Novel DNA Detection Methods Based On Porphyrins And Two-Dimensional Nanomaterials

As the main component of chromosome in cell nucleus,DNA plays an important role in genetic information storage and life function regulation.With the development of molecular biology,especially the accomplishment of the Human Genome Project and the study of gene function,the quantitative detection of sequence-specific DNA fragment shows its great significance in the areas of disease diagnosis,food safety,environmental monitoring and judicial identification.Developing highly sensitive and selective DNA detection methodologies are crucial due to the low abundance of the sequence-specific DNA fragment in real sample as well as the complexity of the real sample composition.This thesis focuses on exploring novel DNA detection methodologies,which utilizes the peroxidase activity of porphyrins to amplify the detection signal,as well as develops signal transduction/output modes based on two-dimensional nanomaterials.The thesis includes the following sections:1.Graphene-supported Ferric Porphyrin as a Peroxidase Mimic for Electrochemical DNA BiosensingAs a typical ferric porphyrin molecule,Iron(III)meso-tetrakis(N-methylpyridinum-4-yl)porphyrin(FeTMPyP)possesses outstanding peroxidase activity.Here a novel peroxidase mimic was designed by assembling FeTMPyP on graphene via π-π stacking and then labelling graphene with streptavidin by an amidation reaction.The prepared peroxidase mimic was characterized by atomic force microscopy,UV-vis spectroscopy and Fourier transformation infrared spectroscopy,which confirmed the successful loading of FeTMPyP and streptavidin on graphene.The peroxidase mimic showed a good peroxidase activity due to the high loading of small FeTMPyP molecules on graphene.The DNA biosensor was prepared by immobilizing the biotinylated molecular beacon(MB)on a gold nanoparticles-single-walled carbon nanohom composite modified glassy carbon electrode.In the presence of target DNA,the MB probe would hybridize with target DNA and make its biotin end easily accessible to the streptavidin on the peroxidase mimic.Thus an electrochemical biosensor was developed for the highly sensitive and specific detection of target DNA with a detection limit down to attomolar level.2.Fluorescence Quenching of Carbon Nitride Nanosheet for Versatile Fluorescence SensingThis work investigated the interaction of carbon nitride nanosheet(CNNS),a recently developed two-dimensional nanomaterial,with DNA and its fluorescence quenching mechanism on fluorophore labelled single-stranded DNA probes.The static quenching through the photoinduced electron transfer(PET)from the excited fluorophore to the conductive band of CNNS was identified.Utilizing the affinity change of CNNS to DNA probes upon their recognition to targets and the PET-based fluorescence quenching effect,a universal sensing strategy was proposed for design of several homogeneous fluorescence detection methods with short assay time and high sensitivity.This strategy is versatile and can be combined with different amplification tools for quick fluorescence sensing of DNA and extensive DNA related analytes such as metal cations,small molecules,and proteins.As examples,two simple fluorescence detection methods for DNA or Hg2+,one facile detection method coupled with Exo-Ⅲ mediated target recycling for sensitive DNA analysis,and a ratiometric fluorescence protocol for DNA detection were proposed.This work provided an avenue for understanding the interaction between two-dimensional nanomaterials and biomolecules and designing novel sensing strategies for extending the applications of nanomaterials in bioanalysis.3.Regulative Peroxidase Activity of DNA-Linked Hemin by Graphene Oxide for Fluorescence DNA SensingThis work designed a hemin labelled single-stranded DNA probe,which showed superior peroxidase activity than free hemin in aqueous solution.Non-fluorescent substrates could be catalytically oxidized into fluorescent products by H2O2 in the presence of hemin labelled probe.The probe could be absorbed on the graphene oxide(GO)surface due to the π-π interaction between the exposed nucleotide bases of ssDNA and GO,which brought hemin to the GO surface and inhibited its catalytic property.The inhibition effect of GO toward the peroxidase activity of DNA-linked hemin could be mainly attributed to the decreasing diffusion rate of the probe,the steric hindrance of GO,the interaction of GO with the oxidation intermediate,and the hydrophobicity of π-conjugated GO center.The inhibition effect could be relieved by hybridizing the probe with a complementary DNA target to detach the formed double-stranded DNA and thus the linked hemin from the GO surface.Based on the tunable peroxidase activity of DNA-linked hemin,a simple,quick and highly sensitive homogeneous fluorescence DNA sensing strategy was developed.4.Catalytic Activity of Dual-Hemin Labelled Oligonucleotide:Conformational Dependence and Fluorescence DNA SensingThis work designed a novel "DNA switch" to achieve target DNA regulated biomimetic catalysis for fluorescent DNA sensing.The "DNA switch" was prepared by covalently labeling two ends of oligonucleotide with two hemin molecules.It could spontaneously form an intramolecular hemin dimer due to the flexible structure of single-stranded oligonucleotide,which greatly minimized the peroxidase activity of hemin.The formation of hemin dimer was reversible,and it can be dissociated upon the formation of double-stranded DNA,leading to a DNA regulated catalytic activity.Using fluorescent oxidation product of tyramine as tracing molecule,the hemin dimer could not catalyze the oxidation of tyramine by hydrogen peroxide,leading to a low fluorescence background.In the presence of target DNA,the peroxidase activity of hemin was recovered due to the dissociation of hemin dimer,which led to the formation of fluorescent dityramine.Both the dimer dissociation and the catalytic reaction could be completed within 10 minutes,respectively.The proposed fluorescence method for homogenous target DNA detection showed high sensitivity with a detection limit of 19 pM.The good analytical performance endows the designed simple,convenient,fast and homogenous detection format with promising potential in high-throughput DNA detection.5.Strand Displacement Activated Peroxidase Activity of Hemin for Fluorescence DNA SensingThis work designed a double-stranded DNA probe containing an intermolecular dimer of hemin,whose peroxidase activity could be activated by a DNA strand displacement reaction.Using fluorescent oxidation product of tyramine by H2O2 as a tracing molecule,the low peroxidase activity of hemin dimer ensured a low fluorescence background.The strand displacement reaction of target DNA dissociated the hemin dimer and thus significantly increased the catalytic activity of hemin to produce a large amount of dityramine for fluorescent signal readout.Based on the strand displacement regulated peroxidase activity,a simple and sensitive homogeneous fluorescent DNA sensing method was proposed.The detection ccould conveniently be carried out in a 96-well plate within 20 min.This method showed high specificity,which could effectively distinguish single-base mismatched DNA from perfectly matched target DNA.