Microcystin-LR detecting method based on enzyme-free immuno-sensor

A microcystin and sensor technology, applied in the field of immunoanalysis chemistry, can solve the problems of undrinkable, complicated processing process, high detection cost, etc., and achieve the effect of online detection, high sensitivity and low cost

Inactive Publication Date: 2016-05-04
CHANGZHOU UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In May 2007, the concentrated outbreak of blue-green algae in Taihu Lake caused the tap water in some areas of Wuxi to stink and become undrinkable
[0003] At present, the detection technology of MC-LR in water mostly adopts high-performance liquid chromatography, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry based on laboratory analysis. The sample pretreatment process is complex and time-consuming, requiring professional and technical personnel to operate. , the detection cost is high

Method used

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  • Microcystin-LR detecting method based on enzyme-free immuno-sensor
  • Microcystin-LR detecting method based on enzyme-free immuno-sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation of carboxylated carbon nanofibers

[0040] a. Disperse 40 mg of carbon nanofibers with a diameter of 60 nm in 60 mL of HNO with a solute mass fraction of 30% 3 In the solution, mix and ultrasonically stir evenly;

[0041] b. Reflux the mixed solution obtained in step a in an oil bath at 140°C for 24 hours;

[0042] c, filtering and washing until the pH is 7.0 to obtain carboxylated carbon nanofibers with better water solubility;

[0043] d, preparing a dispersion of carboxylated carbon nanofibers prepared in step c of 1 mg / mL;

Embodiment 2

[0045] Preparation of gold nanoparticles and its labeled goat anti-mouse antibody

[0046] a. Heat 50mL of chloroauric acid solution with a mass concentration of 0.01% to boiling, and quickly add 1.75mL of trisodium citrate solution with a mass concentration of 1% to it under vigorous stirring, and continue the reaction for 10 minutes to obtain a brick with a particle size of 13nm Red gold nanoparticle colloidal solution;

[0047] b, get the gold nanoparticle colloidal solution 1mL that step a obtains and use 0.1M K 2 CO 3 After adjusting the pH to 9.0, add 25 μL of 1 mg / mL goat anti-mouse antibody, and stir at room temperature for 2 h;

[0048] c. First filter the above mixture, centrifuge and wash the obtained precipitate three times with 0.01M (pH7.4) phosphate buffer solution, and disperse it with 300 μL of phosphate buffer solution with a concentration of 1% bovine serum protein to obtain the labeled goat anti-mouse Antibody gold nanoparticles (gold nanoparticle probes...

Embodiment 3

[0050] Preparation and detection steps of MC-LR immunosensor

[0051] Glassy carbon electrodes with a diameter of 3mm pass through 0.3μm and 0.05μm Al 2 o 3 After the powder is polished, it is thoroughly ultrasonically washed in ethanol and deionized water in turn to obtain a smooth and clean electrode.

[0052] After dripping 5 μL of the dispersion of carboxylated carbon nanofibers prepared in Example 1 on the dried above-mentioned electrode, then drop-coating 5 μL of 2 mg / mL polyethylene glycol-20000 dispersion, and saturated water at 4 ° C Place it in a steam environment overnight, and form a layer of polyethylene glycol film by physical adsorption;

[0053] 10 μL of 500 μg / L MC-LR antigen was added dropwise on the surface of the modified electrode treated above, and after physical adsorption in a 25°C wet box for 1 hour, the modified electrode was rinsed with phosphate Tween buffer (0.05% Tween-20) with a pH of 7.4. After the electrode, 10 μL of bovine serum albumin sol...

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Abstract

The invention relates to a preparation method of an enzyme-free immuno-sensor for detecting microcystin-LR (MC-LR) and a detection method thereof, and belongs to the technical field of immuno-analytical chemistry. According to the method, carboxylated carbon nano fiber and polyethylene glycol are assembled on the surface layer of a glass-carbon electrode to construct a high density MC-LR antigen sensing interface; gold nano particles are synthesized, a gold nano particle probe is prepared, the indirect competition immunoassay principle is adopted, embedded MC-LR antigen and MC-LR target antigen compete for MC-LR antibody to form an immune complex, the gold nano particle probe reacts with the immune complex; the gold nano particles bonded on the surface of the electrode are oxidized to generate AuCl<4->, the value of the peak current of the reduction peak of AuCl<4-> can be measured by an electrochemical method, and finally the MC-LR is detected through the value of the peak current. The provided method can online detect MC-LR high sensitively, the purification treatment of a water sample is not needed, the detection is simple and rapid, the detection system does not need any enzyme, and thus the detection cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of immunoanalysis chemistry, in particular to a method for detecting microcystin-LR. Background technique [0002] In 1996, more than 100 acute liver failures occurred in Brazil, and at least 50 people died of the acute effects of algae toxins within 7 months, which attracted worldwide attention. In May 2007, a concentrated outbreak of blue-green algae in Taihu Lake caused the tap water in some areas of Wuxi to stink and become undrinkable. Cyanotoxins in fresh water have become a global environmental problem, and cyanotoxin poisoning incidents often occur around the world. The most common cyanotoxin is a cyclic heptapeptide toxin, Microcystin-LR (MC-LR). Due to the significant inhibitory effect of this toxin on protein phosphatases 1 and 2A, it may cause profound disturbances in liver function and structure. MC-LR is the most toxic toxin. In 1998, the World Health Organization (WTO) stipulated that the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/405
Inventor 张静孙玉峰陈智栋王文昌
Owner CHANGZHOU UNIV
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