The Design And Application Of Nano-Fluorescent Probes Based On DNA Templated Copper Nanoclusters And Gold Nanoclusters

Due to its convenient optical signal transduction,high sensitivity and rapid response ability,fluorescence-based sensing is currently by far the dominant analytical tool and is widely used in the field of chemical analysis,environmental monitoring and biological imaging.Organic dyes are the most commonly used fluorescent labels,but their low absorption coefficients and weak signal lead to the reduced sensitivity and response of the targets.Their poor photobleaching resistance also limits their use in long-term tracking.In recent years,a series of novel nanomaterials-based fluorescent probes have been developed for chemical and biological research.The introduction of nanomaterials into fluorescence analysis has opened up new venues for fluorescence analysis.Among them,few-atom metal nanoclusters(or nanoparticles)have been gradually developed as ideal alternatives to traditional organic dyes due to their strong and robust fluorescence emission.They are usually prepared by a biomineralization manner,in which biomolecules(e.g.small thiol-molecule,polymer,protein and nucleic acid)are used as templates to mediate the formation of metal nanoclusters.Unlike successful synthetic strategies and fluorescence tunability of silver nanoclusters(AgNCs)with different DNA templates,the use of DNA as a template for preparing fluorescent copper nanoparticles(CuNPs)and gold nanoclusters(AuNCs)has been reported in relatively few studies.In this thesis,aiming at the understanding of the relationship between the sequence composition and the formation of DNA-CuNPs and DNA-AuNCs,a series of DNA of different sequence composiston were systematically investigated for the formation of DNA-CuNPs and DNA-AuNCs.On this basis,a series of new fluorescent sensors for enzyme,small molecule and heavy metal ions have been developed based on DNA templated CuNPs and AuNCs.The detailed description is listed as follows:1.dsDNA-templated fluorescent copper nanoparticles:poly(AT-TA)-dependent formationTo identify the specific DNA sequence for the formation of the dsDNA-templated fluorescent CuNPs,we here focus on the relationship between the sequence composition of dsDNA and the formation of dsDNA-CuNPs.A series of dsDNA of different sequence compositions were systematically investigated for the formation of CuNPs,the fluorescence intensity and fluorescence quantum yield were measured to determine the effect of each dsDNA.Through systematical investigation on the formation of the fluorescent CuNPs templated by dsDNA,poly(AT-TA)was found as the specific dsDNA sequence which can act as a highly-efficient template for the formation of the fluorescent CuNPs,while other sequence compositions have little or no function for supporting the formation of the fluorescent CuNPs.The fluorescence intensity is highly-dependent on the polymerization degree and the length of poly(AT-TA).The reaction conditions are benign and the fluorescence response is very fast.This finding will be helpful for the understanding of the relationship between sequence composition and dsDNA-CuNPs,and useful for the applications of CuNPs,such as constructing of DNA-templated nanodevices and designing of biochemical nano-probes.2.dsDNA-specific fluorescent copper nanoparticles as a "green" nano-dye for polymerization-mediated biochemical analysisTaking advantages of dsDNA-CuNPs like strong fluorescence intensity,facile synthetic process and fine biological compatibility,a novel,sensitive and label-free fluorescent method for polymerization-mediated biochemical analysis has been developed based on the sequence-dependent formation of CuNPs.In the presence of the corresponding target,polymerization is triggered,generating dsDNA which can efficiently template theformation of fluorescent CuNPs.The strategy has multifaceted excellences:simplicity,environment-friendly manner and good detection capabilities.Morever,the proposed strategy is applicable for broad targets which can induce the formation of a complementary primer-template complex and subsequent primer-extension reaction.Thus,this work will expand the way to apply the dsDNA-CuNPs as a "green" nano-dye for signal transducing in a wider range of fields,such as aptamer-based detection of non-nucleic acid targets,molecular biology and biomedicine.3.Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repairIn view of the features of DNA-CuNPs which could be prepared by single-stranded poly(T)or double-stranded poly(AT-TA)DNA,a smart and high efficient dumbbell-shaped DNA was designed and employed for the template to induce DNA-CuNPs.This dumbbell-shaped DNA probe templated CuNPs(DP-CuNPs)displayed good fluorescence emission and stability.Then,this highly-efficient DP-CuNPs was employed as a label-free nano-fluorescent probe for evaluating T4 ligase and T4 polynucleotide kinase activity based on the target-dependent locking of the dumbbell-shaped DNA probe.The locked DP could resist the digestion of exonucleases and then served as a highly efficient template for synthesizing high fluorescence CuNPs.However,in the absence of analyte,the nicked DP would undergo enzymatic digestion to mononucleotide by exonucleases and then failed to template fluorescence CuNPs.Therefore,the fluorescence changes of CuNPs could be used to evaluate these enzymes activity.Furthermore,the proposed method was also extended to detecting alkaline phosphatase activity,which illustrates the versatility of this strategy and holds the potential for other DNA repair enzymes detection.Thanks to its excellent analytical performance,this sensing system was successfully applied in the detection of analyte in diluted cell extracts,which indicates its great potential for practical application.4.Oligonucleotide-templated rapid formation of fluorescent gold nanoclusters and its application for Hg2+ ions sensingIn order to understand the relationship between the sequence composition and the formation of DNA-AuNCs,we developed a simple,rapid and mild strategy for synthesis of DNA-templated fluorescent gold nanoclusters(AuNCs)through association of gold iorns to DNA templates and reduction with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES).After systematical investigation on the formation of fluorescent AuNCs by using different DNA templates,C5 DNA was found as the best template for the formation of fluorescent AuNCs in this system.This process could be completed withirn 5 min after the reaction beginning under ambient conditions.The prepared C5-AuNCs displayed good blue emission and photostability.Furthermore,this C5-AuNCs could be applied to detecting Hg2+ ions specifically based on the specific and strong interaction between Hg2+ and AuT.The C5-AuNCs provided excellent selectivity for Hg2+ ions over other metal ions,and also high sensitivity,with a detection limit of 50 nM.As biocompatible,environmentally-friendly,and synthesis-rapid,this C5-AuNCs probe appears to be promising candidate for biochemical sensing via simple modification of template DNA.5.A high-selective nanosensor for ultrafast detection of Cu2+ ions based on DNA-templated gold nanoclusters and Fenton-like reactionInspired by the distinct features and great potential in many fields of fluorescent C5-AuNCs,we herein report a simple,fast,and sensitive fluorescence method for detecting Cu2+ ions by Fenton-like reactions mediated quenching of AuNCs.Under aerobic conditions,ascorbic acid not only is involved in the reduction of Cu2+,but also reacts with O2 to produce H2O2.Then,hydroxyl radical(OH),yielded by H2O2 and Cu+,can seriously quench the fluorescence of AuNCs.Thus,Cu2+ ions can be detected by the fluorescence change of AuNCs.Attributed to the unique Fenton-like between ascorbic acid and the C1uLi ions,high selectivity was achieved for Cu2+ ions monitoring by this nanosensor,with practical applications in real water and blood samples.