The synthesis, functionalization of magnetic nanoparticles and gold nanoparticles, and their applications in nucleic acid/protein purification and identification

• Main Authors: Wen-hao Deng, 鄧文豪
• Other Authors: C. P. Lee
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/66473032561444609816

碩士 === 慈濟大學 === 醫學研究所 === 95 === The magnetic nanoparticles with an average size of 12 nm were synthesized by chemical co-precipitation of ferric chloride and ferrous chloride (molar ration = 2:1) in a basic solution of ammonium hydroxide (pH 10), followed by hydrothermal treatment (80℃, 30 min). Silica was then coated on the surface of the magnetic nanoparticles by hydrolysis of tetraethylorthosilicate (TEOS). The magnetic nanoparticles or silica-coated magnetic nanoparticles were used successfully in purification of E. coli genomic DNA from culture. The purified genomic DNA was with good OD260/OD280 ratio and the yield was about 10-17.5 ug from 1 ml of E. coli culture by using 0.16-2.5 mg of magnetic nanoparticles. Hydrophilic poly(methacrylic acid) (PMAA) coated magnetic nanoparticles with an average size of 50-100 nm were prepared by Chung-Shan Institute of Science and Technology, Armaments Bureau. M.N.D. The resultant magnetic nanoparticles contain carboxyl groups (-COOH) on their surface. The dT25 oligonucleotides, Oligo(dT)25, were then immobilized on the magnetic nanoparticles via carbodiimide activation (2% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDAC). The magnetic nanoparticles coated with Oligo(dT)25 (Oligo(dT)25-MP100) were used successfully in purification of mRNA from human total RNAs. The yield of mRNA was about 7-9 ng mRNA/μg total RNAs by using 0.12 mg Oligo(dT)25-MP100. The magnetic nanoparticles with an average size of 19 nm has also been synthesized by oxidizing an aqueous suspension of ferrous hydroxide (Fe(OH)2) with H2O2 at pH 7.8 and room temperature. Then, the surface of the magnetic nanoparticles was modified by reaction with 3-(aminopropyl) triethoxysilane (APTES) in ethanol at room temperature. The aminosilane-coated magnetic nanoparticles contain active amino groups (-NH2) on their surface. Streptavidin were then immobilized covalently on the amino-modified magnetic nanoparticles using glutaraldehyde as a cross-linking reagent. The streptavidin-coated magnetic nanoparticles were used to purify biotinylated green fluorescent protein (GFP) to confirm its function in biotin capture. The 22 nm gold nanoparticles were prepared by reduction of hydrogen tetrachloroaurate with sodium citrate. Two 5’-thiol oligonucleotide probes with length of 12 nt, specific to the HBV YMDD region, were conjugated to the gold nanoparticles by thiolation. The thiol oligonucleotide probes in pH 6.5 solution were immobilized on the surface of gold nanoparticles by strong sulfur-Au adsorption. Hybridization of the gold nanoparticle probes with synthetic target oligonucleotides resulted in the formation of an extended polymeric gold nanoparticle/polynucleotide aggregate, which triggers red to purple color change in solution. The aggregates exhibited characteristic, exceptionally sharp “melting transitions” which allow us to distinguish target sequences that contain single point mutations. When the reaction mixtures were spotted onto a C18 reverse-phase thin-layer chromatography plate (C18 RP-TLC plate), color differentiation was enhanced and a permanent record of the test was obtained. Observing the color change with precise temperature control via direct visual colorimetry or one-spot colorimetric detection allowed us to detect the HBV mutations in the YMDD region. This method can detect one picomole (1011 copies) of an oligonucleotide and successfully differentiated two single point mutations in the HBV YMDD locus (G741T, Met550→Ile550; C728T, Ala546→Val546) by their different melting temperatures.