Enzyme Instructed Self-Assembly Induced Excimer Formation And Its Applications In Bioimaging

How to induce excimer formation of fluorophores at low molecular concentrations and how to construct high-performing excimer-based luminescent analytic tools in the dynamic and complex biological environments remain challenges.Here,we first reported that enzyme instructed self-assembly(EISA)enabled the monomer-excimer transition of a coumarin dye(Cou)at low molecular concentrations,and the resulting higher ordered luminescent supramolecular assemblies(i.e.,nanofibers)efficiently recorded the spatiotemporal details of alkaline phosphatase(ALP)activity in vitro and in vivo.During the laser scanning confocal microscopy,the unconjugated Cou could be well excitated by a laser(405 nm)and its monomer fluorescence could be well collected due to its absorption wavelength λabs=409 nm and emission wavelength λem=470 nm.However,no Cou excimer fluorescence could be detected even at an 8 mM molecular concentration.This result might be associated with the formation of the twisted intramolecular charge transfer(TICT)state in the unconjugated Cou and the steric hindrance caused by the 7-diethylamino group in the unconjugated Cou.To some extent,this also helped to explain why the report about Cou excimer in solutions is rare.Conjugated Cou to short self-assembly peptides with a hydrophilic ALPresponsive group that could be cleaved by external or endogenous ALP yielded precursors pYD and pYDD,both of which could emitted Cou excimer fluorescence(λabs=430 nm,Xem=575 nm)at high molecular concentrations.After ALP treatment,the amino acid pY in the precursors was converted into Y,which greatly increased the self-assembly ability of the precursors and leaded to aggregates/nanoparticles-nanofibers transition of precursors that were dissolved in phosphate buffered saline(PBS,pH=7.4).The Cou group within the nanofibers adopted H-type stacking and the concentration-dependent fluorescent spectra showed that Cou excimer formed in solutions at low molecular concentrations.Structureproperty relationship revealed that the self-assembly motif was a prerequisite for peptides to induce the monomer-excimer transition of Cou.The results of fluorescent spectra confocal images,cellular ALP activity inhibiting experiments,and real-time cellular ALP activity tracking experiments showed that pYD rapidly,selectively,and quantitatively responded to external or cellular endogenous ALP activity.The imaging signals ofp YD increased exponentially(≈10000-folds)compared to those of the unconjugated Cou,and the imaging signals and selectiveness of pYD also increased significantly in contrast to its control peptides without in-situ EISA ability(Ac and YD).pYD illuminated the intercellular bridge membrane whereas they disassembled intracellularly and subsequently traversed the bridge including the midbody,a densely packed structure that many immunofluorescent antibodies cannot penetrate.pYD served as an efficient ALP imaging tool that directly identified cancer cells(tissues)from normal cells(tissues),which could be a feasible tool for early diagnosis of cancer.This work,for the first time,demonstrated that EISA induced excimer formation of a fluorophore at low molecular concentrations and that the in-situ EISA constructed high-performing excimer-based luminescent analytic tools in the dynamic and complex biological environments,thus extending the functions of EISA and providing a new application of supramolecular chemistry.