Study On Exonuclease-assisted Recycling Amplicationg Technique For Protein Detection And Its Application In Molecular Logic Gates

Exonuclease-assisted recycling amplification technique has been developed forsensitive and selective detection of platelet-derived growth factor BB (PDGF-BB),which has also been applied in the operation of molecular logic gate system.This thesis can be summarized as the following two parts.1. Detection of PDGF based on exonuclease-assisted cascaded recyclingamplificationHere an exonuclease III (Exo III)-assisted cascade autocatalytic recyclingamplification (Exo-CARA) strategy is proposed for label-free chemiluminescent (CL)detection of platelet-derived growth factor BB (PDGF-BB) by taking advantage ofboth recognition property of aptamer and cleavage function of Exo III. Functionally,this system consists of a duplex DNA (aptamer-blocker hybrid), two kinds of hairpinstructures (MB1and MB2), and Exo III. Upon recognizing and binding withPDGF-BB, aptamer folds into a close configuration, initiating the Exo III cleavagereaction，Finally, numerous “caged” G-quadruplex sequences on DNAzyme1andDNAzyme2release that intercalate hemin to catalyze the oxidation of luminol byH2O2to generate an amplified CL signal, achieving excellent specificity and highsensitivity with a detection limit of6.8×10-13M PDGF-BB. The proposed strategyhas the advantages of simple design, isothermal conditions, homogeneous reactionwithout separation and washing steps, effective-cost without the need of labeling, andhighly amplification efficiency, which might be a universal and promising protocol forthe detection of a variety of biomolecules whose aptamers undergo similarconformational changes.2. Label-free detection of DNA and its application in logic gates based on exonuclease-assisted recycling amplificationHere exonuclease-assisted recycling amplification has been proposed forultrasensitive and highly selective detection of DNA, which has also been applied tothe operation of molecular logic gate system. In this study, the sequence of DNAzymesare “caged” by complementary DNA, which thus cannot intercalate hemin. Upon theintroduction of trigger DNA, the exonuclease-assisted recycling amplification reactionis initiated. As a result, the DNAzyme sequences are released. The amplied signal isdetected by chemiluminescent reader. The proposed exonuclease-assisted recyclingamplification strategy is successfully applied to the operation of molecular logicgates.