Research On New Methods Of Fluorescent Biosensing Based On Copper Nanoclusters

The application of organic fluorescent dyes in fluorescent biosensors is limited due to their complex synthesis,high background and poor biocompatibility.In recent years,fluorescence metal nanoclusters have attracted the attention of researchers in the field of biosensing because of their low toxicity,good biocompatibility and simple synthesis.In this paper,using DNA-templated copper nanoclusters(CuNCs)as fluorescent probes and the properties of CuNCs whose fluorescence properties were closely related to the template DNA sequences,a low-background,highly sensitive fluorescence biosensing method was constructed by designing DNA sequences.The specific research content is as follows:1.Design of high signal-to-noise ratio copper nanocluster fluorescence biosensing methodDNA is an effective template for the synthesis of fluorescent CuNCs,and the fluorescence of CuNCs is highly dependent on the template DNA sequence.In this study,KF polymerase was used as a model analyte.The DNA of the substrate was obtained through polymerization reaction to obtain a double-stranded DNA(dsDNA)product.CuNCs were then synthesized using the dsDNA as a template to achieve fluorescence detection of KF polymerase activity.We studied in detail the effects of primer DNA and polymerase template DNA sequences on system fluorescence to established a low background,high signal biosensing method.The detection limit of KF polymerase activity was 3.7 × 10-7U/μL.Compared with the fluorescent method using SYBR Green I(SG)as a signal substance,the sensitivity was significantly improved.In this study,the strategy of controlling the fluorescence of CuNCs was designed through the design of template DNA sequences,and a high signal-to-noise ratio fluorescence biosensing method for copper nanoclusters was constructed.2.Copper nanocluster fluorescence biosensing assay T4 polynucleotide kinase phosphatase activityT4 PNKP catalyzes the hydrolysis of 3’-phosphoryl DNA,which plays an important role in DNA replication,damage repair,and recombination.In this study,we proposed a novel label-free method for determining T4 PNKP activity using CuNCs as a fluorescent indicator.In this method,a short 3’-terminal phosphorylated DNA strand hybridizes to a long DNA strand to produce partially dsDNA as a T4 PNKP substrate.The addition of T4 PNKP triggers the dephosphorylation and polymerization of the substrate to generate long-chain dsDNA which be used to in situ synthesize fluorescent CuNCs.By rationally designing DNA sequences,a sensitive detection of T4 PNKP activity was achieved with a detection limit of 0.07 U/mL.The method has the advantages of simple operation,low cost,convenient operation and good application prospect.3.Copper nanocluster fluorescence biosensing for high sensitivity DNA detection through isothermal cycling cascade signal amplificationCascade isothermal exponential amplification reaction is an efficient signal amplification technique.In this study,an isothermal cycling cascade signal amplification combined with fluorescent CuNCs to construct a new non-labeled highly sensitive DNA detection method.The method was divided into two phases:In the first phase,the hybridization reaction between the target DNA and the DNA template 1 could initiate an isothermal double cycle and generated a large amount of short single-stranded DNA;in the second phase,the short single-stranded DNA was hybridized with the DNA template 2,a large amount of long dsDNA was produced by the polymerization of KF polymerase,and then a large amount of fluorescent CuNCs could be produced by using the long dsDNA as a template.Finally,by detecting the fluorescence intensity of the generated CuNCs,non-labeled sensing of the target DNA could be achieved.In this study,the rational design of the DNA template 1 and the DNA template 2 improved signal amplification efficiency and reduced background,thereby achieving highly sensitive detection of target DNA.The limit of detection for the target DNA was 7 pM.This method combined the advantages of cascaded isothermal exponential amplification signal amplification with the sensing advantages of CuNCs.It has the advantages of simple operation,low cost,and high sensitivity.