Method for detecting organophosphorus pesticide by flora-based sensing system

A technology for organophosphorus pesticides and sensing systems, which is applied in the field of detection of organophosphorus pesticides based on bacterial flora-based sensing systems. The requirements of the instrument, the effect of high sensitivity

Active Publication Date: 2018-09-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A problem associated with surface display is that prolonged incubations require proper target protein translocation, leading to delays and non-linear signal accumulation

Method used

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  • Method for detecting organophosphorus pesticide by flora-based sensing system
  • Method for detecting organophosphorus pesticide by flora-based sensing system
  • Method for detecting organophosphorus pesticide by flora-based sensing system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. The bacterial strains, medium and reagents used in this embodiment

[0044] Escherichia coli XL1-Blue (Clontech, USA) and Escherichia coli DH5α (Invitrogen, USA) were used for all cloning and protein expression steps. Bacteria were cultured in 2YT medium (16 g / L tryptone, 10 g / L yeast extract and 5 g / L sodium chloride).

[0045] Dissolve p-nitrophenol (PNP) and Ops (such as ethyl paraoxon, methyl paraoxon, ethyl parathion, methyl parathion, fenitrothion and EPN) in acetonitrile to prepare concentrations 1×10 -1 M stock solution.

[0046] Test chemicals were purchased from Sigma-Aldrich Company. Whatman Universal filter paper (medium speed, creped, diameter = 9.0 cm), animal tissue peptone, beef extract, gelatin, L-sodium ascorbate, D-(+)-sodium pentafluoride, L-glutamic acid monosodium salt Hydrate, ampicillin, kanamycin, tetracycline and anhydrous N,N-dimethylformamide (DMF) were purchased from Promega (Madison, WI, USA). HPLC grade acetonitrile, sodium chlorid...

Embodiment 2

[0074] Embodiment 2, the preparation of filter paper sensing strip

[0075] Sensing paper strips were prepared according to the aforementioned flora-based biosensing system. Briefly, the two cell types were suspended in a 1:1 ratio in sterile dry protection solution pre-warmed at 37°C. 5 μL of the cell suspension was spotted on Whatman filter paper strips (0.6×4 cm), and the strips were dried in a laminar air oven for 10 minutes, followed by vacuum drying at 20° C. in a lyophilizer. The filter paper sensor strips were then stored at 4°C until use.

Embodiment 3

[0076] Embodiment 3, the mensuration of the dose-response OPs curve on the filter paper sensing strip

[0077] Dilute ethyl paraoxon (concentration of 1×10- 3 -1×10 -9 M). Take 100 μL of each standard solution and add to the culture tube containing 900 μL 2YT medium and prepared filter paper strips. The culture tubes were then incubated statically at 28°C for 2 hours. The paper strips were then removed from the culture tubes and kept between layers of plastic wrap to prevent drying. Subsequently, 10 μL of X-gal substrate solution (50 mg / mL) dissolved in DMF was carefully added to the site containing the sensor cells, and color development was allowed to proceed for 90 minutes. Such as Figure 7 As shown in A, the cell-retained area of ​​the paper strip changes from colorless to blue as the concentration of ethyl paraoxon increases.

[0078] In addition to visual observation of the developed blue color, color intensity was also measured using ImageJ software (NIH, Bethesd...

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Abstract

The invention provides a method for detecting an organophosphorus pesticide (OP) by a flora-based sensing system. The sensing system comprises an OPH signal sensing module and a genetic engineering flora containing a signal generating module. One of the modules is used to hydrolyze OPs into p-nitrophenol (PNP), and the other one converts a PNP signal into beta-galactosidase generation for colorimetric detection. Through optimization, the sensing system can detect ethyl paraoxon at a concentration of 1*10<-9>M within 3.5 hours of induction at 28 DEG C, and is about 200 times more sensitive thana single-cell whole-cell sensor. In addition, the sensing system can detect various types of OPs commonly used in agriculture. Besides, the sensing system can be prepared into filter paper for detection of organophosphorus pesticides, showing the prospect of portable on-site detection. The invention provides a sensitive, fast and portable biological sensing platform for pollutant detection, and also shows a practical novel environmental application of an engineering microbial ecosystem.

Description

technical field [0001] The invention relates to the technical field of pesticide detection, in particular to a method for detecting organophosphorus pesticides with a bacterial flora-based sensing system. Background technique [0002] Organophosphorus pesticides (OPs) are widely used in insect control, accounting for approximately 38% of global pesticide use. As potent inhibitors of acetylcholinesterase in the nervous system, they can also cause acute neurotoxic poisoning in humans and animals. Therefore, despite the substantial agricultural benefits of OPs, the large-scale application of OPs raises serious public concerns about health, environment, and food safety. Therefore, there is an urgent need to develop methods for rapid, sensitive and portable OPs detection. Many analytical methods, such as liquid chromatography or gas chromatography and mass spectrometry, have been developed for the detection of OPs. However, they are often expensive and require extensive logist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12Q1/6897C12R1/19
CPCC07K14/21C12N9/16C12N15/70C12Q1/6897C12Y301/08001
Inventor 冯雁
Owner SHANGHAI JIAO TONG UNIV
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