DNA Electrochemical Biosensors Based On DNA Concatamers And Bimetallic Nanoclusters For The Detection Of MicroRNA And VEGF

Electrochemical DNA biosensor is a novel biosensor which developed rapidly in recent years. As it has advantages of fast detection, simplity, sensitivity, low price,electrochemical DNA biosensor has far-reaching significance and applicable value in the study of clinical medicine and genetic project. There is growing concern about the detection of the tumor markers. By detecting the level of tumor markers, they have an important significance in the monitoring, prognosis and treatment of cancer. Therefore,there is an urgent need to develop an new analytical technology to achieve the several common tumor markers of high sensitive detection and improve the accuracy of the cancer diagnosis. Inspired by the research of medical nanotechnology, we developed some DNA electrochemical biosensors combining with magnetic beads,DNA-templated Ag/Pt bimetallic nanoclusters to the high sensitivity and specificity detection of miRNA and vascular endothelial growth factor(VEGF).In chapter 1, an electrochemical sensor based on long-range self-assembled DNA nanostructures by using the p19 protein facilitated magnetic beads(PFMBs) specific enrichment was introduced for the detection of circulating microRNA. To construct the long-range self-assembled DNA nanostructures, the sequence of the bifunctional Janus probe contains two regions. One of which is the complementary RNA oligonucleotide sequence of miRNA-21 and the other is DNA oligonucleotide sequence, which can be used as primer for long-range self-assembled DNA nanostructures. DSA made by ourselvers have the specific identification of dsDNA,which could be readily monitored via voltammetry. The results indicated that, under the optimum conditions, the peak current was linear with the concentration of microRNA in the range of 20 ~ 100 amol/L with a detection limit of 6 amol/L.In chapter 2, we developed a facile one-step approach to synthesizeDNA-templated Ag/Pt bimetallic nanoclusters. The obtained DNA-templated Ag/Pt bimetallic nanoclusters(DNA-Ag/Pt NCs) have about 2 ~ 4 nm in diameter and display highly-efficient peroxidase-like catalytic activity which can catalyze hydrogen peroxide(H2O2)-mediated oxidation of the substrate, 3,3',5,5'-tetramethylbenzidine(TMB), to produce a blue color reaction.Then we measured the mimic enzyme's structure, elemental analysis, kinetic parameters by the UV-Vis spectrometry,transmission electron microscopy(TEM) method. It points out that this kind of Ag/Pt bimetallic nanoclusters have stronger peroxide enzyme activity by comparison to the other artificial enzymes. Moreover, this DNA-Ag/Pt bimetallic nanoclusters have some advantages, such as simple, low cost, good stability and so on. They are also expected to be an indicator used in colorimetric analysis(such as small molecules,proteins, DNA) and in the field of biosensors.In the third chapter, as the previous chapter, we combined DNA-Ag/Pt NCs with the aptamer sequence of vascular endothelial growth factor(VEGF) to design a DNA electrochemical sensor for VEGF detection with high sensitivity and selectivity. A13-mer VEGF aptamer with an amino group at its 3' terminus was covalently attached to a GCE through amide linkage. An ssDNA containing template DNA and a 12-mer VEGF aptamer was used as a template agent for the synthesis of aptamer-functionalized Ag/Pt NCs. In the presence of VEGF, the specific binding of the two aptamers to the target produced a VEGF-Ag/Pt NCs complex, which was fixed on the GCE plates. Then the restricted DNA-Ag/Pt NCs, as signal detection probes, catalyzed the oxidation of TMB, resulting in a significant increase of current intensity and a blue color change in the solution. The current intensity increased with the increasing of VEGF concentration, which provided the quantitative determination of VEGF.Under the optimum condition, the sensor showed a good linear relationship in the range of 6.0 pmol/L ~ 20 pmol/L with a detection limit as low as 4.6 pmol/L and exhibited ultra-high discrimination ability for VEGF. Then we used this sensor for the detection of VEGF in serum of patients with breast cancer. The results are in good agreement with the ELISA method. All above properties make it a promising candidate for VEGF detection in clinical real samples of cancer patients.