Biological Analysis Detection Based On Silver Nanoclusters Stabilized By A Dumbbell-Shaped DNA Template

Fluorescent nanotechnology has rapid development in recent years and a large number of nanomaterials were found out which was applied to cells in vivo imaging,biosensors,gene diagnosis and clinical treatment.Silver nanoclusters(AgNCs)which is one of nanomaterials was brought into sharp focus by its unique properties.Fluorescent silver nanoclusters templated by DNA(DNA/AgNCs)is a kind of molecular aggregation-like core/shell structure with core containing a few to tens silver atoms and shell containing hydrophilic nucleic acid,leading to a number of advantages,such as controllable fluorescence emission wavelength,high photostability,excellent brightness,facile synthesis(one-pot preparation),good biocompatibility and so on.Compared with the fluorescent molecular beacon,DNA/AgNCs has better absorption coefficient and fluorescence quantum yield,which are expected to be a new type of biosensing materials and fluorescent materials.In this paper,we developed a kind of dumbbell DNA template to synthesis of silver clusters to detect NAD+ and miRNA.We developed a novel method for detecting nicotinamide adenine dinucleotide(NAD+)based on fluorescent silver nanoclusters(AgNCs)stabilized by a dumbbell-shaped DNA template containing two cytosine-loops joined in a dsDNA stem.The reaction system consists of two necessary components,dumbbell-shaped DNA template and three enzymes(E.coli DNA ligase,Exo ?,and Exo ?).In the presence of NAD+ as a cofactor,Escherichia coli DNA ligase(E.coli DNA ligase)catalyzes template ligation to generate a sealed dumbbell-shaped structure,preventing digestion by exonuclease ?(Exo ?)and exonuclease I(Exo I).The loop regions of the intact template serve as sites for deposition of highly fluorescent AgNCs.In the absence of NAD+,the ligation reaction does not occur,and the unsealed dumbbell-shaped template is digested into mononucleotides via cooperation of Exo III and Exo I.The destruction of the DNA template results in agglomeration of AgNCs into silver nanoparticles with low fluorescence.The fluorescence enhancement depends on ligation and digestion of the DNA template,allowing quantitative detection of NAD+.Our method is advantageous with low cost,no fluorescent label and can detect the NAD+ with the lever of nM,which can apply to detection of biological samples.The surface-enhanced fluorescence between AgNCs and AuNPs coupled with DNA polymerase and nicking enzyme can be used to detect miRNA based on recyle of target molecular with isothemol amplification.The DNA probe consists of three domains,the complementary sequence of miRNA,the specific site of nicking enzyme and the self-assembly sequence for AgNCs.The 3' end of DNA probe is modified with thiol as a binding site to AuNPs.The SEF of AgNCs and AuNPs was inhibited when miRNA was added to produce the dumbbell shaped template by polymerase which can synthesise the AgNCs and the target molecular was replaced and recycled to realize the signal amplification coupled with DNA polymerace and niking enzyme.Compared with the traditional method of miRNA detection with commercial kit RT-PCR,our method avoids the process of reverase transcription and operations is simple.In addition,our proposed method is cost-effective,label-free,highly selective for detecting miRNA,which can also apply to analyze the miRNA in blood and cell samples.