasmosis. The I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N27/26
Inventor 蒋姝婷谢国明罗鹏花尔辉赵朝辉马翠霞
Owner CHONGQING MEDICAL UNIVERSITY
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