The Study Of Nucleic Acid Amplification Strategy Sensitized Fluorescent Biosensor For Small Molecules Detection

Fluorescent biosensor can convert the target concentration into fluorescent signal based on the specific recognition elements,to realize the qualitative and quantitative detection of small molecules.Due to the homogenous detection environment,fluorescent biosensor is widely used in the clinical sample analysis and drug concentration monitoring with the merits of facile experimental operation,fast signal response,and excellent stability.However,conventional fluorescent biosensors can hardly meet the challenge of critical demands of the trace target detection in the practical application.Therefore,the nucleic acid amplification strategy,which can continuously enlarge the physical and chemical information generated by the target,has become an important part of the construction of highly sensitive fluorescent biosensors.According to the above,we utilized organic dyes and quantum dots as fluorescent indicators,and combined with rolling circle amplification,catalytic hairpin assembly and hybridization chain reaction to construct a series of novel fluorescent biosensor for the detection of heavy metal ion and miRNAs.The specific works are as follows:1.A Sensitive Fluorescent Biosensor Based on a Rolling Circle Amplification Assisted Multisite Catalytic Hairpin Assembly Reaction for Pb²⁺DetectionAs a representative of enzyme assisted nucleic acid signal amplification strategy,rolling circle amplification?RCA?can achieve the exponential amplification of DNA or RNA molecules in a short time.However,traditional rolling circle amplification usually directly assembled the signal probes on the RCA products as the fluorescent reporter,which would reduce the detection sensitivity because of the aggregation induced quenching property of general fluorescent dyes.Therefore,this work utilized the RCA product to provide multiple sites as the primers for the catalytic hairpin assembly?CHA?reaction between the signal probes,which not only increased the reaction efficiency,but also avoid the self-quenching of signal probe assembled on the RCA product to improve the detection sensitivity.As a proof of concept,we utilized this strategy in the construction of fluorescent biosensor for the heavy metal ions detection.The experimental results presented a good performance for the determination of Pb²⁺with a linear range from 0.1 to 50 nmol/L and a detection limit down to 0.03 nmol/L.Furthermore,the proposed fluorescent biosensor could be also expanded for the detection of other biomolecules and proteins,offering a novel avenue for environmental assays and clinical diagnostics.2.A Novel Fluorescent 3D DNA Nanomachine Based on the Biomimetic Interface for MiRNA DetectionThree dimensional?3D?DNA nanomachine has obtained great attentions in recent years due to its high controllability and functionality.However,traditional 3D DNA nanomachine usually immobilized the substrates in the specific position of hard surface of AuNPs or silica via the time-consuming covalent action,thus limited the movement efficiency of nanomachine.Therefore,this work proposed a biomimetic 3D DNA nanomachine based on free DNA walker movement on soft and fluidic lipid bilayers supported by silica@CdTe quantum dots?SiO₂@CdTe?.The lipid bilayers not only rapidly immobilized the DNA substrates through the cholesterol-lipid interaction,but also kept it with a favorable fluidity to improve the walking efficiency and shorten the reaction time within 30 min.Finally,this novel 3D DNA nanomachine presented a good performance for the fluorescent detection of miRNA-141 with a linear range from 1pmol/L to 2.5 nmol/L and a detection limit of 0.21 pmol/L.Furthermore,this work provided a new avenue for the construction of highly efficient DNA nanomachine and exhibited a great potential for the ultrasensitive analysis of tumor related biomarkers and early clinical diagnoses of disease.3.A Label-Free Fluorescent Biosensor Based on Self-Disassembly of DNA Micelles via Nucleic Acid Amplification Induced Hydrophilic-Hydrophobic Regulation for MiRNA DetectionThe common DNA nanostructures?DNA tetrahedrons or cubes?have been challenged in the practical application due to the disadvantages of the complicated DNA sequences,time-consuming assembly and difficulty in loading substrates.Thus,this work designed a new DNA nanostructure with facile DNA sequences,rapid assembling and tunable DNA diblock copolymer micelles,and applied it in the construction of a novel lable-free fluorescent biosensor in combination with the nucleic acid amplification.In this strategy,the amphiphilic DNA diblock copolymers could self-assemble into DNA micelles with a hydrophobic core in the aqueous,which could encapsulate the fat soluble fluorescent dyes with the help of sonication.Once the target was added to trigger the hybridization chain reaction?HCR?of the hydrophilic DNA head,the hydrophilicity of the DNA micelles could be enhanced by the HCR amplification,which could destroy the stability and induce the self-disassembly of DNA micelles,resulting in the releasement of fluorescent dyes to generate the change of fluorescent intensity.The experimental results demonstrated that the novel fluorescent biosensor based on the DNA micelles has a good performance for the detection of miRNA-21,with a linear range from 20 pmol/L to 20 nmol/L and a detection limit of6.9 pmol/L.In addition,this work may provide a new avenue for the construction of DNA nanostructures,and a novel technology for biosensors,drug delivery and other fields.