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Method for detecting escherichia coli in water by in-situ forming Raman enhanced substrate

A technology of Escherichia coli and Raman, which is applied in the field of in-situ formation of Raman-enhanced substrates to detect Escherichia coli in water, to achieve strong specificity, promote economic development, and ensure water safety

Active Publication Date: 2015-02-04
嘉善县临沪新城实业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Routine culture and detection technology requirements are simple, but the pathogenic bacteria in the water body need to be cultivated first, this process takes at least 48-72 hours to complete, and the detection is time-consuming and laborious
Molecular biology detection usually extracts E. coli DNA or RNA, uses PCR technology to specifically amplify the extracted nucleic acid, and then detects E. coli by detecting nucleic acid. This method has high detection sensitivity and strong specificity, but it cannot be achieved. On-site analysis and testing cannot evaluate the status of water pollution in real time
Although the detection specificity of Escherichia coli is strong, the detection sensitivity is low

Method used

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  • Method for detecting escherichia coli in water by in-situ forming Raman enhanced substrate
  • Method for detecting escherichia coli in water by in-situ forming Raman enhanced substrate
  • Method for detecting escherichia coli in water by in-situ forming Raman enhanced substrate

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Experimental program
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Effect test

Embodiment 1

[0018] 1. Escherichia coli selection:

[0019] Escherichia coli O157:H7 was selected as an example to detect target bacteria. Escherichia coli O157:H7 was cultured in LB medium overnight at 37°C. Take 1mL of bacterial culture solution, centrifuge at 4000rpm / min for 5min, remove the culture medium, then use pH7.4 PBS buffer to resuspend the bacteria, dilute the bacteria, and the final concentration of the bacteria is 10 4 cfu / mL, 10 5 cfu / mL, 10 6 cfu / mL, 10 7 cfu / mL for easy detection.

[0020] 2. In situ formation of Raman enhanced substrate:

[0021] 0.1 μg / mL graphene oxide modified with 1 μg / mL glucose oxidase and 0.1 μg / mL anti-E. coli O157:H7 antibody binds to E. coli O157:H7. Centrifuge at 2000rpm / min for 5min, collect the bacteria, and resuspend in pH7.4 PBS buffer.

[0022] Add 0.5 μg / mL silver nano-seeds, glucose oxidase on the surface of graphene oxide catalyzes 1 μg / mL glucose to generate hydrogen peroxide; hydrogen peroxide reacts with 10 μg / mL silver ions ...

Embodiment 2

[0025] 1. Escherichia coli selection:

[0026] Escherichia coli O157:H7 was selected as an example to detect target bacteria. Escherichia coli O157:H7 was cultured in LB medium overnight at 37°C. Take 1mL of bacterial culture solution, centrifuge at 4000rpm / min for 5min, remove the culture medium, then use pH7.4 PBS buffer to resuspend the bacteria, dilute the bacteria, and the final concentration of the bacteria is 10 4 cfu / mL, 10 5 cfu / mL, 10 6 cfu / mL, 10 7 cfu / mL for easy detection.

[0027] 2. In situ formation of Raman enhanced substrate:

[0028] 1 μg / mL graphene oxide modified with 10 μg / mL glucose oxidase and 20 μg / mL anti-E. coli O157:H7 antibody binds to E. coli O157:H7. Centrifuge at 3000rpm / min for 5min, collect the bacterial cells, and resuspend in pH7.4 PBS buffer.

[0029] Adding 50 μg / mL silver nanoseeds, the glucose oxidase on the surface of graphene oxide catalyzes 50 μg / mL glucose to generate hydrogen peroxide; hydrogen peroxide reacts with 50 μg / mL s...

Embodiment 3

[0032] 1. Escherichia coli selection:

[0033] Escherichia coli O157:H7 was selected as an example to detect target bacteria. Escherichia coli O157:H7 was cultured in LB medium overnight at 37°C. Take 1mL of bacterial culture solution, centrifuge at 4000rpm / min for 5min, remove the culture medium, then use pH7.4 PBS buffer to resuspend the bacteria, dilute the bacteria, and the final concentration of the bacteria is 10 4 cfu / mL, 10 5 cfu / mL, 10 6 cfu / mL, 10 7 cfu / mL for easy detection.

[0034] 2. In situ formation of Raman enhanced substrate:

[0035] 0.5 μg / mL graphene oxide modified with 5 μg / mL glucose oxidase and 10 μg / mL anti-E. coli O157:H7 antibody binds to E. coli O157:H7. Centrifuge at 2500rpm / min for 5min, collect the bacterial cells, and resuspend in pH7.4 PBS buffer.

[0036]Add 30 μg / mL silver nanoseeds, and the glucose oxidase on the surface of graphene oxide catalyzes 30 μg / mL glucose to generate hydrogen peroxide; hydrogen peroxide reacts with 30 μg / mL ...

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Abstract

The invention discloses a method for detecting escherichia coli in water by in-situ forming a Raman enhanced substrate. The method comprises the following steps: specifically binding oxidized graphene commonly modified by glucose oxidase and escherichia coli antibodies to the surface of escherichia coli; and then adding a silver nano seed, catalyzing glucose to generate hydrogen peroxide by using the glucose oxidase on the surface of the oxidized graphene, reacting hydrogen peroxide with silver ion to promote the growth of the silver nano seed so as to form silver nanoparticles, adsorbing the silver nanoparticles on the surface of the oxidized graphene, in-situ forming a Raman enhanced substrate; adsorbing the Raman reporter molecule on the surface of the oxidized graphene; combining the common Raman enhancing effects of the silver nanoparticles and the oxidized graphene so as to detect the escherichia coli.

Description

technical field [0001] The invention relates to a method for forming a Raman enhanced substrate in situ to detect Escherichia coli in water. Background technique: [0002] Every day, 1.1 billion people around the world wait for safe water. In developing countries, tens of thousands of people are sick because of E. coli contamination in drinking water. During the Eleventh Five-Year Plan period, 70% of my country's environmental emergencies involved drinking water safety, many of which were polluted by Escherichia coli, affecting drinking water safety, endangering human health, and hindering sustainable economic and social development. The harsh reality forces researchers to develop new methods and technologies for efficient, rapid, and specific detection of E. coli in water, so as to provide technical support for ensuring people's water safety and protecting human health. [0003] At present, the detection methods for E. coli in the water environment mainly include conventi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65
Inventor 陈章李会东李志贤陈国梁
Owner 嘉善县临沪新城实业有限公司
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