The Enrichment Of Escherichia Coli And Construction Of Paper-based Detection Device

In recent years, with the rapid development of industry and agriculture, the living standard of people is increasing day by day, meanwhile, food and water security problem have been widely concerned, among these issues, pathogen pollution in water is one of the major problems. E. coli is the major bacteria in intestine of humans and animals, which is easy to pollute water with faeces. Thus, E.coli is the hygiene standard of water and food. If we found E.coli in water, we can ensure that water has been polluted. E. coli does not cause disease normally, but it can induce severe and at times life threatening diseases in some conditions, such as bloody diarrhea, hemorrhagic colitis, renal failure and meningitis. Therefore, rapid and sensitive detection of E.coli in water is a prerequisite for the timely and effective control and prevention of water pollution.At present, common methods for detecting E.coli include bacterial culture and biochemical identification, enzyme and substrate detection, enzyme linked immunosorbent assay（ELISA）, lateral flow immunoassays（LFA） and polymerase chain reaction（PCR） et al. These methods can be used for E.coli detection, however, they are limited by the need for long-time, costly instrumentation and complicated operations, thus, they are not suitable for point-of-care testing. Recently, microfluidic paper-based analytical devices（μPADs） have attracted a great deal of attention since they are a new class of point-of-care testing devices that are inexpensive, easy to use, and designed specifically for use in developing countries.We prepared a microfluidic paper-based analytical device for E.coli detection specifically, in the meanwhile, magnetic nanoparticles were used to enrich bacteria, which improved the detection sensitivity. Using this method we achieved low cost, visual and quantitative detection of E.coli. In the end, we obtained following conclusions:1. We used functional magnetic nanoparticles PLL-Fe₃O₄ to enrich E.coli. The nanoparticle surface was negatively charged when p H is less than 9, which could combine with E.coli charged positively through electrostatic interaction, then using permanent magnet to separate bacteria. Using this method we achieved efficient and rapid enrichment, the capture efficiency was 97%, the concentration of PLL-Fe₃O₄ nanopatricles was 3 mg/ml, and the time needed was 4 min.2. we prepared a paper-based analytical device including lysis zone and detection zone, furthermore, we sealed the chip using transparent PDMS film, which could reduce evaporation and pollution of reagent, improving the portability. Through food dye test, we ensured that liquid on paper could flow directionally, and mix with another liquid.3. We deteced E.coli using the prepared paper-based analytical device after bacteria capture. Using 6 m M X-Gluc as substrate, PBS as the wash buffer, with temperature of 50, we achieved lysis and visual detection of E.coli within 2 hours. After that, we took pictures using digital camera, and analyzed results by use of IPP. Moreover, we detected S.aureus, Salmonella and L.monocytogenes, all of them had no color change. In the end, we achieved E.coli detection in spring water, and the limit of detection was 102 cfu/ml. It was indicated that the paper-based analytical device could accomplish rapid, sensitive, specific, visual and quantitative E.coli detection.