Study Of Novel Fluorescent Biosensors Based On DNA Hybridization Chain Reaction

As a new isothermal amplification technology of nucleic acid in vitro,hybridization chain reaction(HCR)has caused wide attention of researchers in recent years.The princple of this technology is that two stable hairpin probes can be cross-opened successively and self-assembled into long nicked DNA nanowires only in the presence of target DNA.The fluorescence resonance energy transfer(FRET)is distance-dependent energy transfer based on the near field dipole-dipole interaction between two fluorophores.Using ratio metric measurement of two fluorescent intensities at different wavelengths,it allows more precise measurements due to reduce the influence of those external factors in practical application such as fluctuation of excitation source and probe concentration.Herein,two novel ratiornetric fluorescent biossensors were developed by combining HCR and FRET.Three chapters were involved in this thesis.In the first chapter,the fundamental and application of nucleic acid amplification techniques in vitro were briefly reviewed.Especially,the application of HCR was emphasized.Simultaneously,the principle and application of FRET technique were briefly introduced.The purpose and main content of this thesis were proposed at the end of this chapter.In the second chapter,a sensitive enzyme-free DNA fluorescent biosensor based on HCR and FRET was developed.Hairpin H1 and H2 which are labelled with FAM as the donor and TAMR A as the acceptor respectively,were designed according to the base sequence of target DNA.In the presence of target DNA,long nicked dsDNA nanowires are self-assembled through HCR process.FAM and TAMRAare brought in close proximity,resulting in a FRET process between them.The linear range for target DNA was covered from 2.0 nM to 40.0 nM with a detection limit of 0.7 nM.This proposed method was successfully applied to detect DNA in serum simulative samples with satisfactory results.In the third chapter,a novel Cu2+ sensor based on Cu2+-dependent DNAzyme was developed by combining HCR with FRET technique.In the presence of Cu2+ion,the substrate strands of Cu2+-dependent DNAzymc immobilized on magnetic beads were specifically cleaved and released.The released strands initiated the HCR process of hairpin H1 and H2 labelled with FAM as the donor and TAMRA as the acceptor,respectively.Long nicked dsDNA structures were self-assembled to bring the donor and the acceptor in close proximity,resulting in a FRET process.The relative ratio of fluorescent intensities of the acceptor and donor was proportional to the concentration of Cu2+.The linear range for Cu2+ was covered from 1.0 nM to 100.0 nM with a limit of detection 0.5 nmol L-1.This proposed biosensor was applied to detect Cu2+ ion in tap water with satisfactory recovery.