| Aptamers, which bind ligands with high specificity and exhibit excellent chemical properties, are considered as ideal recognition elements in biosensors. Peroxidase-like DNAzymes, which can be easily replicated, synthesized and preserved, are generally becoming ideal sensing elements in biosensor constructions. Therefore, aptazyme sensors, integrated of aptamers with DNAzymes, exhibit promising prospects in bioanalytical applications. This thesis can be divided into three chapters.In chapter 1, the principle and catagory of aptamer-based sensors (aptasensors), the signal amplification techniques as well as the theoretical guidance of nucleic acid software packages in aptasensor design was briefly introduced. Especially, the design strategies of recently developed aptazyme sensors and their defects were stressed. Based on some of these problems, the objectives and contents of this thesis were put forward.In chapter 2, study on sensing mechanism and performance of a microfluidic chemiluminescence aptazyme sensor was investigated. By taking a couple of adenosine aptazyme sensors with same duplex stems and similar thermal stabilities, the sensing mechanism from the thermodynamic equilibrium consideration has been studied. Based on the results of UV-vis and PAGE techniques, the DNA sequence arrangement and the cationic condition in buffer solution are found to play important roles in modulating the equilibrium between Watson-Crick duplex structure and Hoogsteen G-quadruplex structure, which ultimately influences the sensor’s response towards sample and background signals. By taking the advantage of microfluidic chemiluminescence assay, the developed aptazyme sensor achieved an absolute detection limit of 12 pmol adenosine with just 2μL of pretreated sample solution consumption, as well as showed satisfactory selectivity towards adenosine.In chapter 3, by taking rs242557, an Alzheimer’s disease related gene fragment, as a target model, the impact of toehold length on strand displacement reaction was predicted by NUPACK software simulation in order to rationalize the design of duplex probe for high genotyping capability. Combined with DNAzyme-catalyzed microfluidic chemiluminescence detection, a novel SNP genotyping method has been developed in this work. The G-rich sequence is initially blocked in the duplex strand DNA structure for preventing DNAzyme formation. With the addition of target gene fragment to trigger the dehybridization of blocked domain of the duplex for toehold-assisted strand displacement reaction, the DNAzyme, which formed in turn, can catalyze the oxidation of luminol to generate chemiluminescence. The developed method is not only label-free, easy to design and exhibited high throughput ability, but also displays a remarkable specificity to target rs242557 against single base mutation, achieving a discrimination factor of 56 as well as a detection limit of 1.7 nmol/L with just 2 μL of sample solution consumption.In chapter 4, by using the strategy of amplification assays based on toehold-mediated DNA strand displacement while coupling with the advantages of DNAzyme-catalyzed microfluidic chemiluminometry, a simple, sensitive, enzyme-free and non-label SNP assay of rs242557 that links to Alzheimer’s disease has been developed. Two hairpins (H1 and H2) with complementary domains can coexist in the solution due to the kinetic hindrance in the absence of target, while the sensor performed consecutive opening of two hairpins and assembles of G-quadruplex DNAzyme with the aid of target DNA. In this way, the proposed sensor can detect a 0.15 nmol/L DNA by a consumption just 2 μL of sample solution with satisfactory SNP discrimination, showing great potential in genetic diagnosis.In chapter 5, the work presented in this thesis was summarized. An attempt was also made to propose the future development of aptazyme sensors related techniques. |
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