New Dna Electrochemical Biosensor And Dendritic Nanoparticles In Which The Applied Research

With further understanding about the genetic root of human diseases, people are becoming more interested in the rapid, fast, reliable nucleic acid assays. Various kinds of DNA biosensors have been researched and reported. DNA biosensor is a comprehensive technology combining several subjects such as biology, chemistry, medical, physics and electronic techniques. The electrochemical biosensor attracted more attention because it is simple, reliable, selective, sensitive and cheap. Meanwhile, it is compatible with the DNA biochip which will have bright future in commercial application.Depending on its designed structure, small scale, mono-dispersing function groups, dendrimers molecules have become the highest materials to research in the nanomaterial field. Since dendimers molecules first came up in the middle of 1980s, a great amount of dendimers with different function groups have been invented in the 20 years. There are several kinds of dendimers such as pure carbon hydrogen chain, other atom mixed, metal containing. In recent years, the focus has turned from synthesis of dendimers to its application, especially in the pharmaceutical. Newkome and co-workers demonstrate that dendimers with positive charge can absorb the bio-substance with negative charge such as DNA. They prove the potential ability of dendimers to be the carrier of bio-active substance. At present, PAMAM is considered to be the best carrier which is used frequently.The paper focuses on the application of PAMAM in DNA biosensor which can be divided into two directions. The first is PAMAM enhanced DNA biosensor with application of PAMAM in DNA immobilization on PAMAM modified electrode. The second is PAMAM application in DNA labeling. We combine the electrochemical DNA arrays technology with dendrimers nanoparticles technology by applying the dendrimers molecules to DNA biosensors. The result shows that the sensitivity, selectivity and reliability have been greatly improved due to the strong uniting ability and compatibility with DNA of dendrimers molecules. The paper can be divided into four sections as followed:I The Preparation of PAMAM Modified Grassy Carbon Electrode and Its Application in DNA Hybridization DetectionWe realized the simple and rapid genetic detection with preparation of DNA electrochemical sensor on PAMAM modified electrode. PAMAM nanoparticle that has 64 amides at periphery was firstly absorbed onto the freshly polished grassy carbon electrode, and then DNA probe was immobilized on the PAMAM modified electrode through static force between negatively charged DNA and positively charged PAMAM in acidic condition. The volume of DNA which was immobilized on the electrode was obviously improved comparing to the direct immobilization on freshly polished grassy carbon electrode. As the result, the detection limit for target DNA was improved. The preparation process of DNA target modified electrode was characterized by electrochemical measurements such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV). We applied the PAMAM enhanced DNA electrochemical sensor to DNA hybridization by using the DNR as indicator. It is demonstrated that the PAMAM enhanced DNA electrochemical sensor has the potential ability to detect DNA hybridization at low concentration with simple and rapid preparation process. The influence of ion intensity of supporting solution on the volume and stability of DNA on electrode was also investigated.II PAMAM Dendrimers-Based DNA Biosensors for Electrochemical Detection of DNA HybridizationAs a critical step of DNA electrochemical biosensor, the mobilization of DNA on the electrode surface is of great significance. In the paper, we prepared a PAMAM enhanced DNA biosensor with high selectivity, sensitivity and stability for the measurement of DNA hybridization. There are three steps for preparation. I Self-assembling of mercaptoacetic acid (RSH); II Covalent immobilization of PAMAM; III Covalent immobilization of DNA on PAMAM modified electrode. The volume of DNA immobilized on the electrode was demonstrated to be greatly improved with the covalent immobilization of PAMAM. And that decreased the space resistance for DNA hybridization which highly improved the hybridization efficiency and hybridization detection ability. The preparation process of DNA probe modified electrode was characterized by electrochemical measurements such as cyclic voltammetry (CV), differential pulse voltammetry (DPV) and EIS. After preparation, it was used in detection of DNA hybridization with indictor, DNR. Experiments carried out with these novel materials not only showed an improved DNA attachment quantity on the dendrimers-modified electrodes compared to DNA sensors with oligonucleotides directly immobilized on Au electrodes, but also exhibited a high selectivity, sensitivity and stability for the measurement of DNA hybridization.III The Preparation of PAMAM/Cu²⁺ Labeling Probe DNA and Its Electrochemical DetectionPAMAM (amido-terminated polyamidoamine, generation 4) with Interior tertiary amine groups is reported to be a good stabilizer and template for preparation of Cu clusters. Here we synthesize novel oligonucleotide DNA probes labeled of PAMAM dendrimers containing Cu²⁺ at its interior (abbreviated to G4-NH₂/Cu²⁺) at certain pH value. The influence of pH value on combination between Cu²⁺and PAMAM was investigated using UV–visible spectroscopy. FTIR was used to examine the absorption peak change after the labeling process and the result showed the characteristic absorption peak appeared after the DNA labeling process.The application of G4-NH₂/Cu²⁺ labeled DNA in target DNA detection was realized electrochemically by hybridizing DNA of complementary sequence immobilized on glassy carbon electrode. The work provides basis for the preparation of PAMAM labeling probe DNA and its application in hybridization detection. VI Preparation of PAMAM Labeling DNA Probe for DNA Hybridization Using EIS MeasurementOn the basis of work in section III, we covalently linked DNA probe with Polyamidoamine Dendrimers with a Trimesyl Core. The 4.5 generation Polyamidoamine Dendrimers with a Trimesyl Core (DT4.5) has 24 in its periphery. The labeling was realized by forming of formation of peptide bonds between DNA and DT4.5. The impedance of electrode was obviously increased by the immobilization of DT4.5 labeled DNA on the electrode through the hybridization with target DNA on electrode surface. The result showed that the impedance value was lined with the concentration of labeled DNA in the concentration ranging from 1×10^-8M to 1×10^-11 M. The detection limit is 7×10^-12 M. The process of labeling and DNA electrode modification were characterized by electrochemical measurements, FTIR, AFM. DNA labeled with DT4.5 exhibited high selectivity, sensitivity and reliability combining with electrochemical impedance technique. There is no need of indicator for the detection of DNA hybridization. The method provided great promise for impedance DNA hybridization analysis.