Construction Of Enhanced Hydrogen Bond-Induced Signal Amplification Nanoprobes And Application In Imaging Of Low Abundance Of Enzymes

In situ signal amplification strategy is an effective mean to detect low-abundance of endogenous targets.However,most of signal amplification strategies rely on exogenous substances,which change the intracellular microenvironment and cause certain interference to the body.ATP is the main energy source for cell life activities,and the concentration maintain stable in cells.Therefore,ATP is an ideal endogenous molecule for signal amplification.At present,several strand-displacement cascade amplification strategies based on ATP/aptamer have been developed for sensing low-abundance miRNAs in cells.However,the operation of strand-displacement cascade amplification was depended on the ATP/aptamer binding.But the interaction between ATP and aptamer was unstable and easily weaken by biomolecule(e.g.,enzyme and proteins)in complicated intracellular microenvironment,causing limited operating efficiency of signal amplification.It is shown that multivalent binding can significantly improve the binding affinity between molecules.With this considertation,and inspired by the intermolecular interactions between ATP and signaling reporter molecules,we proposed an enhanced hydrogen bond-induced fluorescence amplification(EHFA)strategy in this work.As a proof-of-concept,we constructed an enhanced hydrogen-bond-induced signal amplification nanoprobe to monitor the fluctuation of fibroblast activation protein(FAP)in activated hepatic stellate cells.The specific work of this paper shown as follows:Synthesis and screening of enhanced hydrogen bonding fluorescent dyes.In this work,we screened out an enhanced hydrogen bond fluorescent dye(ABF1)from 14 compounds,which can rapidly bind with ATP to enhance fluorescence intensity.Firstly,we designed ABF1 as a small molecule with rotatable arms to reduce its background fluorescence.Secondly,melamine was introduced to enhance its hydrogen bonding ability for ATP.Finally,to further verify the binding mechanism of ABF1 to ATP,we synthesized two other dyes(ABF2 and ABF3)with different degrees of protonation.The experiments show that ABF1 enhances intermolecular binding affinity by increasing the hydrogen bonds.Application of enhanced hydrogen bond-induced signal amplification probes.Based on the previous work,we constructed an enhanced hydrogen bond-induced signal amplification nanoprobe with fluorophore,BHQ2 and polymer,which was used to-image of FAP in activated hepatic stellate cells.The synthesis of polymers and construction of nanoprobes were characterized by Fourier transform infrared spectroscopy,NMR and gel permeation chromatography.Experimental results showed that the nanoprobe can respond to FAP with high sensitivity and specificity,and its detection limit is as low as 0.304 ng/mL.The cell imaging results showed that this nanoprobe can monitor the changes of FAP concentration in activated hepatic stellate cells.