Dendrimer-based Dna Electrochemical Biosensor Research

Deoxyribonucleic acid (DNA) is one of important materials to the life. Studies of its structure and function contribute to the research of genetic variation, and the detection and analysis of DNA is an important basis for clinical diagnosis. Common analytical methods include chromatography, micro-spectrophotometry, absorption spectrophotometry, fluorescence spectrophotometry, light scattering techniques, electrochemical analysis and sensor technology. DNA sensors, according to how the transducer works can be divided into different methods, such as electrochemical sensors, photochemical sensors, piezoelectric sensors, optical fiber sensors. Electrochemical DNA analysis and electrochemical sensor research are the hotspots in recent years. Compared with common markers such as the fluorescent, luminescent markers, its advantage is good selectivity, high sensitivity, testing low-cost, easy miniaturization, etc., without destroying the test system or disturbed by the turbidity and hemolytic interference from biological samples. It can be widely applied to clinical genetic diseases, environmental detection, and identification in the field of Law. DNA electrochemical sensors have gradually become direct DNA sequence detection methods in molecular biology research.Dendrimers developed in the middle of last century are a kind of synthetic polymers of three-dimensional structure. Their structural characteristics are highly branched. As compared with traditional linear polymers, dendrimers have good solubility and low viscosity. With their internal cavity and external to contain a large number of functional groups, dendrimers can disperse or complex with various metal particles. As a new type of molecules in the surfactants, nano-composite materials have shown good application prospects. With an increase of their generation, the molecular structure of dendrimers is gradually close to spherical. With the traditional surfactants in comparison, although their molecular structure is different, the functional groups of the dendrimers also contain pro-oil-based and water-based, and therefore similar to traditional surfactants'nature. At the same time, the structure of dendrimers are much clearer than the traditional polymers, because of non-crystalline, low viscosity and good water-soluble, and the end to import a large number of reactive or functional groups, so dendrimers show better application performance in Biomedicine, Modification of Materials, Industrial Catalysis and the oil industry.The dendrimer can be used as a template polymerization in nanocomposites. After surface-modified, PAMAM dendrimer can also be used to prepare Cu-PAMAM, Ag-PAMAM, Au-PAMAM and CdS-PAMAM Dendrimer metal nanocomposites, etc. Because of the special structure of dendrimers, that is, with the internal cavity, external to contain a large number of functional groups, they are widely used in the preparation of DNA electrochemical sensor.In this paper, the work is committed to developing DNA electrochemical biosensor based on dendrimers. This paper mainly includes the following three aspects: (1) The spectral analysis of PAMAM-NH₂ (G4), Ag/PAMAM-NH₂ (G4); (2) Carboxylated multi-wall carbon nanotubes and PAMAM-NH₂ (G2)-modified DNA electrochemical sensors; (3)Study of DNA electrochemical sensor based on DT4.5/Cu2+ -marked DNA probe. In this paper, the novelty lies in that we applied the composite nanoparticles Ag/PAMAM-NH₂, carboxylated multi-wall carbon nanotubes, PAMAM dendrimer and DT4.5/Cu2+ dendritic complexes to DNA electrochemical sensor, and got comprehensive characterization by ultraviolet - visible spectroscopy, fluorescence spectroscopy, infrared spectroscopy, atomic force microscopy, differential thermal - thermal gravimetric analysis, X-ray photoelectron spectroscopy and some electrochemical analysis methods such as cyclic voltammetry, AC impedance, differential pulse voltammetry, etc. Data show that this new type of DNA electrochemical sensor has high sensitivity and low detection line, good stability, good reproducibility and reproducibility. The main contents of this paper are divided into the following sections:ⅠThe spectral analysis of PAMAM-NH₂ (G4), Ag/PAMAM-NH₂ (G4)This article includes two parts: (1) Through the atomic force microscope (AFM) imaging, we compare PAMAM-NH₂ (G4) with PAMAM-NH₂ (G2) discovering morphology differences. A strong fluorescence emission of NH₂-terminated fourth-generation poly(amidoamine) dendrimers has been studied through changing pH, aging time, concentration of the solution of PAMAM-NH₂(G4)or adding biological moleculars dsDNA to this solution; (2) We take PAMAM as templates to create Ag/PAMAM-NH₂(?G4) dendrimer nanocomposites (DNC). We discuss titration curve of the solution of Ag+/PAMAM-NH₂ (G4). The results showed that different mole ratio of Ag+ to PAMAM and pH strongly influence complexation between Ag+ and PAMAM. The silver-DNCs were characterized by means of UV-visible absorption and fluorescence spectroscopy. Ag/PAMAM-NH₂ (G4) dendrimer nano moleculars in DNA sensors have an extremely wide application prospect.ⅡCarboxylated multi-wall carbon nanotubes and PAMAM-NH₂ (G2)-modified DNA electrochemical sensorsIn this paper, we introduce a new type of high-sensitivity DNA electrochemical sensor, that is, we applied carboxylated multi-wall carbon nanotubes and PAMAM dendrimer to DNA electrochemical sensor. Layer-by-layer films assembled by alternate adsorption of carboxylated multi-wall carbon nanotubes (MWNT-COO-) and NH₂-terminated second-generation poly(amidoamine) dendrimers(PAMAM-NH₂(G2)) onto a glassy carbon electrode were reported. After covalently adsorbed with PO4 probe sequence, PAMAM/MWNT-COO-/GC electrode was immersed in the solutions of complementary sequences of different concentrations, and then we can get Rct-concentration curve by EIS. Layer-by-layer films were characterized by CV, EIS, IR, XPS and TAG. At the same time, the concentration of MWNT-COO- and the timing of the hybridization were discussed. This sensor has demonstrated high sensitivity and wide linear range. Linear range is between 5.0×10^-10 M to 5.0×10^-14 M, and the minimum detection limit is 1.034×10^-14 M. Experiments show that this sensor has good stability, good reproducibility, and at the same time, it has good regeneration capacity. In addition, we use daunomycin as indicator to test the recognition of DNA electrochemical probe on the target DNA. As a result, response signal is significant and satisfactory.ⅢStudy of DNA electrochemical sensor based on DT4.5/Cu²⁺-marked DNA probeWith the complexation between DT4.5 and Cu²⁺, it is the first time that we embed large amounts of Cu²⁺ into the inside of DT4.5, and then the complex is marked to ssDNA. Thus, we get DT4.5/Cu²⁺ labeled electrochemical DNA probe. In this paper, the complexation between DT4.5 and Cu²⁺ is charactered by UV absorption spectroscopy. At the same time, we study the impact of the pH value and mechanism of the complexation between DT4.5 and Cu²⁺, and prove the successful preparation of DT4.5/Cu²⁺ labeled DNA electrochemical probe. We also discuss and optimize a variety of experimental conditions, including the preparation of modified electrode and choosing the various parameters on the process of anodic stripping voltammetry (ASV) detection of Cu²⁺. As a result, we choose to use carbon nanotubes modified electrode, -0.4V as the optimal enrichment potential, and 300s as preconcentration time. As a result of using the highly-sensitive anodic stripping voltammetry and carbon nanotube modified electrode, the sensitivity of sensors on the specific sequence of DNA fragment detection has been greatly improved. The detection limit reachs 1.0×10^-12M. This sensor shows good stability and reproducibility.