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Enzyme immunosensor for detecting Shigella species and its preparation method and application

A Shigella and enzyme immunization technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of complex detection process, long detection time, human and environmental hazards, etc., and achieve simplified preparation steps, short response time, highly specific effect

Inactive Publication Date: 2009-09-16
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The national standard method is convenient to obtain materials, but the sample pretreatment is complicated, the operation process is cumbersome, the detection cycle is long, and the sensitivity is low; ELISA requires special equipment, detection steps are cumbersome, high cost, long detection time, and prone to false positives; PCR , gene chip and nucleic acid probe technology require expensive equipment, complex detection process, high requirements for the detection environment and professional skills of operators, the reagents used are relatively harmful to the human body and the environment, and are also prone to false positives

Method used

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  • Enzyme immunosensor for detecting Shigella species and its preparation method and application
  • Enzyme immunosensor for detecting Shigella species and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of Enzyme Immunosensor

[0041] Ultrasonic dispersion of 2.0 mg of multi-walled carbon nanotubes in 10 mL of chitosan sol with a mass volume percentage of 1%, to obtain a uniform multi-walled carbon nanotube-chitosan composite;

[0042] Fully mix the multi-walled carbon nanotube-chitosan complex with the anti-Shigella flexneri antibody solution labeled with horseradish peroxidase in equal volumes; place the mixture at 4°C for 12 hours;

[0043] Take 3 μL of the above mixed solution and apply it dropwise on the surface of the working electrode of the disposable screen-printed electrode, and place it at room temperature for 3-5 hours to dry and form a film;

[0044] Soak the coated immune electrode in PBS phosphate buffer solution containing 0.2% BSA (bovine serum albumin), place it at 4°C for 1 hour, rinse it with double distilled water, and remove chitosan that is not firmly bound to the electrode Antibody membrane and blocking non-specific adsor...

Embodiment 2

[0046] Embodiment 2: the method for detecting Shigella

[0047] The enzyme immunosensor described in Example 1 was connected to the electrochemical workstation (CHI 1030A) through wires, and the computer was connected to the electrochemical workstation.

[0048] Put the enzyme immunosensor into the electrolytic cell, and the detection bottom solution of the electrolytic cell contains 0.6mmol / L H 2 o 2 And the acetate buffer solution of 1.0mmol / L thionine, pH value is 7.0. Set the CV measurement parameters of the electrochemical workstation: the potential scanning range is -0.1~-0.6V, the scanning rate is 0.1V / s, apply voltage to the working electrode of the enzyme immunosensor, and measure the current value Ip1 between the auxiliary electrode and the working electrode is 1.946μA.

[0049] Anal swabs from patients with vomiting and diarrhea were taken aseptically, after enrichment, isolation and culture, centrifuged at 8000r / min for 5min, the lower layer of bacteria sludge w...

Embodiment 3

[0052] Using the immunosensor described in Example 1 and the detection method of Shigella in Example 2, different concentrations of Shigella were measured, and a standard current-concentration curve was made, specifically using the Shigella flexneri standard sample preparation 10 10 cfu / mL in PBS solution and serially diluted to 10 4 cfu / mL. Create a standard curve, such as figure 2 shown. Its detection limit: 10 4 cfu / mL; linear interval: 10 4 cfu / mL~10 10 cfu / mL; linear correlation coefficient: R 2 = 99.54%.

[0053] Take the anal swabs of patients with vomiting and diarrhea by aseptic operation. The sample processing and detection methods are the same as in Example 2. Finally, ΔI=0.9312 μA is obtained. Compared with the standard current-concentration curve, the Shiga flexneri contained in the tested sample is calculated. Bacteria concentration is 3×10 7 cfu / mL.

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Abstract

The invention discloses a sensitive, rapid, accurate, high-specificity and economical enzyme immunosensor for detecting Shigella species, comprising screen printing electrodes, a sensitive membrane containing enzyme marked Shigella species antibody- carbon nano-tube- shitosan mixture is coated on the working electrode of the screen printing electrodes. In the invention, the chemical amplification function of the multi-wall carbon nano-tube and enzyme is combined with the specificity of the immunosensor, theenzyme immunosensor has the specificity of immunoreactivity and the sensitivity of the electrochemistry analysis and can accurately detect the lower-content substance. The invention also discloses a preparation method and application of the enzyme immunosensor, the Shigella species can be directly detected, with the sensitive, rapid, high-specificity advantages, enzyme immunosensor is cheap and suitable for detecting the Shigella species in the foundation and in situ.

Description

technical field [0001] The invention relates to the technical field of rapid detection of food-borne pathogenic bacteria, in particular to an enzyme immunosensor for detecting Shigella based on an antigen-antibody immune reaction and a preparation method and application thereof. Background technique [0002] Among the hundreds of millions of food-borne disease patients in the world every year, 70% are caused by eating food and drinking water contaminated by various pathogenic microorganisms. Shigella (Shigella) is one of the main pathogens that cause acute infectious diarrhea. As few as 10 bacteria can cause infection, and the infected human body will have symptoms such as nausea, vomiting, abdominal pain, diarrhea (severe pus and blood in the stool), and even endanger the human body. life. Shigella infection kills more than one million people every year, mostly children in developing countries. [0003] The rapid detection of Shigella is one of the most critical measures ...

Claims

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Application Information

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IPC IPC(8): G01N27/333G01N33/569G01N27/48
Inventor 赵广英詹学佳
Owner ZHEJIANG GONGSHANG UNIVERSITY
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