High Sensitivity Lysozyme Sensor Of Aptamer Recognition And DNA Looping Reaction Amplification Network Integration

In this thesis, several novel aptameric biosensors with high sensitivity and highselectivity were developed based on the specificity of aptameric recognition,nanoparticle technology, DNA molecular hybridization and DNA duplication cycle.Several biomolecules such as thrombin, adenosine and lysozyme can be selectivelyrecognized and determined on these biosensors, providing theoretical basis for theearly diagnosis of several diseases. The following two biosensors were developedand studied in this thesis:1. In this study, a complex of various autonomic cycling DNA-related processeswas established and used to detect lysozyme with prominently enhanced sensitivity.The complex was triggered by aptameric recognition of lysozyme (the target), andwork in3groups of cyclingDNA-related processes which provide cascading signalamplifications: a main cycle includingstrand-displacement polymerization andtarget–displacem-ent polymerization, a downstream cycleof strand-displacementpolymerization, and a series of DNA nicking-polymerization cycles. A total of8autonomic and interrelated cycles were enclosed in these3groups, while only2probes,2primers, and2enzymes were needed as raw feeds, and the complex can beoperated simply in one-pot mode. With this complex, lysozyme could be quantifiedwith remarkably enhanced sensitivity in the range from1.0×10-14M to1.0×10-12M. A detection limit of3.6×10-15M wasobtained according to3σ rule, which was7orders of magnitude lower than that (1.8×10-8M) obtained withoutamplification.In this cyclic system, multiple cycles was realized in multi layers, while thewhole system can only be triggered by the target–lysozyme. Despite of itscomplexity, this cyclic system can be performed in “one-pot” mode by simplymixing the related DNA strands, enzymes and dNTP. The displacement of DNAstrand and the displacement of macromolecular target in DNA polymerization were all realized in this system, and these displacement processes were fully verified.The detection sensitivity was greatly enhanced by the multiple reaction cycles inthis system. Good selectivity was found in this system, which was based on thespecificity of the aptameric recognition. The realization of macromoleculedetection such as protein indicates that the application of this sensor can beextended to the biology research, medical research or clinical diagnosis.2. This study is based on the first study.a complex of various autonomic cyclingDNA-related processes was also established and used to detect lysozyme withprominently enhanced sensitivity. The complex was also triggered by aptamericrecognition of lysozyme (the target), and work in3groups of cycling DNA-relatedprocesses which provide cascading signal amplifications: The amplificationconsisted of three amplification layers: the fundamental amplification was providedby rolling-circle amplification; the further amplification was provided bytarget-displacement polymerization; and extra amplification was provided bynicking-polymerization cycle. Despite of its complexity, this cyclic system can beperformed in “one-pot” mode by simply mixing the related DNA strands, enzymesand dNTP. The displacement of DNA strand and the displacement ofmacromolecular target in DNA polymerization were all realized in this system, andthese displacement processes were fully verified. The detection sensitivity wasgreatly enhanced by the multiple reaction cycles in this system. Good selectivitywas found in this system, which was based on the specificity of the aptamericrecognition. The realization of macromolecule detection such as protein indicatesthat the application of this sensor can be extended to the biology research, medicalresearch or clinical diagnosis.