| Summary: | 博士 === 淡江大學 === 化學學系博士班 === 95 === In this report we demonstrated to find appropriate and convenient separation conditions for fast mutation DNA detection and analysis the nanoparticles synthesis by different ways.
First, we find the micellar gel structure of triblock copolymer F127, EO99PO69EO99 (EO and PO being ethylene oxide and propylene oxide, respectively.), could be adjusted to modify the pore size and its distribution with the additions of Dextran which could be the separation mediums for high separation resolution of DNA. Besides, we investigated the effect of cation of the separation medium additions and for reannealing of denature DNA. Then we also demonstrated to the separation effect on the separation temperature, detection length and detection mode of wild type and mutation type DNA. In our result we were able to resolve the single point mutation of the DNA sequencing result in variants human papillomavirus type 16 (HPV-16) E6 proteins, when gel composition of 30% F127 + 0.1% Dextran2M containing 50µM Co2+ at 20℃, detection length was 20cm and electric field strength of 600V/cm. This variant made the Glutamin variant to Histidine was induced the degradation of p53 protein in vitro. If we used the LIF detection mode and increase the detection length to 30cm, electric field strength adjust to 700V/cm, we can obtain the optimums separation conditions.
Besides, we also find the capillary electrophoresis (CE) was used to separate gold nanoparticles (AuNPs) modified with neutral surfactants. This method provided us a simple and fast approach to the analysis of particle size and size distribution of AuNPs having different surface properties. The surface of AuNPs synthesized by different ways usually adsorbed or bonded different stabilizers or product molecules of redox reactions. Therefore, besides the particle size, the surface properties of AuNPs would also influence their electrophoretic behaviors in CE experiments. In order to homogenize the surface properties of differently synthesized AuNPs before proceeding to electrophoretic analysis, we used a series of surfactants to modify the surface properties of these AuNPs. In so doing, we expected to obtain the information regarding particle size and size distribution of AuNPs directly from the electrophoresis results. In this experiment we used 10mM Sodium tetraborate and 10mM Sodium phosphate containing 200mM Sodium dodecyl sulfate (SDS) as electrophoretic run buffers (pH 8.8), and AuNP Standards were treated with the synthesized compound hexyl-oligo(p-phenyleneethyny-lene)-poly (ethyleneoxide) as the sample and separated by CE. The optimum linear relationship (R2 > 0.995) between electrophoretic mobilities and sizes for AuNP standards could be achieved. The results were all in agreements with those obtained from TEM measurements. In this report we demonstrated that CE combined with surface modifying reagent could provide a convenient and efficient method for nanoparticle analysis.
The Au nanoparticles can be obtained in air-saturated aqueous solutions that contain triblock copolymers F127 but not any other reducing agent, these block copolymers act as both reductants and colloidal stabilizers and prove very efficient in both functions. In F127 aqueous solutions the process of AuNPs synthesis was step by step, then we can obtain the AuNPs of small particle size, narrow distribution and good reproducible. Because the temperature was influence to the micelle behavior of F127, we synthesized the AuNPs at 4℃,25℃and 95℃, respectively, and observed the particle size and distribution of AuNP was not influence by temperature. When the concentration of F127 increase from 5% to 13%, the particle size was decreased from 26nm to 12nm, as the F127 concentration up to 40% the particle size of AuNP was not changed. Use our synthesis method, we can obtain the AuNP of different particle sizes and narrow distribution to different F127 concentration.
With the same step by step synthesis method, Au nanoparticles (NPs) were synthesized by added reductant in different alkyl length of anionic surfactants (CnH2n+1SO4Na, n=8, 10, 12 and 14, SOS, SDeS, SDS and STS) solutions could also be characterization by capillary electrophoresis. When synthesis temperature at 25℃, a very broad peak of AuNP was observed, it means the particle distribution of AuNP was very widespread but when the synthesis temperature adjust to 4℃, we could find out the particle size decreasing and narrowing in 5mM or 20mM SDS solution. Then the pyrene was added in all anionic surfactant solutions, the chemical reaction between pyrene and gold complexes was attributed to the formation of AuNPs inside the micelles core at the embryonic stage. Further, pyrene has the extraordinary effect in decreasing the size and narrowing the dispersity of AuNPs, besides, the particle size was also decreasing with the increasing concentration of anionic surfactants. Compared with the experimental results in particle size and distribution it seems that particle size smaller and narrower in the more hydrophobic surfactant. In this report we demonstrated that CE combined with surface modifying reagent could provide a convenient and efficient method for nanoparticle analysis.
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