Detection Of Bacteria Labeled By Novel Nano Fluorescent Probes Based On Microfluidic Chip System

Some incident of bacterial food or water poisoning had seriously affected the public security,and alerted that the technologies for bacterium detection was still faced with some challenges, such as longer consuming time, multifarious operations, lack of miniaturization for detecting device, immaturity of research method and so on. In order to cover the shortage, the microfluidic chip technology was coupled with fluorescence detection technology combines with the advantages of fluorescent nanoparticles labeling technology to detecte the food-borne pathogenic bacteria. These new methods could describes a rapid, convenient and sensitive detect bacterium. And it also offered important research value and potential application prospect for bacteria detection of food safety, environmental safety and clinical diagnosis.The main research work and results are as follows:?1? Acridine orange labeling for the Detection of Pathogenic Bacteria on a microfluidic chipWe have developed an acridine orange labeling for the simultaneous detection of E.Coli JM109 bacteria with the fluorescent spectrometry and a self-designed and built microfluidic chip system, respectively. The result was assessed both by fluorescence spectroscopy and LED fluorescence detector and found to enable detection of E.Coli JM109 at concentrations down to 10² cfu·mL^-1 and 103 cfu·mL^-1, respectively. Finally, method was applied in bacteria detection of frozen pork sampling samples. The results were consistent with national standard, and the detection time from national standard method of 48 h shorten to less than 4 h, which greatly shortened the time of detection of foodborne pathogenic bacteria and provided a new idea for rapid detection of food safety.?2? Detection of Staphylococcus aureus using acridine orange-doped silica nanoparticles as a fluorescent labelA sensitive, simple and rapid method was proposed for Staphylococcus aureus detection by using acridine orange?AO?-doped silica nanoparticles probe that covalently coupled with bacteria using glutaraldehyde as the crosslinker. Firstly. an amino-modified AO@SiO₂ nanoparticles were synthesized by self-modified inverse-microemulsion method by TEM and fluorescent spectra and so on. These nanoparticles were monodisperse and possess uniform diameter of ⁸0 nm. Fluorescence studies indicate that the photostability of the nanoparticles was improved compared to that of AO. Fluorescence intensity and the total number of S. aureus are linearly correlated within the range of 103 to 107 cfu·mL^-1, and the limit of detection is 500 cfu·mL^-1. The recovery rate for a spiked sample ranged from 96.5% to 102.7%, and the RSD was lower than 7%?for n = 7?. This method shows a bright application prospect for detection of the total bacterial count due to its several advantages such as rapid,technically simple and high sensitivity while compared with the conventional plate counting and organic dye-based method.?3?A microfluidic chip integrated with aptamer-functionalized graphene oxide nano-biosensors for multiplexed detection of Salmonella typhimuriumWe described here that a microfluidic chip integrated with aptamer-functionalized graphene oxide nano-biosensors was designed, in which one end was labeled with the CDs-Apt as the energy donor and GO acting as the energy acceptor. Based on the chip and laboratory made LED induced fluorescence detector, a microsystem for fast and sensitively detecting Salmonella typhimurium was constructed. Firstly, the water-soluble monolayer graphene oxide and carbon points were prepared by the modified Hummers method and hydrothermal method, respectively. Then, the electrodeposition of r GO/ITO on-chip was conducted in a conventional three-electrode system using cyclic voltammetry. Finally, based on the fluorescence resonance energy transfer?FRET? of r GO and CDs-Apt on this microfluidic chip system, Salmonella typhimurium was used as a bacterium model to develop the microfluidic platform with a detection limit of 100 cfu·mL^-1. Under the optimum conditions of 1.0 mM CDs-Apt, 1.0 ?L·min-1 flow rate and 10 min for fluorescence quenching and 30 min for fluorescence recovery. The method was selective in that fluorescence was not much enhanced in case of other bacteria. The detection platform not only has a lower limitation and good selectivity, but also can detect its targeted bacteria in a short time. Furthermore, the CDs-GO FRET-based detection can be used for targets with fluorescence nanoparticles of which emission spectra can be distinguished as donors.