The Study On The Preparation And Performance Of Rapid Detection Chip For Enterohemorrhagic Escherichia Coli

The safety of food and drinking water directly relates to people's health and life safety.In order to guarantee the quality of food and drinking water,developing fast detection technology is extremely essential.Bacterial infection is one of the main causes of food poisoning,in particular,pathogenic bacteria can cause more severe consequences.EHEC O157:H7 used in this thesis can cause serious diseases and even death.Low dose of EHEC O157:H7 could infect human and leads to slight diarrhea,hematochezia,even acute kidney failure,rapid detection and identification of EHEC O157:H7 is the key to preventing and treating effectively.Conventional methods of bacterial detection and identification of EHEC O157:H7 rely on the submission of samples to laboratories for culturing in selective media.The cell culture-based assays are reliable but is lengthy and generally takes up several days to obtain a confirmed result.In order to quickly detect and identify the EHEC O157:H7 in samples,in this thesis,the biosensors based on the planar substrate and the substrate with three dimensional micro-nano structures were constructed by layer-by-layer technology and nanoprinting technique.The proposed biosensors can specifically capture the EHEC O157:H7 through the specific recognition between antigen and antibody.The vortex mixing effect of PDMS chip with herringbone structure was further introduced to improve the efficiency of the biosensors for capturing EHEC O157:H7.Finally,a lab on a chip was developed by combining the proposed biosensors and microfluidic chip-based loop-mediated isothermal amplification(LAMP)for the selective capture,culture,release,lyse and recognition of the EHEC O157:H7.The main research contents of this thesis are as follows:This thesis prepared the biosensor based on the planar substrate with carbon nanotubes by layer by layer technology,studied the capture efficiency and detection limit of EHEC O157:H7,the results showed that the capture efficiency was about 60% and the detection limit was 1 CFU/m L.In addition,the contructed biosensor possessed the specificity,biocompatibility,reusability,stability and practical applications.In order to increase the surface area for antibodies' binding and bacterial adhesion,three dimensional micro-nano structures were fabricated on the substrate using nanoimprinting technique.The nanostructured substrates with 3D microarray efficiently improve the capture efficiency of EHEC O157:H7 and the morphology of 3D microarray can influence the bacterial capture efficiency significantly.Finally,it was found that the squared 3D microarray with side length of 6 ?m,the height of 2 ?m and the interval of 2 ?m had the highest capture efficiency in our experiment.The capture efficiency is 82.13% for EHEC O157:H7 and the detection limit is 1 CFU/m L.In addition,this biosensor also had good specificity,biocompatibility,stability and practical applications.To further increase the contact time of EHEC O157:H7 with the biosensors,the herringbone structure embedded microfluidic chip was combined with smooth biosensor and biosensor with three dimensional micro-nano structure.The PDMS chip with herringbone structure was used as the upper layer of microfluidic chip.The experiment results proved the herringbone structure had vortex mixing effect and enhanced the capture efficiency of EHEC O157:H7.The microfluidic chip which combined PDMS chip with herringbone structure and the biosensor with three dimensional micro-nano structure had the highest capture efficiency(95%),and the detection limit was 1 CFU/m L.This microfluidic chip not only had the superior bacteria capture performance,also had the better specificity and good prospect of practical application.To meet the requirements of detecting the EHEC O157:H7 qualitatively and quantitatively and reducing the use of expensive equipments,the DNA of the captured EHEC O157:H7 was analyzed using a PDMS microfluidic chip-based LAMP coupled with an optical turbidity sensor.A PDMS microfluidic chip used in this work had an 8 parallel lines layout with brief channels geometry.This microfluidic chip-based LAMP allows a qualitative and quantitative detection of EHEC O157:H7.This microfluidic chip-based LAMP was further combined with above biosensors for the capture,culture,release,lyse and recognition of the EHEC O157:H7.The proposed sensing platform was capable of detecting concentrations as low as 1 CFU m L-1 without complicated instrumentation in less than 3.5 h.Finally,a lab on chip was constructed by integrating above units in one chip,which could realize the integrative detection of EHEC O157:H7.In conclusion,this research increases the capture efficiency of EHEC O157:H7 to 95%,improves the detection limit of EHEC O157:H7 to 1 CFU/m L and realizes the qualitative,quantitative and integrative detection of EHEC O157:H7 rapidly and sensitively.