Microfluidic Chip Analysis For Bacteria Based On In-situ Fluorescence And Impedance Detection

It is self-evident that a detection method for bacteria with high efficiency is extremely important in the field of food safety,environmental health,and bio-medical treatment.Microfluidic chip plates integrated with fluorescence and impedance techniques are potential strategies for achieving rapid,efficient and sensitive bacteria detectionandmicromationofdetectionsysytem,whichhas a broad development prospects.In this thesis,the limitation of current bacteria detection methods,such as time-consuming,complicated process,lower sensitivity and relying on large test instruments,were analyzed and researched,and in-situ fluorescence and impedance techniques were integrated with microfluidic chip plates and nanotechnology to build a microsystem for bacteria analysis.In order to avoid the shortcomings that traditional fluorescent marker reagents are easy to be quenched and are toxic,the fluorescent nanoparticles with high fluorescence yield and good biocompatibility were designed and prepared.By simulating the flow field and electric field of each function zone,the main functional structures of microfluidic chip were determined.And the enrichment,nanoparticals labeling on microchip were studied to develop the microfluidic chip based highly efficient bacteria detection method,which have significant research value and application prospect in the field of integration and miniaturization of repid bacteria detection.The main research work and results are summarized as follows:?1?The requirement and significance of bacteria detection,and the developing situation and the trend of microfluidic chip techniques used in bacteria detection and other biochemical field were reviewed.The research purpose and contents were proposed by further analysing the mean problems presented in current bacteria detection.?2?Based on the studise of the inhibiting effects of D-limonene emulsion on biofilm,the requirement for bacteria detetction method were analysied and presented.It was of great importance to develop high efficient and sensitive bacteria detetction methods,because of the research results of the inhibiting effects of D-limonene emulsion on biofilm by using organic fluorescence reagents based labeling method.And the integration of nanotechnology and multi-function microfluidic chip was an effective way to develop the new methods for bacteria detection.?3?The novel in-situ fluorescence detection methods were developed for bacteria test by using the newly designed and prepared multi-function microfluidic chips integrated with immunocapture-CdSe/ZnS QDs labeling and DEP enrichment.The selectivity of the bacteria analysis system was guaranteed by the microfluidic chips with immunocapture function.And owning to DEP enrichment and in-situ fluorescence detection on microfluidic chip,the sensitivity of bacteria detetcion was enhanced and the visual observation of bacteria were achived on the mirochip.Salmonella typhimurium captured in detection zone of the microfluidic chip were labelled by hydrophilic CdSe/ZnS QDs by which the quantitative analysis of Salmonella typhimurium were achived,and the detection limit was down to 37 cfu mL^-1.In order to make full use of the superiorities of hydrophobic CdSe/ZnS QDs in the field of bacteria detection,CdSe/ZnS@SiO₂-NH₂ nanoparticles were prepared by using modified reverse microemulsion method,and then were labeled to the surface of bacteria by glutaraldehyde in two-step strategy to establish the new quantitative testing methods.Salmonella typhimurium labeled by CdSe/ZnS@SiO₂–NH₂ still were dielectric,and the CdSe/ZnS@SiO₂–NH₂ labeled bacteria?5cells/25?L?could be captured along the electrode edges from the continuous flow by p DEP.Hence,with the help of bright fluorescence of FNPs,the visualization of bacteria counting was attained under a fluorescence microscope.?4?A new fluorescence probe CQDs-apt and DEP technique were used to develop the microfluidic chip based bacteria detection methods.CQDs with the advantages of simple preparation and high compatibility were conjugated with aptamer?apt?to develop the new fluorescence probe which was of superior fluorescence property and specificity.Under optimized conditions,a rapid and sensitive bacteria detection method were developed by using CQDs-apt as fluorescence probe,and without any sample enrichment process,the detection limit was down to 50 cfu mL^-1.On the basis of aptamer recognized Salmonella typhimurium specifically,microfluidic DEP chip was integrated with this fluorescence probe to sort and quantitatively test the viable Salmonella typhimurium in sample.The developed method could shorten the detection time to less then two hours and has the advantages of efficiency,sensitivity and easy for integration,which has great applied potential in the field of biochemical analysis and drug screening.?5?By integrating DEP enrichment and in-suit impedance technique,the new E.coli detection methods were developed on microfluidic chips.We designed and prepared a microfluidic chip with DEP enrichment and in-suit impedance detection function,based on that an impedance analysis microsysytem was developed.The problems about improving sensitivity of bacteria detection and on-line testing were solved by integrating interdigital electrodes on microchip to conduct pre-enrichment and in-suit imoedance detection,and the new methods of bacterial quantification were established with the help of DEP enrichment.Using E.coli as test sample,experimental results showed that the detection limits of E.coli was 5×10⁴ cfu mL^-1,and testing time was only 6 min under the optimized condition.The method was successfully used to detect E.coli in synthetic chicken synthetic samples,which proved the proposed method has the potential to be useful for detecting bacteria in practical samples.Based on the full play of impedance detection,“Tesla”structure mixing zone and interdigital microelectrodes were further integrated on microfluidic chip to reduce the bacteria detection limits.An detection method for bacteria which can improve the sensitivity of impedance detection were developed by taking the advantagees of the electronic signal amplification effects of AuNPs@Ag complexes,and the impedance detection limits for bacteria on microfluidic chip were down to 5×10² cfu mL^-1.