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A Magnetic Molecularly Imprinted Sensor for Detecting Signal Molecules of Gram-negative Bacteria

A magnetic molecular imprinting, gram-negative bacteria technology, applied in the direction of material electrochemical variables, can solve the problems of high cost, complex detection process, long detection time, etc., and achieve strong electrode regeneration, simple operation and high accuracy. Effect

Active Publication Date: 2017-10-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods include enrichment culture screening and subsequent counting detection, biochemical reaction identification or serological identification, etc. These traditional methods are mature in technology, high in accuracy, and require simple equipment, but the experimental operation is cumbersome and the detection cycle is long. Preparation and Heavy finishing work, insufficient specificity, low sensitivity, and the need for professional operators, etc.
ELISA requires special equipment, the detection steps are cumbersome, expensive, long detection time, and prone to false positives
Although the development of molecular-level detection methods of multiplex PCR and real-time fluorescence quantitative PCR has advantages over traditional methods, the equipment required is expensive, the detection process is complicated, the cost is high, and the detection environment and operators have high requirements for professional skills. The reagents used are very harmful to the human body and the environment, and the specificity of this method is not good due to the complexity of the toxin-producing genes
DNA probe technology is fast and highly sensitive, but this technology has certain limitations: First, the DNA of bacteria that have died or sublethal bacteria after food processing may still exist and be detected, but this does not mean that there must be Live pathogenic microorganisms are in; second, food poisoning caused by some pathogenic bacteria is caused by the ingestion of toxins produced by bacteria, so the positive results obtained by DNA probes or PCR methods for toxin genes only mean that there are The existence of pathogenic microorganisms with these gene sequences has the potential to produce toxins, but it does not necessarily mean that there are living cells, or that the genes have been expressed, or that toxins have been produced
However, different bacterial biosensors have different sensitivities to AHLs with different acyl side chain lengths. For example, Agrobacterium tumefaciens is most sensitive to AHLs substituted by the C-3 carbonyl of acyl side chains, while Chromobacterium violaceum ) is more sensitive to short-chain C-3 AHL without substituents, which shows that a bacterial biosensor cannot detect all AHL molecules

Method used

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  • A Magnetic Molecularly Imprinted Sensor for Detecting Signal Molecules of Gram-negative Bacteria
  • A Magnetic Molecularly Imprinted Sensor for Detecting Signal Molecules of Gram-negative Bacteria
  • A Magnetic Molecularly Imprinted Sensor for Detecting Signal Molecules of Gram-negative Bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of Magnetic Molecular Imprinted Electrochemical Sensor

[0041] The schematic diagram of the preparation and use of magnetic molecularly imprinted electrochemical sensors is shown in figure 1 shown.

[0042] Step 1, Preparation of Magnetic Molecularly Imprinted Polymers: Preparation of Fe by Solvothermal Method 3 o 4 MNPs, Fe prepared by the Stober method 3 o 4 @SiO 2 , Fe was obtained by grafting amino groups on the surface of "core-shell" magnetic spheres by silane reagent KH550 3 o 4 @SiO 2 -NH 2 . Mix the template molecule furanone: functional monomer methacrylic acid: crosslinking agent ethylene glycol dimethacrylate according to the molar ratio of 1: (1-8): (10-30), add an appropriate amount of acetonitrile, and ultrasonically ; Add 100-300mg of Fe per millimole template molecule 3 o 4 @SiO 2 -NH 2 Magnetic nanoparticles, 10-30 mg of initiator azobisisobutyronitrile, sonicated until uniform; 2 Under the atmosphere, react in an ...

Embodiment 2

[0046] Example 2 Study on the electrochemical performance of magnetic molecularly imprinted electrochemical sensors

[0047] In this experiment, the performance of the magnetic molecularly imprinted electrochemical sensor was studied in 2.5mM potassium ferricyanide buffer solution of 1.0M KCl, and the results are as follows: Figure 4 shown.

[0048] Step 1, due to the molecularly imprinted polymer microsphere Fe 3 o 4 @SiO 2 -MIP (MMIP for short) is a non-conductive material, and furanone and AHL are non-electroactive substances, so potassium ferricyanide-potassium ferrocyanide is selected as the probe between the base solution and the electrode to characterize magnetic molecular imprinting Electrochemical performance of electrochemical sensors. After MMIP is eluted, the imprinted holes left behind can provide mass transfer channels for probe ions. When the electrode is placed in the C4-HSL solution for adsorption, due to the specific recognition of C4-HSL by the imprinte...

Embodiment 3

[0051] Example 3 Detection of C4-HSL Standards by Magnetic Molecular Imprinted Electrochemical Sensor

[0052] Add appropriate amount of molecularly imprinted polymer microsphere Fe 3 o 4 @SiO 2 -MIP, adsorption at room temperature for 5-10min; the pretreated magnetic glassy carbon electrode is inserted into the above solution, adsorbed for 1-10min, taken out from the solution, carefully washed with double distilled water for 20s to remove the physically adsorbed substances on the electrode surface ( including unbound samples). The magnetic molecular imprinted electrochemical sensor was placed in the electrolytic cell, and the DPV was measured in 2.5mM potassium ferricyanide buffer containing 1.0M KCl. DPV test conditions: the voltage sweep range is -0.1V to 0.65V, the pulse amplitude is 25mV, the pulse width is 50ms, the pulse period is 500ms, and the potential increment is 5mV. All tests are carried out at room temperature. There is a quantitative relationship between th...

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Abstract

The invention discloses a magnetic molecular imprint sensor for detecting signal molecules of Gram-negative bacteria, belonging to the technical field of rapid detection of pathogenic bacteria. The invention combines surface molecular imprinting technology, magnetic separation technology and electrochemical sensing technology to construct a new type of magnetic molecular imprinting electrochemical sensor, which has the specificity of molecular imprinting technology, the reproducibility and separation of magnetic separation technology The rapidity, rapidity and sensitivity of electrochemical analysis can accurately detect low-level Gram-negative bacteria signal molecules. The invention also discloses the preparation method and application of the magnetic molecular imprinted electrochemical sensor, which can realize the indirect detection of some Gram-negative bacteria, has the advantages of sensitivity, rapidity, high specificity, and low price, and is suitable for the food industry and Detection of Gram-negative bacteria in the medical industry and other fields.

Description

technical field [0001] The invention relates to a magnetic molecular imprint sensor for detecting signal molecules of Gram-negative bacteria, belonging to the technical field of rapid detection of pathogenic bacteria. Background technique [0002] Enteropathogenic bacteria in food is one of the main causes of human foodborne diseases and a major killer of human health. Cooked meat, bread and cakes are very easy to directly cause food-borne diseases, while aquatic products are also a major hidden danger of human food-borne diseases due to the pollution and invasion of various microorganisms in the water. Most of the intestinal bacteria in food belong to Gram-negative bacteria (Gram-negative), the most common ones are Salmonella, Shigella, diarrhea-causing Escherichia coli, Clostridium aerogenes and Vibrio parahaemolyticus, etc. . Gram-negative bacteria, generally refers to bacteria that are red in the Gram stain reaction. They produce endotoxins, which make people sick by ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/26
Inventor 孙秀兰蒋卉张银志
Owner JIANGNAN UNIV
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