| In recent years,food safety incidents caused by foodborne pathogens have occurred occasionally and seriously threatened public health security.Food safety has become a prominent issue that restricts the development of the food industry.Rapid screening of foodborne pathogens are of great important to prevent and control the outbreak of foodborne diseases.However,the existing methods for the detection of foodborne pathogens,such as traditional culture-based identification methods,nucleic acid amplification-based PCR methods and antigen-antibody reaction-based methods have either longer detection time or complex sample pretreatment or lower sensitivity and cannot meet the demand for risk warning of food safety.Therefore,it is urgent to develop new technologies and new equipment for rapid,sensitive and simple detection of foodborne pathogen.Biosensor is a new technology for biological detection with the advantages of rapid detection,good sensitivity and high integration.Combined with immunomagnetic separation,enzyme-catalyzed reaction and microfluidic,biosensors can further improve their specificity and sensitivity.Therefore,this dissertation aims to integrate biosensors,immunomagnetic separation,enzyme-catalyzed signal amplification and microfluidics to explore rapid,sensitive,and simple methods for the detection of foodborne pathogens,which could provid technical support for rapid screening of foodborne pathogens to ensure food safety.The specific research contents mainly include:(1)An impedance biosensor based on immunomagnetic separation and urease catalysis:To solve the existing problems of our previous impedance biosensor,such as the time-consuming and complicated antibody immobilization onto the surface of the microelectrode,the low efficiency of the solid-liquid phase immunological reaction and the unable reuse of the microelectrode in this dissertation,a new impedance biosensor based on immunomagnetic separation and urease catalysis was developed.The proposed method was validated and evaluated using Listeria monocytogenes as a model.First,Listeria monocytogenes in the sample was separated and enriched by the immunomagnetic beads(MBs),to obtain the MBs-bacterial complexes(magnetic bacteria).Then,the immune colloidal gold nanoparticles(AuNP)modified by the urease was combined with the magnetic bacteria to form the MB-Listeria-AuNP-urease sandwich complexes(enzymtic bacteria).Finally,urease on the enzymtic bacteria was used to catalyze the hydrolysis of the non-conductive urea to produce the conductive ammonium and carbonate ions,resulting in a decrease on the impedance of the solution.The quantitative results of the target bacteria could be obtained by measuring the impedance change of the solution using the interdigital microelectrodes.The experimental results showed that this proposed biosensor could achieve the quantitative detection of Listeria monocytogenes within 2 h with the linear detection range of 102-105 CFU/mL.The detection limit of 3×102 CFU/mL.The urease could effectively amplify the impedance signal and increase the detection sensitivity about 10 times.Besides.the electrode did not require time-consuming and complicated biochemical modifications,and could be reused after a easy cleaning procedure,which greatly reduced the detection cost.However,the biosensor still could be affected by the residual salt ions and needed very strict working steps,which greating increases the difficulty in experimental operations.(2)An improved impedance biosensor based on immunomagnetic separation,urease catalysis and microfluidic chip:To avoid the interference of residual salt ions in this previous impedance biosensor,an impedance biosensor based on microfluidics chip was developed,and the electrochemical response of the impedance phase angle was deeply studied.First,using 3D printing and plasma bonding,a separation chip for immunomagnetic separation of target bacteria and a detection chip for impedance measurement of the catalysate were respectively designed and developed.Then,the procedures of immunomagnetic separation,colloidal gold nanoparticles binding,background washing and urease catalysis were achieved in the separation chip under magnetic stirring.Finally,the impedance amplitude and phase angle of the catalysate(ammonium ions and carbonate ions)was measured in the detection chip.The experimental results showed that the biosensor had a linear detection range of 102-105 CFU/mL for Listeria monocytogenes with the detection limit of 1.6×102 CFU/mL.The introduction of microfluidics accelerated the immunoreaction,improved the washing efficiency,and shorten the detection time to 1 h.In addition,the response of the electrochemical impedance phase angle was found that the phase angle spectrum shifted towards the high frequency direction when the concentration of the catalysate increased.As a result,a new method for the detection of bacteria using the impedance phase angle was proposed.(3)An optical biosensors based on immunomagnetic separation,urease catalysis and pH indication:To avoid the requirement of complex instrumentation in the previous biosensors,a novel optical biosensor based on pH indication was further developed and Listeria monocytogenes was used as a model for method validation and performance evaluation.The target bacteria was first separated and enriched by the MBs to form the magnetic bacteria and then combined with urease-modified AuNPs to form enzymtic bacteria,the urease was used to hydrolyze the urea to produce ammonium ions and carbonate ions,resulting in an increase on the pH value of the solution,the pH indicator was used to show the color change and quantitatively analyze the target bacteria.Bromocresol purple was compared with phenol red and bromothymol blue and selected as the optimal pH indicator.The experimented results showed that this optical biosensor could achieve the quantitative detection of Listeria monocytogenes within 2 h with a linear detection range of 102-106 CFU/mL,and the detection limit was 1×102CFU/mL.In addition,this optical biosensor did not the need the complex instrumentation with simple result indication and had shown the potential for on-site applications.(4)An improved optical biosensors based on immunomagnetic separation and microfluidic particle separation:Since most the biosensors still rely on paired antibodies to form the "sandwich" structure for the detection of target bacteria,a novel optical biosensor based on microfluidic particle separation,immunomagnetic separation and enzyme-catalyzed immunoassay was developed,and Salmonella was used as a model for method validation and performance evaluation.First,the biotinylated antibody and biotinylated horseradish peroxidase(bio-HRP)were combined with streptavidin magnetic beads(SA-MBs)to prepare an enzyme labeled immunomagnetic beads(HRP-MBs);The HRP-MBs were used to separate and enrich the Salmonella in the sample to form the enzyme-labeled magnetic bacterial(HRP-MB-bacteria).Then the microfluidic particle separation chip was used to separate the HRP-MB-bacteria from the free HRP-MBs.Finally,the HRP on the HRP-MB-bacteria was used to catalyze the TMB substrate for color change to achieve quantitative analysis of Salmonella.In this dissertation,2.2 μm and 150 nm particles were used to simulate the HRP-MB-bacteria and the free HRP-MBs to optimize the particle separation parameters.The polyvinyl pyrrolidone(PVP)solution with the concentration of 1%and the molecular weight of 360 kD was found to have the best viscoelastic focusing effect for 2.2 μm particles in a flow rate range of 0.2-1.4 mL/h.When the sheath flow rate was 1 mL/h and the particle flow rate was 0.1 mL/h,these two particles could be completely separated.The results showed that this optical biosensor could achieve the quantitative detection of Salmonella within 1.5 h with a linear detection range of 103-106 CFU/mL,and the detection limit was 2×103 CFU/mL.This biosensor provided a novel method for separating the HRP-MB-bacteria from the free HRP-MBs,making it possible to use the MBs as a detection labed and this could greating decrease the dependence of the biosensors on the paired antibodies,and have the protential to shortenthe detection time. |
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