Cascade Nucleic Acid Amplification For Efficient Detection And Intracellular Imaging Of Biomarkers

The sensing and analysis method based on cascade nucleic acid amplification technique is a new type of sensing technology that utilizes nucleic acid molecules as components to build functional modules.Moreover,there are different methods of target recognition and cascade signal amplification for different targets.Many biomarkers,including short-chain nucleic acids,organic small molecules,and low-molecular weight proteins,are involved in the regulation of many biochemical reactions and cell metabolisms in living organisms.And their abnormal expression levels are related to a variety of diseases,so they can be considered as an analytical target in the field of cascade nucleic acid amplification analysis.However,the expression levels of some biomarkers in cells and tissues are relatively low,so it is challenging to have a rapid detection assay and intracellular imaging for biological targets at low concentration with high sensitivity,specificity and stability.The development of new sensing and analysis technology based on cascade nucleic acid amplification techniques,by the assistance of high-order nucleic acid hybridization circuits to achieve higher signal gain,is of great significance for the detection and imaging of some biomarkers with low content.Herein,we successfully designed two kinds of cascade nucleic acid amplification analysis methods with 1:N² signal gain to have an application in detection assay in vitro and intracellular imaging of ATP and mi R-21,respectively.The research work is mainly carried out from the following two aspects:（1）An aptasensor based on nonenzymatic isothermal cascade hybridization chain reaction（Ca-HCR）was constructed,and the aptasensor achieved the amplification detection of ATP by obtaining the FERT signal output of the whole reaction process.In this aptasensor,the aptamer sequence in the ds DNA hybrid Apt C/Apt Inh can specifically identify ATP target to release the initiator sequence,which can activate the continuous hybridization of hairpins involved in the upstream HCR-1 reaction.The self-assembly product in HCR-1 hybridization reaction,ds DNA polymerized nanowires,contains many repetitive initiator sequences that can trigger the downstream HCR-2 reaction,so the downstream HCR-2 reaction can be promoted to generate a"signal-off"readout signal with amplification ratio of 1:N²based on FRET mechanism.Meanwhile the designed aptasensor can take advantage of the intracellular imaging of ATP and monitoring of its regulation in dynamic level.And the reaction mechanism of Ca-HCR aptasensor was studied by kinetics model derivation.In addition,this method can also build a universal aptasensor to detect other biomarkers by introducing different aptamer and corresponding inhibitors sequences as modular component.（2）A multi-functional DNAzyme amplifier based on target regeneration assisted by Exo-III and signal recycling catalyzed by DNAzyme,was constructed to achieve quantitative detection and intracellular imaging of micro RNA-21.In the designed detection platform,the opened arm region of multifunctional hairpin HP-Dz binds with mi R-21 to form a double-stranded hybrid,and mi R-21 can be continuously released from the hybrid through Exo-III digestion.Subsequently the released mi R-21then hybridizes with another HP-Dz to start a new cycle.At the same time,the closed DNAzyme structure in the multifunctional hairpin HP-Dz is activated,which can initiate the cleavage of the fluorophore-labeled substrate to promote an amplified signal output.Finally,a large number of fluorescence signals are generated to achieve dual signal amplification.In order to achieve the naked eye detection of mi RNA,by replacing the Mg²⁺-dependent DNAzyme sequence in the multi-function hairpin HP-Dz with the G-quadruplex sequence,in which Hemin/G-quadruplex HRP minicking enzyme can catalyze H₂O₂ oxidation of ABTS^2-to have a colored signal output,so a non-label multi-functional hairpin HP-G4 amplifier system was constructed.Therefore,the designed multi-functional DNAzyme amplifier is a universal,simple and ultrasensitive signal amplification method,which can be successfully utilized in the mi R-21 detection assay and intracellular imaging,and also plays a significant role in understanding the relevant pathological processes.