Design Of Multifunctional Nanoprobes And Their Applications In Cell Analysis

Cell imaging is a very important analysis method in the field of disease diagnosis and treatment because it can monitor small molecules in living bodies in real time,on site,and clearly.In recent years,cell imaging methods based on nucleic acid-functionalized nanomaterials have been extensively studied.Due to their unique optical properties,good biocompatibility,and strong loading capacity,nucleic acid nanoprobes have been used in cell imaging and disease.The diagnostic and other fields have broad application prospects.However,because complex biological systems have the disadvantages of multiple components,low content,complex distribution,and multiple barriers,it is urgent to design smarter nucleic acid nanoprobes for time-space accurate biological detection to better realize the early diagnosis of disease.Based on the powerful composite nanoprobes and fluorescence imaging technique,the work mainly focuses on the construction of sensitive and stimuli-responsive intracellular detection probes.The details are summarized as follows:1.Target triggered self-powered DNAzyme-MnO₂ nanosystem:towards imaging miRNAs in living cellsThe self-powered nanosystems are receiving more and more attention due to their unique advantages(such as ultra-small size,environmentally friendly,sustainable supply,and high efficiency).The use of self-powered nanosystems for the sensitive detection and quantification of in situ genetic molecules,especially micro RNA(miRNA),is of particular importance.Based on MnO2 nanosheets and nucleic acid molecules,we designed a versatile DNAzyme-MnO2 nanosystem to achieve target-induced self-powered intracellular miRNA imaging.MnO2 nanosheets serve as a transport carrier and quencher,and are reduced to Mn²⁺after entering the cell.Mn²⁺can be used as a cofactor for the subsequent DNAzyme catalytic amplification reaction,and finally achieve ultra-sensitive miRNA imaging of different types of cells and can be used to distinguish tumor cells and normal cells.The nanoprobe integrated delivery capability,quencher ability,target recognition,self-supplying of cofactors and signal amplification all-in-one.These outstanding performances demonstrated the DNAzyme-MnO2 nanosystem as a promising self-powered nanosensor for future applications of monitoring disease-related genetic molecules in living cells and in vivo.2.Smart engineering of self-powered and entropy-driven DNA motor-MnO₂nanocomposite for intracellular micro RNA imagingMany DNA motors have been synthesized and operated in living cells during the past two years but there are still challenges to design more integrated DNA motor which are self-powered and initiated by specific molecules in living cells.Here,we report a smart strategy based on a DNA motor-MnO2 nanocomposite that meet these requirements and operated in living cells for specific miRNA imaging.This motor system once delivered into desired cells,the reduction of MnO2 nanosheets by intracellular GSH(glutathione)not only released the DNAzyme motor and then activated by intracellular target miRNA but also can generated a cofactor,Mn²⁺,which as fuels for the automomous and progressive walking.Furthermore,the false-positive signal generated by GSH on DNA motor destruction can also be greatly reduced due to the consumption of GSH during this process.Our motor system combines the advantages of gold nanoparticle-based motor with the merits of more integrated,lower background fluorescence,entropy-driven and self-powered.The smart design has been applied to intracellular miRNA imaging and will be applied to the detection of other intracellular small molecules.3.NIR remote-controlled“lock-unlock”nanosystem for imaging potassium ions in living cellsDespite great achievements in sensitive and selective detection of important biomolecules in living cells,it is still challenging to develop smart and controllable sensing nanodevices for cellular studies that can be activated at desired time in target sites.To address this issue,we have constructed a remote-controlled“Lock-Unlock”nanosystem for visual analysis of endogenous potassium ions(K⁺),which employed dual-stranded aptamer precursor(DSAP)as recognition molecules,SiO2 based gold nanoshells(Au NS)as nanocarriers and near-infrared ray(NIR)as the remotely applied stimulus.While triggered by NIR,the increased local temperature of Au NS induced the dehybridiztion of DSAP,realized the“Lock-Unlock”switch of the DSAP-Au NS nanosystem,activated the binding capability of aptamer,and then monitored intracellular K⁺via the change of fluorescence signal and the outflow of K⁺in He La cells at any time within 3 h under drug stimulation can be monitored.This DSAP-Au NS nanosystem not only allows us to visualize endogenous ions in living cells at desired time,but also paves the way for fabricating temporal controllable nanodevices for cell research.4.Intracellular microenvironment responsive nanoprobe for imaging multiple metal ions in living cellsMetal-assisted deoxyribozyme catalysis(DNAzyme)has been a general platform for constructing highly sensitive and selective detection sensors of metal ions.However,the“always on”mode of the traditional DNAzyme sensors greatly limits their application in visual analysis of endogenous metal ions in complex physiological microenvironment.To overcome this obstacle,a smart acid-switchable DNAzyme nanodevice is designed for controlling DNAzyme activity in living cells and achieves simultaneous visualization of metal ions(Zn²⁺and Pb²⁺)in situ.This nanodevice ensures that the DNAzyme is locked in the extracellular environment by introducing p H-sensitive DNA nanostructures,and its activity is inhibited.After entering the cells,it is restored to its activity after being driven by the acidic p H in the cell's acidic organelles,and finally realizes the metal ion(Zn²⁺and Pb²⁺)visual analysis at the same time.Moreover,this strategy has broad prospects as a powerful platform for constructing various acid-switchable nanodevices for visual analysis of multiple biomolecules in living cells.5.Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cellsWith the development of brain science,the transmission of information in the nervous system has attracted widespread attention.The basic element of information transmission in the nervous system is the transmission of electrical and chemical signals.In this work,we proposed an approach coupling ultra-thin microelectrode array(MEA)with total internal reflection fluorescence microscopy(TIRFM),which served as a powerful platform to visualize both AP and vesicular exocytosis in a neuronal circuit model formed by neuron-like PC12 cells.Taking advantages of fluorescent false neurotransmitter(FFN),the transient neurotransmitter transport down an axon could be visualized with high spatial and temporal resolution.The MEA/TIRFM platform proved that neuronal PC12 cells have AP-dependent release and AP-independent release after acute hypoxic stimulation.This work provides a new platform for deeper research of the nervous system information transmission mechanism.