Construction Of DNA-Templated Nanocopper-Based Molecular Beacons And The Applications In Biochemical Sensing

Compared with classical molecular beacons,nanomolecular beacons have the advantages of high sensitivity,good biocompatibility,simple operation,and label-free,which can be better and more conveniently applied to the rapid and accurate detection of target,and thus have received widespread research attentions.DNA-templated nanocopper possess advantages including faster synthesis,more cost-effective,environmentally-friendly,lower toxicity and larger Stokes shift,etc.Therefore,DNA template copper nanoparticles become an ideal signal source in biochemical analysis.Inspired by the mentioned above,we construct a novel,intelligent,multifunctional and label-free nanomolecular beacon based on the in situ synthesis of nanocopper.In this work,based on fluorescent nanocopper and nanomolecular beacons targeting nucleic acids,proteins,small molecules,and metal ions,respectively,we carried out the following three aspects:1.Construction of nanomolecular beacons based on DNA-templated nanocopper and nucleic acid detectionIn this work,a specific recognition method for DNA and micro RNA was developed based on the construction of fluorescent nanocopper molecular beacons.In the buffer solution environment,through the denaturation and annealing process of DNA molecules,the single-stranded DNA is induced to self-assemble into a probe with a hairpin structure.The recognition sequence hybridizes with the target DNA when the target DNA molecule is present,inducing a change in the hairpin structure conformation,and the two template sequences hybridize with each other to form double-stranded structure,allowing in situ synthesis of nanocopper and outputting fluorescent signal,realizing the detection of the target DNA.The linear response of the nanometer beacons to DNA detection was in the range of 0.1 n M?0.5?m,and the linear response of micro RNA detection was in the range of 0.1 n M?1.0?m.This high sensitivity is mainly attributed to the strong fluorescence emission of Nanocopper,as well as the reasonable design of the hairpin DNA.2.Rapid homogeneous analysis of non-target nucleic acid moleculesBased on the work in which DNA-templated copper nanomolecule beacons were used for nucleic acid-specific recognition,to explore the applications of the nanobeacon in the detection of non-nucleic acid targets,thrombin and ATP were selected as target models of protein and small biological molecule and detected by utilizing the nanobeacon,respectively.After the specific recognition of the target and analyte,the structural conformation of the probe is induced to change,and the two template sequences hybridize with each other to form a double-stranded structure and allow in situ synthesis of nanocopper and output of fluorescence signal.The linear response of the nanometer beacons to DNA detection was in the range of 0.1 n M?0.5?m,and the linear response of micro RNA detection was in the range of 0.1 n M?1.0?m.The method is highly sensitive and selective without complex labeling and expensive instrumentation,and therefore has great potential for clinical diagnostic applications.3.Fluorescence analysis based on enzyme-assisted isothermal cycle signal amplificationTo further demonstrate the versatility of nanomolecular beacons and explore their potential in isothermal cycling signal amplification,a slightly modified hairpin DNA was designed in this work and combined with an Exo III-assisted enzymatic cleavage cycle reaction to establish a strategy for isothermal cycling signal amplified DNA detection involving copper nanomolecular beacons and an analytical method for highly sensitive DNA detection.A new method for highly sensitive analysis of Hg²⁺was constructed by combining the established signal amplification strategy with the precise design of probe sequences for nanobeacons.The nanobeacon combined with enzyme-assisted assisted isothermal cycle amplification,under the optimized conditions,the detection limit of DNA recognition by nanobeacons was as low as0.83p M,and the recognition of Hg²⁺had a wide linear range of 7 orders of magnitude.Which is two orders of magnitude lower than DNA detection with enzyme-free nanobeacon mentioned above.These results clearly confirm the versatility and robustness of this nanobeacon in enzyme-aided ultrasensitive bioanalysis.Therefore,this work developed a novel homogeneous biosensing method for the enzyme-assisted isothermal cyclic amplified detection of Hg²⁺,which has the advantages of wide linear range,high sensitivity and good selectivity.