| With the development of the Human Genome Project, Gene diagnosis has become an important area in molecular biology and biomedical research. The detection of specific DNA sequence in human body, viral and bacteria nucleic acid is playing a more and more important role in disease diagnose, food pollution, legal medical examination and environment detection. Wide-scale genetic testing requires the development of easy-to-use, fast, inexpensive, miniaturized devices. Many new biological technologies emerged and found their applications in this field. Among them, DNA detection technologies relying on the DNA base-pair recognition are rapidly developed and have received considerable attentions. Electrochemical DNA detection is a novel and developing technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and has been a hot topic in the field of biochemistry and medicine.With the accomplishment of Human Genome Project and the rapid progress of proteomic strategies, protein detection has been a topic of significant interest. Intense research activities are carried out worldwide to develop rapid, simple, specific and sensitive detection devices for protein in medical diagnostics and biomedical research application, while the detection methodologies for protein based on antibodies cannot meet the demand of the human proteome. Now aptamers have been emerging as a new protein recognition element in wide range of bioassays. Aptamers have attracted a considerable attention due to their ability to bind target protein with high affinity and specificity and have many advantages over antibodies, including simpler synthesis, easier storage, reproducibility, and wider applicability. These properties make aptamers ideal candidates as protein recognition elements in a wide range of bioassays and for the development of diseases diagnostics.The emergence of nanotechnology is opening new horizons for the application of nanoparticles in analytical chemistry. Nanoparticles are of considerable interest owing to their unique physical and chemical properties, and offer excellent prospects for biosensor. The power and scope of such nanoparticles can be greatly enhanced by coupling them with the high specificity of the aptamer and the high sensitivity of the electrochemical recognitions.Nucleic acid and protein are the most important biomolecules in life. Nucleic acid is in charge of transferring genetic information and coding other biomolecules, while protein has run through the whole life process. The investigation on the interaction of the nucleic acids and protein has been one of the most important areas. The information of the detailed and fundamental nature of the interaction obtained will help to understand the organization of biological systems and contribute to control and adjust the life process.The goal of the present study is to design and optimize new electrochemical techniques with high sensitivity and selectivity. This paper combines the excellent characteristics of nanoparticles, base-complementary and the specific recognition of protein. The high sensitivity of electrochemical device coupled to their compatibility with modem micro-fabrication technologies, portability, low cost, minimal power requirements, and independence of sample turbidity, made them excellent candidates for DNA and protein detection and also it would broaden the research field of post-genetic time. The dissertation includes seven parts: Chapter One: introductionIn the beginning of this dissertation, the basic composition of a DNA biosensor and its classification were systematically reviewed, especially the design of a DNA electrochemical biosensor was introduced, together with the application and development of DNA biosensor. Secondly, we introduced the concept of the aptamer, emphatically reviewed the operational principle, classification, application and development trends of aptamer biosensor. Thirdly, nanoparticles were introduced, especially the application of gold nanoparticles and magnetic nanoparticles in the biosensor was presented. At last, the purpose and the significance of the dissertation were pointed out.Chapter Two: Study on an electrochemical detection for thrombin based on aptamers and nanoparticlesA high specific, sensitive electrochemical recognition of thrombin based on aptamers and nanoparticles was presented. Two different aptamers were chosen to construct a sandwich manner for detecting thrombin. Aptamer I was immobilized on nano magnetic particle for capturing thrombin, and aptamer II labled with nano gold was used for detection. The electrical current generated from gold after the formation of the complex of magnetic particle, thrombin and nano gold, and then an electrochemical cell designed by ourselves was used for separating, gathering, and electrochemical detecting. Through magnetic separation, high specific and sensitive detection of the target protein, thrombin, was achieved. Linear response was observed over the range 5.6 × 1012 mol/L - 1.12 × 10-9 mol/L, with a detection limit of 1.42×10-12 mol/L. The presence of other protein as BSA did not affect the detection, which indicates that high selective recognition of thrombin can be achieved in complex biological samples such as human plasma.Chapter Three: An Amplified electrochemical biosensor for thrombin based on the enrichment of gold nanoparticles through the hybridization with the complementary oligonucleotideAn amplified electrochemical biosensor for thrombin based on the enrichment of the gold nanoparticles through the hybridization with the complememtary oligonucleotide was presented. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle-aptamerI / thrombin / gold nanoparticle-aptamerII was fabricated. And the signal amplification was further implemented by the enrichment of gold nanoparticles through the hybridization with the gold labeled complementary oligonucleotide, the resulting hybridization is capable of realizing more gold nanoparticle markers attaching for one target protein, thrombin, and hence offer a remarkable amplification of detecting thrombin. A significant sensitivity enhancement was obtained for detection of thrombin with the signal amplification. This new strategy allows the detection of the target protein down to the 4.52 ×10-15 mol//L. The relative standard deviation of eight replicate determinations of 7.47×10-14 mol/L thrombin was 3.0%.Chapter Four: A new amplification strategy for ultrasensitive electrochemical detection with network-like thiocyanuric acid/gold nanoparticlesAn ultrasensitive and highly specific electrochemical detection for thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamer I was immobilized on the magnetic nanoparticles, aptamer II was labeled with gold nanoparticles. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle /thrombin / gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid /gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical detection is expected to have wide applications in protein monitoring and disease diagnosis.Chapter Five: An ultrasensitive electrochemical approach for detection of p53 sequence based on the aggregation of gold nanoparticlesA new approach has been developed for the detection of human tumor suppression p53 gene. Two DNA sequences were designed, and the combined sequences are complementary to that of the target p53 sequence, one was immobilized on the magnetic nanoparticles for capturing the target p53 sequence, the other one is labeled with gold nanoparticles as probe, and the sandwich format was formed for detecting the p53 sequences. The ultrasensitive detection was achieved by the aggregation of gold nanoparticles with introducing thiocyanuric acid into the system, and the detection limit could go down to 2.24×10-17 mol/L. The mutant type p53 DNA sequence could be obviously distinguished from the wide type p53 DNA sequence. Chapter Six: A displacement assay for protein based on gold nanoparticels and magnetic nanoparticlesA nanoparticle based displacement protocol for detection of thrombin is described. The assay relies on hybridizing the magnetic nanoparticle immobilized aptamer with gold nanoparticles labeled complementary DNA, followed by an incubation assay with the target protein. In the presence of thrombin, aptamer prefers to form G-quarter structure with the target protein, resulting in the release of the gold nanoparticle labeled DNA. A high sensitive detection of thrombin was achieved by the excellent electrochemical signal of gold, and its detection limit could go down to 1.22×10-11 mol/L. The assay responds rapidly and specifically to the target protein andrequires no label of the protein and without other reagents.Chapter Seven: A Thermodynamic investigation into the binding affinitiesbetween aptamer-DNA and aptamer-protein based on the displacement reactionA thermodynamic investigantion into the binding affinities between aptamer-DNA and aptamer-protein based on the displacement reaction was presented. Some thermodynamic parameters such as the equilibrium constant, enthalpy and entropy of the dissociation reaction and the displacement reaction were evaluated. The results showed that the change of entropy played an important role in the conversion of the duplex DNA into aptamer-protein binding. And it was also deduced that the binding reaction of aptamer toward thrombin is exothermic, its high affinity and specificity are generally achieved by a combination of complementary molecular shapes, hydrogen bonding, and stacking interactions. This has been the first attempt to compare the stability of the duplex DNA and the DNA-protein complex, and it will provide an insight into how the conformation changes. The obtained thermal parameter will direct us to find out the basic principle for designing the aptamer based biosensor and to deeply understand the thermal properties of the aptamer toward protein, and it will be great importance of developing new methods for disease diagnosis.
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