| Ultrasensitive and accurate acquisition of cellular biological/chemical information through molecular imaging is of utmost significance to elucidate the detailed mechanisms of physiological and pathological processes.However,the traditional fluorescence probe that based on signaling occurs in an equivalent reaction ratio can not used for detecting the low-abundance biomolecule,so realizing the in situ signal amplification in living system is critical for acquiring the signal.More and more amplification systems have been reported for biosensing,but most of them are only suitable for in vitro assays owing to the dependency of assistant tools,such as enzyme,also,the reported work that realizing the intracellular signal amplification was limited for ss DNA detection.Therefore,it is important to develop the intracellular signal amplification strategy that with the endogenous protein as signal-amplifier and used for detecting other active biomolecules.In this acticle,with the intracellular protein as signal-amplifier and the mesoporous silica nanoparticle or micelle as nano-carrier,a series of signal-amplifying fluorescent nanoprobes were fabricated.The intravital imaging studies demon strate that the cytoplasmic protein-self-helped signal amplification strategy can be used for detecting various low-abundance targets,which will solve the problems exist in signal amplification.1)Construction of PEI-?CDP/SQ@BHQ2 Near-infrared fluorescent probe for detecting?OHThe solvent-sensitive dyes are a class of small organic molecules that can change the chemical property in different solvent.We have synthesized a series of squaraine dyes with similar molecular structure and investigated the optical properties in buffer and buffer with FBS.Then we select one of the SQs which can enhance fluorescence intensity in buffer solution with FBS.O n the basis of interaction between SQ and PEI-?CDP,we fabricated PEI-?CDP/SQ@BHQ2 by electrostatic adsorption between BHQ2-ss DNA and PEI-?CDP.While encountered with?OH under complicated conditions,BHQ2-ss DNA will be cleaved to liberate the BHQ2,thus resulting in light the fluorescence to detect the?OH.In this section,the elect fluorophor SQ1 can enhance the fluorescence intensity in organic phase,also,PEI-?CDP/SQ@BHQ2 can respond?OH demonstrate that the ss DNA can be designed as a trigger,which establishes the foundation for the second work in this paper.2)Cytoplasmic protein-assisted in situ fluorescence amplification for ultrasensitive imaging of hydroxyl radical generation in vivo?OH is the low-abundance(?27?10-9 mol/L)and short-lived??27?10-1212 s?chemical inimical and with high reactivity,exploration of the behaviors and functions of this crucial species in biosystems is certainly desirable.We investigated the mechanism of interaction between SQ and protein and designed a f luorescent signal-amplifier.The signal-amplifier was designed with the mesoporous silica nanoparticle?MSN?as the protective vessel to load SQ and further quenching the fluorescence of SQ trapped in pores with appropriate blocker.When the probe was used in vivo,single target molecules can open the pore and lead to release of a large amount of S Q,the free dyes restore the fluorescent signal resulting in the first step of amplification.Next,the unrestrained SQs can bind with cytoplasmic protein in cell and exhibit a large fluorescence enhancement for the in situ duplex signal amplification.Because of this delicate design,the probe exhibits high sensitivity than other fluorescence probes.Aslo,we investigated the generation of?OH in cells with this probe,which have been proved to imaging the?OH in vivo.3)Constructionofcytoplasmicprotein-assistedquadratic signal-amplifying nanoprobe for detecting hypoxia and Imaging of Inflammation in vivoHypoxia represents a physiological status that the oxygen levels decreased and cannot support metabolism,which share a correlative relationship with inflammation and is a typical hallmark of it.However,at the present stage,the hypoxia-response fluorescence probe is limited by low sensitivity and cannot be used for inflammation imaging.To solve this problem,herein,we design a hypoxia-activated signal amplifying near-infrared fluorescent probe by using biological matrix to amplify the fluorescence signal.This probe has been proved that not noly imaging the hypoxia in cells,but also can monitor the development of acute inflammation in mice model.As far as we know,this is the first effort to image inflammator y region with hypoxia condition as the target based on the signal amplification strategy,which solve the problem in the inflammation imaging.4)Construction of cytoplasmic protein-assisted mulriple signal-amplifying nanoprobe for imaging the intracellular m RNA:avoiding the singal fluctuations under rigorous environmentThe intracellular signal amplification that based on the nucleic acid hybridization chain reaction will be faced with a serious problem,which is the signal fluctuation in the complex intracellular environment.To this point,we design a mulriple signal amplifying probe that with MSN as vector and SQ as signal reporter,simultaneously the hairpin DNA probes that can react with target as blockers.The presence of target can initializes the hybridization between the blockers,and then the duplex conformation will release from MSN and lead the SQ bind with protein to amplify the signal.We have demonstrated that the probe can imaging target in cells with high sensitivity.More importantly,compared with other intracellular signal amplifying probes,this probe that with the SQ as s ignal amplifier unit can remain the signal stable for a long time,which is because the nuclease can not degradate the protein.With intracellular protein as signal-amplifier and nanocarrier to load signal reporter,numerous signal-amplifying fluorescent nanoprobes were fabricated for detecting the low-abundance targets,which solve problem that other signal-amplifying fluorescent nanoprobes'dependency of assistant tools,and this is important for signal-amplifying fluorescent nanoprobes used for biosnesing. |
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