Determination Of Hrp Using A Rls Technique And The Preparation And Application Of Magnetie Nanopartieles

The detection of DNA hybridization and immunoassay play a very important role in life sciences. They have been widely used in biochemistry, molecular biology and medical diagnostics. With the development of the life sciences, the disadvantages of the routine methods, such as the radioactive assay, enzymatic assay, have already been revealed. Then, it is very necessary to develop new analysis methods with high sensitivity, simple operation and universal applicability.Horseradish peroxidase (HRP) is widely used as the labels for bioassay. A novel assay for detection of HRP using resonance light scattering (RLS) has been established. The method is based on that 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized to form macromolecule polymer by H2O2 with HRP catalysis, which results in the intense RLS signal. This assay is simple, rapid and sensitive. The optimum analysis conditions have been investigated, including acidity, the concentration of TMB and H2O2, and reaction temperature. There is a good linear relationship between the intensity of RLS and the concentration of the HRP in the range of 1×10-7～1×10-6 g/L and the detection limit is 1.8×10-8 g/L.With 1,6-hexanediamine as amine-funtionlization reagent, a facile one-step strategy is put forward to prepare amine-functionalized Fe3O4 nanoparticles with good magnetism, suitable sizes, good dispersibility and crystallinity.The oxidation of 3,3',5,5'-tetramethylbenzidine by the Fe3O4-nanopaticles/H2O2 system yields colored products. When adding H2SO4, the products finally become yellow from blue. The maximum absorbance peak is at 450 nm and there is a linear relationship between the maximum absorbance and the concentration of Fe3O4. So we design a method to detect specific sequence DNA indirectly with utilizing solid hybridization. By using specific sequence oligonucleotide modified Fe3O4-nanoparticles, the sandwich-type hybrids can be formed after the hybridization reaction. The amount of Fe3O4-nanoparticles was proportional to the concentration of target oligonucleotides. When the Fe3O4-nanoparticles are quantitatively determined by using UV method, the concentration of the target oligonucleotides can be obtained.