Synthesis And Application Of Silver Nanoclusters Templated By DNA

MicroRNAs?miRNAs?,a group of non-coding,short-strand RNAs with 18-25nucleotides,regulate the expression of protein-encoding genes by binding non-coding parts of messenger RNA.MiRNAs regulate a series of biological processes,including cell differentiation,proliferation and apoptosis,by regulating related gene expression or preventing protein synthesis.Recently,abnormal expressions of miRNAs have been found being associated with various cancers,including the up-regulation in breast cancer,ovarian cancer,non-small cell cancer,and colorectal cancer.Thus,it is promising to use miRNAs as biomarkers for clinical diagnosis and prognosis.Although there are many methods currently used for miRNAs analysis,including Northern blotting,real-time reverse transcription polymerase chain reaction and miRNA microarrays,these methods are all time-consuming and very expensive,which limit their applications in the biological field.Therefore,exploring analytical techniques for accurate quantification and qualitative analysis of miRNAs is crucial.The combination of the unique optical/electrical properties of nanomaterials and the highly specific recognition ability of biomolecules has been used to develop new tools for bioanalysis.In addition,the structure of DNA is diverse,containing B-form,G-quadruplex,i-motif and etc.In this study,single-strand cytosine-rich DNA sequences were employed to guide the synthesis of DNA/AgNCs,which is used for the miRNAs-21,miRNAs-182,and miRNA-195 detection through fluorescence changing.In experiments,the regulation of the DNA/AgNCs optical properties was initially proceeded by changing the ratio of DNA and Ag⁺,pH value,various environmental metal cations and surface oxidation states.For deficiency,the overall formation of AgNCs was not enough.For excess,too many silver clusters would lead the aggregation and the fluorescence self-quenching.Therefore,only the appropriate DNA/Ag⁺ratios guarantees the high quantum yield of AgNCs.The results indicated that the fluorescence properties of DNA/AgNCs were easily affected by over-alkaline or acidic conditions,which might deriving from the protonation or deprotonation of DNA bases in different pH values.For acidic environment,the protonation of DNA bases promoted the formation of"i-motif"structures,thus weakening the interaction with DNA and Ag⁺.For alkaline environment,the interaction between the Ag⁺and phosphate groups of DNA might result in a decrease of Ag⁺concentration,thus weakening the interaction of C-Ag⁺-C and leading a decrease of fluorescence intensity.We performed elemental analysis of silver nanoclusters surface by X-ray photoelectron spectroscopy,which showed about 30%of Ag⁺content in DNA/AgNCs.Therefore,the surface oxidation state of the silver nanoclusters is essential for maintaining fluorescence properties.The experiments of various environmental metal cations showed that divalent metal cations can maintain the stability of DNA/AgNCs in a significant degree.In particular,Mg²⁺can prevent the aggregation of AgNCs and avoid the fluorescence quenching.We have three hypotheses about the mechanism Mg²⁺ions role:Firstly,they interact with specific bases of the DNA template to change the stability of the nucleic acid structure or to promote the influence of bases on AgNCs.Secondly,as Mg²⁺can form hydrated hexahydrate compounds,they might interact with nitrogen or oxygen of DNA through hydrogen-bonded backbones and modulate the fluorescence properties.Thirdly,Mg²⁺might serve to maintain the presence of Ag⁺on the surface of silver nanoclusters,thus maintaining fluorescence emission.However,the actual mechanisms requires further studies.Here,the DNA/AgNCs probes successfully performed the qualitative and quantitative detection of miRNA-21,miRNA-182,and miRNA-195 with lowest detection limits of 19.3 nM,15.6 nM,and 47.1 nM,respectively.In order to evaluate the specificity of the detection,a selective experiment of miRNA-21 was performed.The experimental results showed that the probe responded well only to the target miRNAs.Furthermore,the recovery rates of miRNA-21 with three different concentrations were ranged from 93.0%to 110.0%.