| The accomplishment of Human Genome Project proclaimed the post genome era coming, which made proteomic strategies and protein detection become a topic of research interest in life science. Proteins are the fundamental components of human body and play an important role in life activities. So. they are closed related to human health. The research on protein structure and protein function is quit meaningful for human nutriology, drug screening, disease diagnosis and clinical analysis. Intense research activities are carried out to develop simple, fast, high throughput, sensitive and specific detection techniques for protein in bioanalysis area.Protein quantities analyses is the base of protein study. The traditional detection methodologies for protein was based on the interaction between antigens and antibodies, which could not meet the demand of human proteome. Now aptamer as a new protein recognition element has been extensively used for specific detection of protein in biosensors. G-quadruplex-based DNAzymes is a new-type DNAzyme with mimics horseradish peroxidase catalytic activities. Owing to its easy construction and high catalytic activities, they as a novel catalytic label have shown great potential for the development of various biosensors. In addition, nanoparticles have received great attention because of their low-cost, easy synthesis and big specific surface area. And their favorable biocompatibility and unique optical and electrochemical properties made them become a convenient tool in biosensing assay. Therefore, it is of great value to exploit novel nano-biosensors for fast, sensitive and specific detection of protein.The goal of the present study is to design novel colorimetric nano-biosensors with high selectivity and sensitivity. This paper combines the excellent performance of gold nanoparticles and magnetic nanoparticles, G-quadruplex DNAzymes and the specific recognition of protein, which provide a new biosensing technology for protein detection and would broaden the research fields of post-genetic time. The dissertation includes three parts:Chapter One:IntroductionIn the beginning of this dissertation, the basic composition of a biosensor and its classification were systematically reviewed, especially the design of a enzyme biosensor was introduced, together with the application and the development of enzyme biosensors. Secondly, we introduced the concept of G-quadruplex-based DNAzymes, emphatically reviewed its formation, properties and application for DNA and protein detection in biosensors. Thirdly, nanoparticles were introduced, especially the application of gold nanoparticles and magnetic nanoparticles in biosensors was presented. At last, the purpose of the significance of the dissertation were pointed out.Chapter Two:High sensitive colorimetric assay for thrombin based on gold nanoparticles-DNAzyme dual catalyst and magnetic nanoparticles seperationA novel colorimetric assay for thrombin with high sensitivity and specificity was presented. In the strategy, we designed AuNPs-DNAzymes as a dual catalyst for thrombin detection. AuNPs were modified with TBA (thrombin binding aptamer) and peroxidase mimicking DNAzymes sequence at a ratio of 1:10. The complementary oligonucleotides of TBA were immobilized on magnetic nanoparticles (MNPs), then they were hybridized to form DNA duplex structure as the aptasensor. In the presence of thrombin, the TBA prefers to form the G-quarter structure with thrombin, resulting in the dissociation of the aptasensor and releasing the modified AuNPs. The released AuNPs with unique peroxidase mimicking activity are able to catalyze ABTS to generate a blue-green colored product in the presence of H2O2, thereby providing the amplified colorimetric readout signals for thrombin detection. This assay is low cost, facile, fast and high specificity and sensitivity for thrombin determination, with a detection limit of 0.1 nM. On the basis of results, this method shows great potential in other protein analysis and in disease diagnosis.Chapter Three:An sensitive colorimetric aptasensor for thrombin based on a symmetrically split peroxidase DNAzymeCompare with the normal signal peroxidase DNAzyme, the symmetrically split peroxidase DNAzyme exhibited higher sensitivity in nucleic acid and protein detection and was widely used in biosensors. In this protocol, based on a symmetrically split G-quadruplex DNAzyme, two colorimetric biosensors for thrombin assay have been elaborated with high sensitivity and specificity. The first one was a "signal-off" strategy that was a sensitive and fully DNA-structured aptasensor, which was built by integrating thrombin binding aptamer (TBA) for high specific recognitions and split G-quadruplex DNAzyme for signal amplification. Although the aptasensor was easy and cost-effective, in the presence of thrombin, a decreased colorimetric signals were obtained and false positive signals easily interfered the target detection. So, a "signal-on" strategy for thrombin assay was proposed based on the target-induced strand displacement reaction and magnetic nanoparticles separation, avoiding the interference of the false positive signals. This assay has favorable linear relationship in the thrombin concentration from 1 to 40nM and the detection limit was 1nM. This assay was facile, rapid, sensitive and highly specific. On the basis of results, this method will serve as a versatile tool for proteins and other biomolecule detection. |
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