The Biosensor Based On Aptamer And Continuous DNA Duplication

In this thesis, several novel aptameric biosensors with high sensitivity and high selectivity 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 selectively recognized and determined on these biosensors, providing theoretical basis for the early diagnosis of several diseases. The following biosensors were developed and studied in this thesis:1. A cyclic reaction amplification system involving two DNA duplication processes based on aptameric recognition was designed using CdS nanoparticles (4 nm in diameter) as the tag, and a thrombin biosensor was established based on this system. Good selectivity was found in this biosensor. The detection limit was found to be 5×10^-14 M, and thrombin could be quantified in the range from 2×10^-14 M to 10-12 M.2. A cyclic reaction amplification system involving two DNA duplication processes based on aptameric recognition was designed using PbS nanoparticles (5 nm in diameter) as the tag, and an adenosine biosensor was established based on this system. Good selectivity was found in this biosensor. The detection limit was found to be 5×10-11 M, and thrombin could be quantified in the range from 2×10-11 M to 10-9 M. Adenosine in human plasma sample can be determined using this method, and the results was verified using high performance liquid chromatography. Accuracy and practicality was testified in this method.3. A new cycle system was established that utilize nicking endonuclease and polymerase. Multi-layer amplification and eight cycles were involved in the cyclic system. The aptameric sensor exhibited good selectivity in lysozyme detection, and the detection limit was found to be 8.1×10-15 M. Lysozyme in human plasma and human urine can be detected using this system, showing good accuracy and practicality.In this cyclic system, multiple cycles was realized in multi layers, while the whole system can only be triggered by the target– lysozyme. Despite of its complexity, this cyclic system can be performed in"one-pot"mode by simply mixing the related DNA strands, enzymes and dNTP. The displacement of DNA strand 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 in this system. Good selectivity was found in this system, which was based on the specificity of the aptameric recognition. The realization of macromolecule detection such as protein indicates that the application of this sensor can be extended to the biology research, medical research or clinical diagnosis.