Functional DNA-based Signal Amplification And Novel Fluorescent Detection Strategy

Deoxyribonucleic Acid(DNA)is the carrier and transfer of genetic information.The qualitative and quantitative analysis of sequence-specific DNA shows its great significance in gene diagnosis,food safety and environmental monitoring.With the development of molecular biology,DNA has been found to have specific recognition with some biological molecules.Studying the interaction between DNA and biologi-cal molecules is important for biomolecular detection especially for design of anti-cancer drugs.Therefore,developing new methods for DNA and other biological mol-ecules is desiderated.Well-designed functional DNA can be applied to expand the de-tection objects by interaction with biological molecules.Besides,the porphyrin mimic enzyme can be ultilized as a signal amplification tool to develop catalytic fluores-cence methods because of its good catalytic activity.This thesis focuses on functional DNA and porphyrin mimic enzyme for further assembly to construct new fluores-cence strategy.This work includes two parts as follows:1.Label-free triple-helix aptamer as sensing platform for "signal-on" fluorescent detection of thrombinThe design of a label-free aptamer for separation of recognition sequence from sig-nal reporter is significant to ensure the high-efficiency affinity between aptamer and target.This work develops a label-free triple-helix aptamer(THA)as sensing platform for "signal-on" fluorescent detection of thrombin.THA was composed of aptamer se-quence and help DNA 1(H1),which contained the complementary sequence of hexa-chloro-fluorescein(HEX)labeled help DNA 2(H2).The specific recognition event between aptamer and thrombin triggered the dismission of THA to release H1.The released H1 then reacted with the signal probe of H2/graphene oxide(GO)nanocom-posite to form H1-H2 duplex,leading to the fluorescence recovery of H2 due to the detachment of H1-H2 duplex from the surface of GO.With employment of THA as a signal transducer and GO as a "superquencher",this method shows a sensitive re-sponse to thrombin with a wide concentration range from 5 to 1200 nM.The limit of detection is 1.8 nM(S/N=3)with excellent selectivity.Considering the universality of THA,the proposed aptasensor would provide a platform for homogeneous fluorescent detection of a wide range of analytes.2.Target-triggered assembly of dendritic DNA-porphyrin as mimic enzyme for amplified fluorescent detection of DNAA target-triggered dendritic DNA has been successfully in situ assembled with aux-iliary DNA on the modified slide via hybridization chain reaction.The gigantic den-dritic double-stranded DNA(dsDNA)could not only maintain the activity of iron(III)meso-tetrakis(N-methylpyridinum-4-yl)porphyrin(FeTMPyP)molecules,but also serve as the excellent carrier to enrich abundant FeTMPyP molecules.By inserting FeTMPyP into dsDNA with groove binding interaction,the dendritic DNA-FeTMPyP was constructed and acted as the mimic enzyme of peroxidase.Compared with those harsh proteases,it owned excellent biomimetic catalytic activity with simpler prepara-tion,easier modification and especially better stability.An amplified fluorescene strategy based on the H2O2-mediated catalytic oxidation reaction of tyramine by the DNA-FeTMPyP mimic enzyme was proposed for DNA detection.Under optimized conditions,it had a detection limit of 103 pM and showed high sensitivity and good specificity.Combining HCR and catalytic reaction along with fluorescence analysis,the strategy will provide a promising application for designing and developing novel biosensors.