Detection Of Salmonella Typhimurium Based On Fluorescent Nanoparticles Labeling And Microfluidic Chip-Dielectrophoresis Technology

Pathogen detection, as an essential index, has played an important role in foodsafety, disease diagnosis and environmental monitoring. Recently, various assaymethods for pathogen have been developed, such as media culturing, molecularbiology, enzyme-linked immunosorbent assay, flow cytometry, and so on, which makea great contribution to the development of pathogen detection method. However, mostof these methods are still time-consuming, laborious or discontinuities. Therefore, itis extremely necessary to develop a sensitive, continuous, and real-time pathogendetection method. Because of their unique advantages, nanomaterials have beenwidely used in the field of biomedical analysis. In particular, as a result of the highfluorescence intensity and optical stability, the fluorescent nanoparticles-based signalamplification has been frequently reported and utilized for improving detectionsensitivity. Meanwhile, the microfluidic chip-dielectrophoresis technology,integrating the dielectrophoresis principle and microfluidic chip technology, not onlycan enrich and separate cells, bacteria and other complex biochemical sampleseffectively and rapidly, but also can be easily combined with functionalizednanoparticles. This presents a novel idea for the real-time, continuous andultrasensitive detection of samples with small amounts. In this thesis, by selectingSalmonella Typhimurium as the target, we carried out the research work focused onthe continuous, rapid and sensitive pathogen detection based on fluorescentnanoparticles labeling and microfluidic chip-dielectrophoresis technology. The mainworks are summarized as follows:1. Rapid and ultrasensitive detection of Salmonella Typhimurium based onfluorescent nanoparticles amplification and microfluidic chip-dielectrophoresistechnology.A rapid, ultrasensitive and convenient method for S. Typhimurium detection wasdeveloped based on dielectrophoresis-integrated microfluidic device on-lineenrichment and fluorescent nanoparticles (FNPs) amplification. In the protocol, S.Typhimurium was firstly incubated with the anti-S. Typhimurium antibody conjugatedFSiNPs on the basis of investigating dielectric properties of S. Typhimurium. And theS. Typhimurium cells were labeled with FNPs via antibody-antigen interaction. Thenthe reacted mixture of S. Typhimurium and Ab-FNPs bioconjugates was directly introduced into the pDEP-based microfluidic device. The FNPs labeled S.Typhimurium moved to the electrodes and accumulated in the electrode gap byapplying an AC voltage in a positive dielectrophoresis (pDEP) frequency region,whilst the free Ab-FNPs were flowed away. This new method constructed asimultaneous on-line enrichment and detection platform, which eliminated theseparation and washing-out steps usually required for fluorescent nanoparticles labelinvolved bioassay. Utilizing signal amplification effects produced by FNPs labeling,the detection limit of S. Typhimurium was determined to be56cfu/mL. And detectiontime was reduced to40min since enrichment was accelerated significantly by pDEP.The specificity and application in real sample of this method also have beeninvestigated. The method was expected to become a common method for pathogendetection.2. Detection of Salmonella Typhimurium based on aptamer recognition andmicrofluidic chip-dielectrophoresis technology.Utilizing the advantages of aptamers such as high affinity, high specificity, easysynthesis and labeling, easy storage and low cost, we presented a fast, convenient,continuous and sensitive detection method for S. Typhimurium based on a similarprinciple described above with the substitution of antibody by sal aptamer against S.Typhimurium. Sal aptamer was labbeled with FNPs, and then the S. Typhimurium wasintroduced into the pDEP-based microfluidic chip after incubation with FNPs labelledsal aptamer. The FNPs labelled S. Typhimurium accumulated in the electrode gap byapplying an AC voltage in a positive dielectrophoresis (pDEP) frequency region, andthe signal was collected by inverted fluorescence microscope CCD imaging system.The detection limit of S. Typhimurium was determined to be280cfu/mL. Comparingwith the antigen-antibody reaction, this method can reduce the incubation time by halfrelying on aptamer recognition technology, and was expected to be a more rapid andconvenient detection method for pathogens.