| In the investigation of biosensors, the sizes of nanomaterials are in the same dimensional range as the ones of many primary biomolecules, such as proteins and nucleic acids. Therefor, we can detect the physiological function of biomolecules by nanomaterials and even open out the process of lives. Recently, because gold nanoparticles can increase surface area, improve mass transfer, catalytic properties, good biocompatibility and so on, it has drawn more and more attention in the field of electroanalytical chemistry. In addition, nano-materials synthesised in liquid-liquid interface are ordered and monodisperse. In this paper, liquid-liquid interface methods were used to prepare gold materials, which were applied to fix biological molecules. Immunosensor is constructed based on the ECL of water-soluble CdSe quantum dots modified by thioglycolic acid. The main results are as follows:1. Monodisperse gold nanoparticles were synthesised by liquid-liquid interface method. The first step involved the phase transfer of AuCl4- into the organic phase (toluene,5 ml) by triphenyl phosphine (PPh3). In the second step, cetyltrimethyl ammonium bromide (CTAB) and NaOH were dissolved in deionized water then formed the aqueous solution. Then, the organic phase was added to the aqueous solution. The last step was that hydrazine hydrate was added to the water phase of this device. A slight purple coloration of the interface appeared and indicated the onset of reduction of the gold salt. The reaction was allowed to continue for 24 hours at 30℃. Then, Au nanoparticles were formed in the liquid-liquid interface. As the reaction proceeded, the color of the interface intensified, finally resulting in a robust film at the interface. HRP was added to the aqueous phase and chemisorbed into the prepared monodisperse gold nanoparticles by Au-S bond assembly. Therefor, Au-HRP monodisperse was also formed in the liquid-liquid interface. ITO electrode was used to extract the thin films up, then, based on the ordered Au-HRP monodisperse, a novel H2O2 sensor were obained. The prepared thin film that possessed highly ordered and oriented structure, large surface area, good biocompatibility and good conductivity, providing a good array for fixing HRP and prepared biosensors. Meanwhile, experimental conditions were investigated. Optimization of the experimental parameters was performed with regard to pH, operating potential and amounts of HAuCl4. Under optimal detection conditions, the constructed sensor had a linear response range of 4μM to 1.3 mM and the detection limit was 0.9 p.M at a signal-to-noise ratio of 3. The biosensor showed an acceptable reproducibility, a good stability and high sensitivity.2. A novel H2O2 sensor based on the direct electron transfer of HRP that fixed on ITO electrode modified by monodisperse gold film formed in the liquid-liquid interface was prepared. ITO electrode was used to extract the thin Au films up. The ordered monodisperse Au nanomaterials were soked in fresh HRP solution, then HRP was chemisorbed into the prepared monodisperse Au nanoparticles by Au-S bond assembly, obtained the direct electron transfer of HRP. Then, H2O2 sensor was formed. Monodisperse gold nanoparticles film was used to maintain the activity of HRP and promoted the direct electron transfer between the electrode and HRP. The direct electron transfer of HRP showed a pair of redox peaks with a formal potential of-298mV (vs. SCE) in 0.1 M pH 7.0 phosphate buffer solution. Under optimal detection conditions, the constructed sensor had a linear response range of 1.9μM to1.0 mM and the detection limit is 0.4μM at a signal-to-noise ratio of 3, based on the irreversible reduction of H2O2. The biosensor showed an a good stability, acceptable reproducibility, lower limit of detection and high sensitivity. Therefor, Monodisperse gold nanoparticles film provided a good matrix for protein immobilization and had a promising application in constructing sensors.3. A sensitive and specific immunoassay method for detecting a-fetoprotein (AFP) based on electrogenerated chemiluminescence (ECL) was described. ECL could perform detection for the samples with a series of different concentrations of a target analyte. CdSe quantum dots (QDs) were used as labels and were linked to AFP antibody (anti-AFP, the secondary antibody, Ab2*). Immunoassay was carried out on a modified electrode using a sandwich assay approach, where anti-AFP (Ab1) covalently bound to the surface of Au electrode to be allowed to capture AFP specially. Afterwards, Ab2* was allowed to bind selectively to the captured AFP. In the presence of H2O2, the ECL intensity increased with the increase of AFP, which indicated an immunosensor for AFP was constructed. The detection of AFP based on measuring ECL intensity of CdSe without the enzyme and mediator can promote the stability of the immunosensor. The linear range of the AFP assay was from 0.002 to 32 ng mL-1. Furthermore, the immunosensor showed high sensitivity, good precision, stability, and reproducibility and could be used for the detection of real sample with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assays (ELISA) method. The strategy was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect AFP in practical samples. |
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