Study On The Detection Of Cells System Using Fluorescent Nanoparticle-Based Labeling And Dielectrophoresis Technology

Detection of cells system is very important in food safety detection, disease diagnosis and environmental monitoring. At present, common methods for the detection of cells system including the fluorescence analysis, enzyme-linked immunosorbent assay, biological sensors, and flow cytometry, have contributed much to the improvement of sensitivity and accuracy of the cells system detection. However, these mthods still exhibit deficiencies in some extent, such as preenrichment of samples, time-consuming and lacking sensitivity. Therefore, it is highly desirable to develop new approaches for synchronization of cells system enrichment and detection with improved simplicity, selectivity and sensitivity. Recently, fluorescent nanoparticles with great fluorescence intensity and photostability used in the detection of biomoleculars has attracted a wide spread attention. Compared with the traditional methods for pathogens detection, fluorescent nanoparticle labeled immunoassay contributed to the great improvement of sensitivity and rapid detection. In addition, the rapid development of DEP theory and micro fabrication technologies provide a new opportunity in ultra-sensitive detection for trace amounts of cells. Aiming at the important aspect of cells system detection, this thesis mainly focused on development of rapid, sensitive and multiplexed detection methods for several important cells by using fluorescent nanoparticle, fluorescent aptemer and DEP technology.1. Reaserch on the effect of bio-modified fluorescent silica nanoparticles on dielectric properties of E.Coli0157:H7.Polar electrodes were designed to assemble E. Coli0157:H7cells with different dielectric properties into different region of the electrodes. The assembly behavior of E.Coli0157:H7cells on the polar electrodes were investigated after incubated with bio-modified fluorescent silica nanoparticles. The DEP behaviors of E.Coli0157:H7and E.Coli0157:H7labelled by bio-modified silica nanoparticle had been studied at the frequency range from50KHz to100MHz, we found that E.Coli O157:H7exhibit positive DEP response at the frequency range of50KHz-80MHz, show negative DEP response at the frequency between90MHz and100MHz; E.Coli0157:H7labelled by bio-modified silica nanoparticle exhibit positive DEP response at the frequency range of50KHz-60MHz, and exhibit negative DEP response at the frequency range of70MHz-100MHz. It is found that the bio-modified fluorescent silica nanoparticles could change the DEP critical frequency of E.Coli0157:H7from90MHz to70MHz, indicating that the dielectric properties of the cells had been changed. The result will play important role in the development of a rapid and sensitivity detection method for E.Col O157:H7cells using DEP.2. Rapid and ultrasensitive E.Coli O157:H7detection based on dielectrophoretic enrichment and fluorescent nanoparticles label.A rapid, convenient, and sensitive method for pathogen detection in microfluidic channels using dielectrophoretic enrichment and fluorescent nanoparticles have been developed. E.Coli0157:H7was selected as the detection target and specially labeled with antibody-conjugated RuBpy-doped silica nanoparticles. After applying an appropriate AC frequency and voltage to the electrode, E.Coli0157:H7labeled with fluorescent silica nanoparticles could be specially accumulated and captured in detection region. At the mean time, nanoparticles without label were washed away. E.Coli0157:H7labeled with fluorescent silica nanoparticles to be captured has a large fluorescent signal witch could be detected. Thus, the separation and washing-out steps usually requiring fluorescent nanoparticles label involved bioassay are eliminated in the presented procedure. Utilizing signal amplification effects produced by DEP enrichment and fluorescent nanoparticles label, the fluorescence intensity of the enriched fluorescent nanoparticles labeled E.Coli O157:H7at the detection area increased with E.Coli0157:H7concentration in the range4.2×102～4.2×106cfu/mL. And the limit of detection for E.Coli0157:H7is64cfu/mL in deionized water. The specificity of this method has been investigated by selecting the staphylococcus aureus and bacillus subtilis. Since the E.Coli0157:H7enrichment was accelerated significantly by DEP, the detection of E.Coli0157:H7could be accomplished within80min, after fluorescent nanoparticles labeling. We applied the established approach to detecting in artificially contaminated bottled mineral water samples, E.Coli0157: H7of420cfu/mL were accurately detected. This proposed method may offer application opportunities for rapid, cost effective, convenient, and sensitive analysis of bacteria in foods, water, and environmental samples.3. Rapid and sensitive CEM cells detection based on dielectrophoretic enrichment and fluorescent Aptamer identification.A method for the rapid detection of CEM Cells has been developed based on dielectrophoretic enrichment and aptamer probes. FAM-sgc8c aptamer which was used to generate fluorescent signal for microscopic examination, was used to recognize CEM cells. By applying an appropriate AC frequency and voltage to the electrodes, the FAM-sgc8c-CEM cells could be specially accumulated and captured in detection region. Thus, the captured FAM-sgc8c-CEM cells has a large fluorescent signal witch could be detected by microscope. With this method, CEM Cells in5%sucrose buffer was detected in the range8.1×104～1.2×106cells/mL. As the low as1200CEM cells could be detected in10μL5%sucrose buffer. And the total assay including sample pretreatment was accomplished within4h. This proposed method will be promising for rapid detection of tumor cells in clinical samples.