Synthesis Of Novel Two-photon Excitited Nanoprobes For Caspase-3 Activity Imaging Assay

Apoptosis is an important physiological process that occurs either during the course of development and the normal maintenance of homeostasis,or in pathological circumstances including cancer,neurodegenerative diseases,autoimmune disorders,and heart failure.Cell apoptosis is also one of important mechanisms for transplanted liver ischemia reperfusion injury,and hepatic ischemia-reperfusion injury is an important factor to influence the effects of liver transplantation what has become an effect means for end-stage liver disease.Caspase-3 has been identified as the most important mediator among caspase family for the initiation and propagation of apoptosis.Consequently,the development of intracellular protease sensors toward caspase-3 is essential for understanding the roles of this protease in physiological and pathological processes as well as for therapeutic effect of cancer treatment or screening the new drug candidates for dysregulated apoptosis.Many methods for the analysis of caspase-3 activity,including chemiluminescent detection,bioluminescence detection,Western blot assay,colorimetric assay,fluorescence resonance energy transfer(FRET)-based fluorometric assay,flow cytometric-based analysis,atomic force microscopy,and electrochemical methods.Among these developed technologies,the FRET mechanism based detections have taken a crucial place due to the excellent properties of the fluorescence detection,such as high sensitivity,specificity,fast response,and technical simplicity.Nanomaterials possess good biocompatibility and surface plasmon resonance,which make them appropriate fabrication materials of novel biosensors as a carrier and act as a receptor or donor for quenching fluorescence in the FRET donor and receptor energy pairs.Two-photon fluorescent dyes as a fluorescent molecule,comparing to one-photon fluorescent dyes,with low energy near-infrared light as the excitation source,there are many advantanges such as lower tissue autofluorescence and self-absorption,reduced photodamage and photobleaching,high spatial resolution and deeper penetration depth,etc.Based on this,the research work of this paper is as follows:1.We labeled two-photon dye(TPdye)EBMVC on peptide sequence Cys-Ala-Asn-Leu-Asn-Asp-Glu-Val-Asp-Lys(CANLNDEVDK)contain the caspase-3 specific recognition sequence Asp-Glu-Val-Asp(DEVD),based on the combination of carboxylated oxide graphene(GO-COOH)and the peptide-TPdye containing-NH2 group through amidation reaction,the GO-COOH/peptide-TPdye nanoconjugate was designed as a two-photon nanoprobe to detect the presence of apoptosis protease caspase-3 in virto.First,we modified the GO by chemical method using strong acid,and got characterizations of the GO-COOH.Then,we evaluated the different quenching efficiencies of the GO and the GO-COOH when they impacted on the peptide targeted EBMVC.In the absence of caspase-3,the system kept a lower background fluorescence because of the efficient fluorescence quenching efficiency of GO-COOH.When caspase-3 was added to the system,caspase-3 cut the peptide at the cleavage site DEVD between D and Kfrom D and lysine K,and the TPdye EBMVC was free to the system and the fluorescence was restored,what could achieve the detection of apoptotic protease caspase-3 in vitro.2.Based on the gold nanoparticles(AuNPs)and peptide with free-SH can form a stable spherical structure via the Au-S bond,we develop a detection method of caspase-3 in vivo through AuNP/peptide-EBMVC conjugate as a two-photon nanoprobe.The fluorescence emission spectra of EBMVC have a large overlap with the fluorescence absorption spectra of AuNPs.The FRET effect occurs when the distance between the energy donor and acceptor was sufficient close(7 nm?10 nm),and the two-photon fluorescence of the peptide was quenched.When the AuNP/peptide-EBMVC nanoconjugate probe reacted with caspase-3,caspase-3 specifically cut the peptide chain at DEVD between D and K,the peptide chain fragment targeted with EBMVC away from AuNPs,FRET effect was destroyed and the fluorescence of TPdye EBMVC recovery.The system achieved fluorescence imaging of cells and tissues during apoptosis period.