In-situ Precise Synthesis Of Gold Cluster Nanoenzymes Based On The Confined Space Of Protein Assemblies

Gold cluster nanoenzymes consist of a few to about a hundred of gold atoms,with the diameter of < 2 nm approaching the Fermi wavelength of an electron.Due to ultra-fine size and special structure,gold cluster nanoenzymes exhibit many excellent properties(e.g.catalysis and fluorescence)and have wide application prospects in the fields of enzyme catalysis,biological detection and biological imaging.Generally,gold cluster nanoenzymes are composed of gold nuclei and shell ligands.How to precisely control the growth process of gold nuclei by rational design of ligand molecules is the hardest task in the preparation of gold cluster nanoenzymes.Proteins that have well-defined spatial structures and highly versatile surface properties are considered to be an ideal template in the direct synthesis of gold cluster nanoenzymes,which has become a research hotspot in this field.However,proteins are easily denatured to lose their natural structure and biological activity in response to external factors,making the gold cluster nanoenzymes preparation process difficult to control.The nucleation growth mechanism is also unclear.Therefore,the development of new methods for the controlled synthesis of gold cluster nanoenzymes based on protein templates is extremely challenging.In this paper,a new method has been proposed for the precise synthesis of gold cluster nanoenzymes in situ using the confined space of thermal stable protein(SP1)assemblies.SP1 is a ring-like shaped dodecamer with the inner diameter of about 2.5 nm.By adding a CCY tripeptide sequence to the N-terminal of SP1 protein(CCY-SP1),the gold cluster nanoenzymes CCY-SP1@Au NCs were formed by the synergistic effect of cysteine and tyrosine to achieve the chelation,localization,and in-situ reduction of gold ions.Transmission electron microscopy(TEM)and X-ray energy spectroscopy(EDS)confirmed that uniform-sized gold cluster nanoenzymes were observed in the cavity of CCY-SP1 protein assemblies with the diameter of 2 nm.The results of circular dichroism(CD)showed that the protein in gold cluster nanoenzymes CCY-SP1@Au NCs still retained the original secondary structure without denaturation.Enzyme assay revealed that CCY-SP1@Au NCs have horseradish peroxidase(HRP)-like activity and a ping-pong mechanism,with a wider p H-and temperature-dependent activity than natural enzymes.In addition,gold cluster nanoenzymes exhibited obvious red fluorescence and the average fluorescence life up to microsecond.The influence of protein ligands on the properties of gold cluster nanoenzymes was further investigated and the results indicated that the structural change of CCY-SP1 protein can regulate the fluorescence intensity and the horseradish peroxidase(HRP)-like activity of gold cluster nanoenzymes.