Construction Of Self-Assembled Peptide-Gold Nanoclusters And Its Application For Analyzing Of Intracellular Protease Activity

In natural biological systems,the folding and assembly behaviors are the most common phenomena for biomolecules such as peptides and proteins,which play an important role in physiological functions.This feature also provides powerful inspiration to design biological nanomaterials with specific structures.Recently,the self-assembly of gold nanoclusters protected by peptides has become a hotspot due to its precise structure,maneuverability,stability,and atomically precise assembly direction and mechanism.Designable polypeptide ligands can introduce amino acid sequences with specific functions to achieve excellent targeting.They have received more and more attention in biomonitoring and bioimaging applications.In this thesis,self-assembled gold nanoclusters are constructed based on polypeptide ligands,and a specific cleavage sequence of Asp-Glu-Val-Asp(DEVD)was introduced to response to Caspase-3 protease in apoptotic tumor cells.Specifically,three main aspects of this thesis were carried out as follows:(1)Self-assembled gold nanoclusters(Ac-CG-Au-NCs)with a linear structure of nanofibers were constructed based on N-acetylated dipeptide(Ac-CG)ligands.Through the optimization of synthesis conditions,and structural characterization and analysis,it can be confirmed that the sulfhydryl group on the ligand Ac-CG can reduce Au ions,and then Au(I)-SR complexes-based clusters can be assembled into nanofibers or nanoribbons through metallophilic interaction,hydrogen bonds,and π-π stacking provided by side chain groups to produce aggregation induced emission.In addition,due to the host-guest interactions between hydroxypropyl-β-cyclodextrin(HP-β-CD)and Ac-CG-Au-NCs clusters,the inter-cluster forces are strengthened to enhance fluorescence intensity.(2)The self-assembly of gold nanoclusters exhibits pH-responsive features.The fluorescence intensity gradually decreases with the increasing pH value,with a good linear relationship between 5.0 and 6.8.This is determined by the morphological dependence of the assemblies,i.e.,the clusters self-assemble at acidic pH and are luminescent,while they disassemble at neutral pH environment,accompanied with the disappearance of the fluorescence.The self-assembled gold nanoclusters show low cytotoxicity and can be used to localize lysosomes in He La cells by fluorescence imaging.(3)The peptides Ac-CGDEVDF,Ac-CGDEVDFF,which contain Caspase-3 protease cleavage sequence(DEVD)are designed.In the specific enzymatic cleavage experiments,the linear structure of self-assembled gold nanoclusters changes and disassembles,with simultaneous reducing fluorescence intensity,confirming that they can be specifically cleaved to induce changes in the self-assembled structure.In the apoptotic model of He La cells,the self-assembled gold nanoclusters showed significant decrease in fluorescence signal after coincubation with apoptotic cells due to the high expression of intracellular Caspase-3 protease,confirming their recognition for intracellular protease activity.