Research Of Microfluidic Biosensors For Rapid Detection Of Foodborne Pathogens

Foodborne pathogens have become a great concern for human public health.Every year,millions of people in the world are infected by food and water contaminated with pathogenic bacteria,resulting in various diseases that endanger human health.The traditional culture-based detection of foodborne pathogens usually takes 2-3 days or more.The detection methods based on molecular biology and immunology reduce the detection time compared with the traditional method,but still have problems such as complex operation,dependence on professional laboratories and technicians,high detection cost and low detection flux.Therefore,rapid,high-sensitive,low-cost,and easy-to-use testing methods for food security are needed.Microfluidic technology with flexible operation,high throughput detection,low reagent consumption,and easy integration with miniaturized instruments has been applied in many fields.This dissertation aims to develop microfluidic biosensors with fast detection systems for rapid,high-throughput,and highly sensitive detection of foodborne pathogens based on the principles of immunology and nucleic acid amplification.The developments of microfluidic biosensors are based on microfluidic technology,and integrated light absorption,magnetic separation,nano-material fluorescence quenching and catalytic amplification technologies,and combined with smartphones.This dissertation consists of five chapters.Chapter 1:A brief introduction to the traditional and commonly used detection methods for foodborne pathogens was given.The application of microfluidic biosensors as well as the smartphone assisted microfluidic sensing in the detection of foodborne pathogens were systematically summarized.Then the research purpose and the main research contents of this dissertation were proposed.Chapter 2:A microfluidic biosensor based on Pt-PCN-224 with high catalytic activity was developed.Using the peroxidase activity of nanocomposites and smartphones,a highly sensitive detection of Escherichia coli(E.coli)O157:H7 was carried out.The microfluidic chip integrated the entire detection process including sample injection,immune incubation,magnetic separation and enrichment,and finally collected color signals through a smartphone for quantitative analysis of the target bacteria.Under the high catalytic activity,a linear range from 2.93×10~2 to 2.93×10~8CFU/m L and the limit of detection(LOD)of 2 CFU/m L were obtained.The proposed microfluidic immunosensor could detect water,milk and cabbage samples within 1 h and obtained recoveries between 90 to 110%in the spiked testing,showing high accuracy compared with the standard culture method.Chapter 3:A microfluidic biosensor based on immunoassay and the design of the microfluidic chip including axial symmetry structure and vacuum chamber was developed for multiplex detection of E.coli O157:H7 and Salmonella typhimurium(S.typhimurium),simultaneously.The multiplex immunoassay was conducted with agitation driven by programmed audio signals during the immune-incubation.The programmed music signals were generated by a smartphone and drove the audio speaker,causing the speaker diaphragm to vibrate to provide a stirring power in the incubation process on microfluidic chip.The final fluorescence image signal collection and processing are carried out using a smartphone.Under selected conditions,the linear ranges were 1.9×10~3-1.9×10~7 CFU/m L(E.coli O157:H7)and 6.0×10~2-6.0×10~7CFU/m L(S.typhimurium),and LODs were both below 1 CFU/m L.The microfluidic biosensor successfully detected water samples within 30 min and recoveries ranged90%-110%were achieved in the spiked samples,which were consistent with the detection results obtained by the standard method.Chapter 4:A microfluidic biosensor was developed for one-step detection of multiple foodborne bacteria ss DNA simultaneously.This technology was based on the fluorescence resonance energy transfer(FRET)between the graphene oxide(GO)and fluorescence molecules modified capture ss DNA of the target bacteria ss DNA(ct DNA)for detecting target ss DNAs of E.coli O157:H7,Listeria monocytogenes,Salmonella and Staphylococcus aureus.The fluorescence recoveries were recorded by a smartphone on the corresponding fluorescent detector.Under selected experimental conditions,the microfluidic biosensor realized the detection of four kinds of bacteria ss DNA simultaneously within 5 min with LODs of 0.17,0.18,0.27,and 0.17 n M,respectively.Trace amounts of foodborne bacteria ss DNA were also successfully detected in milk and water samples in the throughput analysis with the recoveries from98.2%to104.6%.Chapter 5:Multiplexed detection of foodborne pathogens using one-pot CRISPR/Cas12a combined with recombinase aided amplification(RAA)on a finger-actuated microfluidic biosensor.Wells on the finger-actuated microfluidic chip for RAA reaction and CRISPR/Cas12a detection were isolated to avoid interference,while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells,realizing the one-pot RAA-CRISPR/Cas12a assay.The final fluorescent signal was acquired and processed by a smartphone.Under selected experimental conditions,seven pathogenic bacteria including Bacillus cereus(1.07×10~2-1.07×10~6CFU/m L),Listeria monocytogenes(8.8×10~1-8.8×10~7CFU/m L),Staphylococcus aureus(4.6×10~2-4.6×10~6CFU/m L),Pseudomonas aeruginosa(3.93×10~1-3.93×10~7CFU/m L),S.typhimurium(6.8×10~1-8×10~7CFU/m L)Cronobacter sakazakii(4.7×10~2-4.7×10~7CFU/m L),and Vibrio parahaemolyticus(1.68×10~2-1.68×10~7CFU/m L)could be tested in about 1 h with LODs all below 500 CFU/m L.Recoveries ranged from 90%to 116%of the spiked samples were readily achieved.