Research Of Functional Nucleic Acid Fluorescent Biosensors Based On Enzyme-free Signal Amplification

With the development of science and technology,people have deeper understanding of the functional information of biomolecules and the pathological mechanism of diseases.As an emerging tool in the field of bioanalysis,fluorescent biosensors targeting important biomolecules have greatly promoted the development of disease diagnosis,drug screening and the research of biological functions due to their advantages of fast response,simple design,high sensitivity and non-destructive analysis.At the same time,with the development of DNA nanotechnology,the functions of nucleic acids are no longer limited to the carriers for the storage and transmission of genetic information,but are also widely involved in the construction of fluorescent biosensors due to their precise programmability,easy functional modification and good biocompatibility.In particular,in the past 20 years,more and more functional nucleic acids such as aptamers with specific recognition ability and deoxyribozymes with catalytic activity,have been developed through in vitro screening technology.And then various functional nucleic acid-based fluorescent nucleic acids have been constructed,which greatly expand the application scope of fluorescent biosensors in the field of biological analysis.At the same time,with the development of enzyme-free nucleic acid isothermal amplification technology,it has been found that this technology is very suitable for the amplification and analysis of low-abundance biomolecules since the achievement of output signal amplification is based on the continuously reacting of nucleic acids under constant temperature conditions.Therefore,in recent years,enzyme-free nucleic acid isothermal amplification technology is often used as a signal amplification element in the design of fluorescent biosensors,which greatly improves the response speed and sensitivity of biosensors,and is applied to the analysis of important biomolecules in living cells and actual samples.Nonetheless,these nucleic acid fluorescent biosensors often face the disadvantages of false positive signals from poor stability of nucleic acid molecules in complex environments and low cellular uptake efficiency.At the same time,the multi-components of these sensors often proceed in the form of free diffusion,resulting in low reaction efficiency and unsatisfactory sensing performance in complex analytical environments such as living cells and practical samples.Therefore,in order to solve the problems mentioned before,this research paper is based on the classical functional nucleic acid fluorescent biosensors,combined with enzyme-free nucleic acid signal amplification technology and DNA nanomaterials etc.,developed a variety of new biosensing tools with high sensitivity,fast response,and reliable signal for the reliable detection of intracellular micro RNAs(miRNAs),high-sensitivity analysis of miRNAs in actual samples,spatiotemporal controllable imaging of metal ions on the cell membrane surface,and accurate identification and imaging of target cells.The specific research contents are as follows:In order to solve the problems of traditional hairpin DNA cascade amplification(HDCA)probes such as difficult to be taken up by cells,low reaction efficiency,and prone to generate false positive signals in complex cellular environments,the spatial confinement effect of DNA nanostructures and fluorescence resonance energy transfer(FRET)-based ratiometric signal readout modes are introduced into HDCA probes to construct a novel FRET-based DNA cube biosensor for fast,efficient,and reliable intracellular miRNA imaging.In this biosensor,the DNA cube nanostructures not only provide the probes with better resistance to nuclease degradation and good cellular uptake,but also serve as a spatially confined scaffold to significantly improve the response performance of the probes.In addition,ratiometric signal readout mode based on FRET can also avoid false positive signals caused by nuclease degradation and system fluctuations,and improve the reliability of detection.This strategy greatly exploits the advantages of DNA self-assembled structures as confinement scaffolds to improve the efficiency of nucleic acid signal amplification reactions,and provides a new idea for the design of reliable and efficient nucleic acid biosensors in living cells.In order to further enhance the response efficiency of the hairpin DNA cascade reaction from spatial confinement effect,we constructed an erythrocyte membrane-based DNA amplification sensing platform using erythrocyte membrane materials that can simultaneously enrich multiple pairs of hairpin DNA.By modifying the DNA stands with cholesterol molecules,the hydrophobic interaction between cholesterol and erythrocyte membrane can achieve the convenient enrichment of large number of DNA strands.On the one hand,the effective enrichment of DNA strands by erythrocyte membrane can greatly enhance the efficiency of target strand cycling in multiple pairs of hairpin DNA reactions,thereby improving the response speed and detection sensitivity of the hairpin DNA cascade reaction.On the other hand,the better biocompatibility and the ability to resist non-specific adsorption of erythrocyte membrane also enable the sensing platform to maintain good reaction performance and stability in complex analysis environments,and realize the sensitive detection of miRNAs in real samples such as cell lysates.The construction strategy of this sensing platform can also be extended to other DNA strand cascade reaction systems.The construction strategy of this sensing platform greatly exerts the potential of erythrocyte membrane materials as confinement scaffolds,and is expected to provide new design ideas for the construction of novel spatial-confined DNA cascade sensing platforms.In order to solve the problem that traditional single-target biosensing is prone to serious false positive signals,we construct an integrated DNA logic gate nanomachines that can achieve the bispecific recognition,logic calculation and signal amplification imaging on the cell membrane surface.Using soft erythrocyte membrane material with good biocompatibility and large load as a platform,the integration of each element of the DNA logic system is realized.Thereby the efficiency of operation and signal amplification reaction in the DNA logic nanomachine is improved through confinement.In the DNA logic nanomachine,two kinds of recognition antennae based on aptamer are selected as the recognition elements to realize the accurate recognition of the target cell through dual proteins,and sequently achieve the logical computation of recognition information through the "AND" logic gate element based on DNA strand displacement.In addition,by combining hybridization chain reaction(HCR),the amplification of the recognition signal of low-abundance target proteins was achieved.This integrated bispecific "recognizing-biocomputing" DNA molecular logic machine realizes high-specificity identification and imaging of target cells.This strategy is expected to provide new ideas for biomedical applications of DNA molecular logic gates.Since metal ions play a vital role in many physiological processes,the understanding of the distribution and concentration fluctuations of metal ions in biological samples has always been a central topic in bioanalysis and biomedicine.Deoxyribozymes(DNAzymes)can catalyze the cleavage of their substrates with high selectivity and sensitivity in the presence of their metal cofactors,achieving the amplification of signal.Hence they are widely used to construct fluorescent probes to monitor various metal ions.However,traditional DNAzyme-based biosensors can respond to their metal cofactors during the delivery to the target site,resulting in serious false-positive signals.To solve this problem,we design and synthesize an activatable light-controlled DNAzyme probe.By replacing the RNA base in the cleavage site with 2’-O-nitrobenzyl adenosine to achieve photoprotection of the substrate strand,the activity of the probe is only restored upon exposure to 365 nm UV light,preventing false positive signal from the interaction of probe with the metal cofactors during delivery.At the same time,stable and efficient cell membrane anchoring can be achieved by modifying cholesterol molecules to light-controlled DNAzyme probes.The probes can realize in situ controllable detection of zinc ions in the cell microenvironment.Meanwhile,we applied a DNAzyme-based sensing method to monitor metal ion-induced Aβ aggregation in brain tissues.The strategies presented in this study are expected to expand the application of DNAzyme-based sensors for metal ion monitoring in complex biological samples and provide a deeper understanding of the role of metal ions in Alzheimer’s disease pathology.