Study Of High-Sensitivity DNA Biosensor Based On Nanoporous Gold

The main idea of the reseach was to explore a novel and sensitive biosensor for the determination of DNA sequence based on the combination of an alternative high surface-area electrode material—nanoporous gold (NPG), nanophase materials and DNA hybridization technology. The DNA biosensor showed high sensitivity, rapidity and simplicity so that it could be widely used in the field of biological analysis. This masteral dissertation consists of four parts besides the preface.1. We have investigated a simple method to dealloy these silver/gold alloys that is to immerse them in nitric acid, in which silver will be dissolved but gold not. NPG leaf has an intrinsically uniform and conductive porous structure. On the basis of this structure, an ideal electrocatalytic nanoarchitecture could be constructed by adsorbing an NPG leaf onto the entire surface of GCE.2. We are particularly concerned with the remarkable properties of NPG with an eye on its potential application as sensitive DNA biosensor. In present work , we studied the advantages of NPG modified GCE for the first time, which was made by a simple dealloying method for the detection of nucleic acids and DNA hybridization. The oligonucleotide immobilized on the surface was hybridized with different concentrations of the complementary sequences and the amount of hybrid was evaluated by DPV analysis using [Co(phen)33+] as an indicator of the hybridization reaction. Electrochemical impedance spectroscopy(EIS)was also applied to monitor the whole procedure in preparing the modified electrodes, which provided useful information for probing the changes of the surface modifiation. This NPG modfied DNA electrochemical biosensor demonstrated an extremely high sensitivity that could detect the presence of a target nucleotide at a concentration as low as 3.82pM, which was much lower than the bulk Au biosensor with sensitivity of about 110 pM. The resulted biosensor should help in the development of specific biosensor assays by rapidly screening detection sequences of interest as well as any testing situation in which pM concentrations of nucleic acid sequences need to be rapidly identified and quantified.3. In the present work, we described a sensitive DNA biosensor based on the use of the NPG electrode as a solid support for the immobilization of probe DNA, which is a critical step in the development of new biosensors and assays. DNA-Au bio-bar-code, which have become increasingly incorporated bioassays with its effective amplification based on AuNPs functionalized with a large number of oligonucleotide strands,was involved in this strategy. Different from the reported AuNPs with one kind of oligonucleotides, we used AuNPs labeled with two kinds of DNA bio-bar-code. One is complementary to the target, while the other is not, reducing the cross-reaction of targets with the complementary DNA loaded on the same one AuNP. Taking advantage of dual-amplification effects of NPG electrode and multifunctional encoded AuNP coupled with Ru(NH3)6+ as an indicator, our DNA biosensor had a limit of detection as low as 28 aM.4.The biosensor was carried out using glassy carbon electrode (GCE) modified with label-free 21-mer single-stranded oligonucleotides related to hepatitis B virus sequence via covalent immobilization and 3-2-pyridyl-5, 6-diphenyl-1,2,4-triazine PDTA as an electrochemical indicator, Electochemtrical detection was performed between DNA and PDTA by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range. The linear ranges from 5.87×10-7 M to 4.63×10-6 M ,with a linear correlation of r = 0.9990 and a detection limit of 3.12×10-9 M.