Study On The Nucleic Acid Biosensors Based On Rolling Circle Amplification Of DNAzyme And Metal Nanoclusters

With the rapid development and improvement of the science and technology,functional nucleic acids have broadened the vision of people,breaking the people of nucleic acid only as a traditional concept of genetic information storage and transport carrier,deepen the further understanding of nucleic acids,and provided a new active material for the study of molecular biology.At the same time,rolling circle amplification has been widely concerned by the virtue of high sensitivity,specificity and easy operation.Due to its excellent optical and optical properties,DNA-AgNCs have become a common fluorescent nano probe.DNAzyme machine and rolling circle amplification were proposed to detect copper ions.DNA-AgNCs were proposed to detect S1 nuclease and L-Cys.The main research is performed as following:A novel rolling circle amplification based fluorescent DNAzyme machine has been devised for the amplified detection of copper(Cu²⁺)ions.In the absense of Cu²⁺ions,the DNAzyme substrates strand introduce the rolling circle amplification reaction as primers.The long single-stranded DNA?ssDNA?product containing tandem repeats was generated in large quantities by the rolling circle amplification and could serve as an excellent template for periodic binding of molecular beacons?MBs?.Finally the MBs hybridize with the long ssDNA product to generate a distinguishable fluorescence enhancement.The enzyme strand carries out hydrolytic cleavage of the substrate strand in the presence of Cu²⁺ ions.The cleaved DNAzyme substrates suspend the rolling circle amplification reaction leading a fluorescence negation.A good linear correlation?R=0.9947?was obtained between fluorescence intensity and the logarithm of the Cu²⁺ ions concentration over the range from 1 nM to 4 ?M.The detection limit was estimated as 0.6792 nM.The combination of the DNAzyme and RCA has a substantial impact on the development of amplified DNAzyme sensors.A label-free fluorescent method for the detection of S1 nuclease activity has been developed using oligonucleotide-templated silver nanoclusters.S1 nucleasehydrolyzes single-stranded DNA?ssDNA?into mononucleotide and oligonucleotide fragment.In the presence of S1 nuclease,the ssDNA as a template is degraded to mono-or oligonucleotide fragments,thereby resulting in a reduction in fluorescence.The proposed sensor was shown a linear response in the range of 5×10^-54×10^-3U/mL of S1 nuclease with a detection limit of 2×10^-6 U/mL.The sensor revealed good recovery rates from 91.8% to 109.5% in RPMI 1640 cell medium,indicating that the sensing system is feasible for the detection of S1 nuclease in practical samples.A fluorescent method for the detection of L-Cys has been developed using oligonucleotide-templated silver nanoclusters?DNA-AgNCs?.In the presence of L-Cys,C-Ag⁺-C complexes are destroyed by the combination of the sulfhydryl?SH?and silver ion?Ag⁺?,DNA-AgNCs shows a decrease in fluorescence.The proposed sensor was shown a linear response in the range of 2 nM¹0 ?M of L-Cys with a detection limit of 0.2 nM.The sensor revealed good recovery rates in serum sample.