| With the implement of the human post-genome project, the development of protein detection methods with simple, rapid, high-sensitive, high-specific and high-throughput has attracted considerable attention in the analytical science. Proteins are involved in every physiological process and activity, and directly related to many human diseases. It is of great importance to develop methods for diagnosis and therapy of diseases with high sensitivity and accuracy.In recent years, two kinds of functional oligonucleotides, aptamers and DNAzymes have been generated by an in vitro selection technique named SELEX (Systematic Evolution of Ligands by EXponential enrichment). The molecular recognition formats of aptamer-target and antibody-antigen are similar, beyond that the aptamers have several advantages over antibodies, such as higher specificity and affinity, allowing a wide range of targets, low or no immunogenicity, extreme stability and can be fabricated into functional nanostructures; DNAzymes refer to single-stranded DNA molecules with high catalytic capabilities under the action of coenzyme factor such as metal ion and amino acid, and featured as ease of synthesis and modification, and high stability. Emerging as two kinds of novel recognition modules, aptamer and DNAzyme raised great attention in the recognition, sensing and detection fields.In combination of high specifity, high affinity of aptamer, and high catalytic activity of DNAzyme, this dissertation focused on the construction of new analytical methods for the detection of target proteins with high sensitivity and high specificity. Five chapters are included.The first chapter is the introduction. Focused on the wide applications of aptamer and DNAzyme in the field of analytical chemistry, a review composed of the definition and features of aptamer, the applications of aptamer in separation and biosensing fields, the definition and functional classification of DNAzyme, the biosensing application of DNAzyme (including allosteric DNAzyme) is summarized.86 articles are cited. The innovation concepts and researches of this dissertation are briefly introduced.In chaper 2, using magnetic beads as separation medium, a novel colorimetric sensing system for thrombin detection was developed with two different aptamers as recognition molecular for sandwich assay. This method was simple and the detection of thrombin can be carried out with naked eyes. Benefited from magnetic beads with large surface area and can be magnetic separated and aptamer specific to the target protein, the thrombin can be detected in 5% or 50% human serum. A linear range was obtained from 10 to 80 nM in 5% human serum, the limit of detection (LOD) was 10 nM. The linker length of G15D has great impact on the detection sensitivity, and the attachment of linker (6 thymidine unit) at the 5'-end of the aptamer can increase thrombin binding capacity significantly. Under the optimized linker length, both aptamers (G15D and 60-18[29]) can be used as capturing modules for quantification of thrombin, respectively.In chapter 3, based on a new fluorescence aptamer probe 5F-39mer, a novel affinity probe capillary electrophoresis-laser induced fluorescence detection (APCE/LIF) method for quantification of recombinant human erythropoietin-a (rHuEPO-a) was establilshed. The mechanism of binding interaction between aptamer and rHuEPO-a was also investigated. After optimization of APCE conditions, the results showed that the existence of Na+in the micro-environment solution was the key factor for the formation of 5F-39mer-rHuEPO-a complex. The presence of Na+in the running buffer significantly improved the resolution between free aptamer and complex. Meanwhile, the existence of Na+in the sample buffer increased the intensity of complex. Additionally, suitable electric voltage and sample buffer additives (bovine serum albumin) were necessary to stabilze the complex. Under the optimized conditions the method was successfully applied for the quantification of rHuEPO-a in physiological buffer, artificial urine and human serum with high sensitivity and high specificity.The linear range of rHuEPO-a covered from 0.2 to 100 nM with a LOD of 0.2 nM. The concentration of rHuEPO-a in the real sample (commercial rHuEPO-a injection) could also be quantified precisely. Using such an aptamer-based APCE/LIF method, we investigated the the role of N-oligosaccharide moiety contribute to the specific aptamer-rHuEPO-a recognition.In chapter 4, self-assembled immobilization of biotinylated DNAzyme-aptamer intergrater was accomplished on the streptavidin (STV)-coated magnetic beads or capillary wall. Using the separation technique such as magnetic bead or CE technique, a novel detection method for proteins based on the specific capturing and enrichment of aptamer, and the catalytic cleavage of DNAzyme was established. First, we thoroughly investigated the factors that influenced the cleavage activity of Cu2+-dependent CA3â…¡-43mer in the free solution and solid support (the surface of magnetic beads.Then the anti-thrombin aptamer was attached to the 5'-end of the substrate chain of CA3â…¡-43mer, and immobilization of CA3â…¡-43mer-aptamer was accomplished on the STV-coated magnetic beads, the capture and detection of thrombin was fulfilled by determining the self-cleavage activity of DNAzyme. Furthermore, the immobilization of DNAzyme-aptamer intergrater was successfully accomplished on the capillary wall using hexadimethrine bromide (HDB)/STV layer-by-layer techniques, and the fluorescecce intensity of the thrombin-aptamer complex that produced by DNAzyme self-cleavage was employed for detection of thrombin down to nM concentration.In the last chapter, with magnetic beads as the separation medium, a novel sensing method for quantification of thrombin protein was constructed on the basis of allosterically aptameric DNAzyme (aptazyme), which composed of an anti-thrombin aptamer and a Cu2+-dependent DNAzyme sequences. After optimizing the main factors, such as the base pair number of stem I, the complementary bases between aptamer and stem I, and the concentration ratio of DNAzyme and aptamer, the self-cleavage activity of DNAzyme had been inhibited by aptamer. In the presence of the thrombin, the formation of a thrombin-aptamer complex resulted in restoration of the catalytic activity of the aptazyme. The self-cleavage activity was coupled to the generation of a fluorescent signal, permitting the quantification of thrombin. This novel sensing system was simple, cost-effective, highly specific and label-free, a linear range was obtained from 0 to 100 nM, LOD was 5.1 nM. It will provide a new analytical platform for the protein detection. |
PDF Full Text Request