Study On The Construction And Applies Of Novel Protein Aptasensors Based On DNAzyme And Nano Materials

With the successful completion of the Human Genome Project (HGP) in June2000, instead of sequencing the nucleic acid, the study of life-science research was focused on the research of functional genomics and proteomics. As one of the life basic materials, protein is the agent of the life information and material basis of life. In vivo, protein performs a variety of physiological functions, such as maintaining the growth and repairing the body and the organization, providing nutrition and energy to the body, catalyzing the metabolic reactions in vivo and defending the body from the invasion of harmful substances and so on. The study of the relationship of protein structure and function and the qualitative and quantitative detection of protein are of great significance in many fields, such as in the treatment of disease, clinical diagnosis, drug screening and human nutrition and health. With the rapid development of life sciences and biotechnology, how to develop portable biological sensors with easy operation, detection sensitivity and practical significance to detect biological protein is currently a hot topic in the field of life science research.The traditional technology to detect proteins is based on the utilization of the antibody-antigen reaction to identify proteins. However, the limitation of this method to detect proteins is that not all protein molecules have their single antibodies to combine with. What is more, the antibody is unstable and sensitive to the external environment. When assembled onto the biosensors, it tends to lose its activity. Thus, it still be a difficulty to build highly sensitive biosensors to detect proteins based on the antibody-antigen reactions. However, the development of many new biotechnologies, such as the use of nano materials and functional nucleic acids, has injected new vitality into the development of bioanalysis methods with high specificity and sensitivity. The development of new biotechnology-based protein sensors is expected to achieve fast and highly sensitive detection of proteins. Aptamer are specific DNA or RNA stands selected from random-sequence nucleic acid libraries by in vitro evolution process called systematic evolution of ligands by exponential enrichment (SELEX). Due to their simple synthesis, high specificity and wide applicability, which can make up for the lack of formal aptamers based on the antibody-antigen reactions, aptamers have been proven to be definitely useful for the detection of protein. At present, the protein detection technology based on aptamer has been developed rapidly, covering the areas of electrochemical methods, colorimetry, fluorescence detection methods and crystal quartz microbalance methods. The related science and technology are growing rapidly as well.As a novel DNA enzyme, G-quadruplex-based DNAzyme has been widely used in biosensors, therapeutic agents and DNA nanodevices due to their high chemical stability, low cost for synthesis, biocompatibility, and ease of structural prediction and modification. The application of DNAzyme can replace the traditional RNAzyme and enzyme to amplify the detected signals. In addition, the nanoparticles have been widely used in the construction of high-performance biosensors because of its unique properties and biocompatibility.The innovation of this paper is combining nanotechnology, the aptamer and the new DNA enzyme to construct and optimize novel protein aptasensors based on DNAzyme and nano materials. It develops new protein detection techniques and new detection methods for biological analysis.Chapter one:introductionIn the beginning of the paper, we introduced the kinds of proteins, the function of proteins, together with the significance of protein detection. Then, the principle of the protein biosensors was briefly introduced. Secondly, some of the functioned nucleic acids, such as the aptamer and nanotechnology were presented, especially the application and research developments in optical sensors and electrochemical sensors, as well as the application value of being the composition of a biosensor. Finally, the application of nanoparticles in biosensors and the development trend of the detection of protein biosensors were presented, such as magnetic nanoparticles. In the end the significance and innovation of this paper have been clarified.Chapter two:A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzymeIn this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H2O2-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV-Visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from0.5to20nM and the detection limit of thrombin was0.5nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins. This assay can be expected to open up new opportunities for clinical diagnostics.Chapter three:A novel electrochemical biosensor for lysozyme detection based on DNAzyme and the aptamer-introduced substitution reactionIn this paper, a novel electrochemical biosensor was reported to detect lysozyme. Aptamer was employed as the recognition element and the HRP-mimicking DNAzyme was utilized as the sensing part of biosensor. The change of electrochemical signals was based on the substitution reaction of lysozyme. Firstly, a DNA stand was assembled on a gold electrode. Then, another long-chain DNA contains recognition element and sensing element was linked to the gold electrode by hybridizing with the former DNA. Because that aptamer was more likely to combine with its target protein, when lysozyme was intoduced to the system, the long-chain DNA may be dissociated from the surface of the gold electrode. Therefore, the catalytic ability of the aptasensor and the electrochemical signals of hemin was decreased. The aptasensor showed favorable recognition for lysozyme and was not affected by other proteins during the detection process. The detection limit was2×10-9mol·L-1. This strategy is stable, fast and low cost. It also eliminates the cumbersome steps of the electrochemical labeling and holds a promise for parallel, easy and cheap detections of more molecular targets after further revisions and elaborations.