Controlled Preparation And Biological Applications Of Nucleotide-protected Metal Nanoclusters

Metal nanoclusters(MNCs)is a novel nanomaterial composed of metal atoms from several to hundreds.Due to their excellent optical,electrical,magnetic and other properties,they have been widely used in analysis and detection,biological imaging,and even catalysis.The structure of nanoclusters is composed with metal core and ligands.By constructing alloy nanoclusters,the composition of metal core can be changed to adjust the spatial structure selectively and their physical and optical properties.Compared with high quantum yield(QY)materials such as quantum dots(QDs),MCs have great limitations in applications.In addition,the protected ligands attached to the surface of MCs,and the free functional groups,potential properties and toxicological properties play the decisive role in the design and application of MNCs.Therefore,it is of great significance to develop novel nanoclusters with high biocompatibility and improve their fluorescence and catalytic properties to solve their limitations in the application field.In this paper,different types of nucleotides with rich structure and high biocompatibility were used as protected ligands to prepare MNCs,and the strategy of aggregation induced emission enhancement(AIEE)was used to improve the fluorescence performance of nanoclusters protected by adenosine monophosphate(AMP).Then,the assembly was applied to the detection of amino acids and protein.Cytidine monophosphate(CMP)-protected gold nanoclusters were prepared by substituting ligand structures and leaving the phosphate group freely outside on the surface of nanoclusters.Therefore,it was used as a direct fluorescent substrate for the quantitative detection of alkaline phosphatase(ALP)activity.Furthermore,the catalytic activity of guanosine monophosphate(GMP)-protected gold nanoclusters was further improved by doping platinum atoms to prepare a high-performance oxidase-like catalyst.Firstly,the fluorescence properties of the adenosine monophosphate-protected gold and silver nanoclusters(Au-Ag NCs@AMP)were improved by constructing assembly to expand their applications in detection.In the presence of Al(?),the dispersed nanodots are merged into large spherical particles with?50 nm in diameter and the fluorescence of Au-Ag NCs@AMP was enhanced and blue shifted.It was confirmed by transmission electron microscope(TEM)that the Au-Ag NCs@AMP transformed into a large assembly,AuAg NC-Al(?).And the enhancement of fluorescence was attributed to AIEE property.The fluorescence of AuAg NC-Al(?)was quenched by cysteine(Cys)and exhibited a high selectivity among 20 different kinds of amino acids.The method can be used for the quantitative detection of Cys in the range of 1.0–16.0?M and the limit of detection(LOD)is 50 n M.The fluorescence probe has a high recovery rate in Cys-spiked mineral water and serum.The fluorescence quenching mechanism was further explored that the partial replacement of ligand AMP by Cys at the surface of Au-Ag NCs@AMP,and the morphology was reassembled from the spherical particles into strips.In addition,the fluorescence of the assembly enhanced significantly by plasmodium vivax lactate dehydrogenase(Pv LDH),a crucial biomarker of malaria,enabling the quantitative detection of Pv LDH.Al(?)was employed as an“assistant agent”,which can significantly improve the identification of the probe,enables it to distinguish the Pv LDH from other LDHs.Further studies showed that the bimetallic nanoclusters inhibited the activity of Pv LDH,suggesting it bound near the active site of Pv LDH with high affinity.Zeta potential and UV-vis absorption showed that there was a strong interaction between Au-Ag NCs@AMP and Pv LDH.The chemical modification by maleimide showed that the free thiol groups in Pv LDH also participated directly and played a key role in the process.Therefore,the interaction between Au-Ag NCs@AMP and Pv LDH are mainly driven by electrostatic interaction and bind to the free thiol groups close to the active site of Pv LDH.In conclusion,we have greatly improved the luminescence property of MNCs and expanded the application through supramolecular assembly.Secondly,the cytidine monophosphate-protected gold nanoclusters(AuNCs@CMP)was prepared by ligand substitution and applied as a direct fluorescent substrate for the detection of ALP activity.The structural characterization of AuNCs@CMP showed that the ligand CMP binds to the gold core via two oxygen atoms of ribose and cytosine,and the phosphate group was freely distributed outside of the nanoclusters,which provides a direct-action site for ALP hydrolysis.In particular,the fluorescence emission of AuNCs@CMP was located at 570 nm,while the cytidine-protected gold nanocluster(AuNCs@Cyt)was located at 485 nm,which laid the foundation for ALP activity detection.When the ALP was introduced into AuNCs@CMP,a new fluorescence emission is indeed generated at 485 nm.In addition,chitosan oligosaccharide(COS)was introduced,which significantly amplified the fluorescence signal of the hydrolysate(AuNCs@Cyt),resulting in a LOD for ALP with 0.00026 U·L^–1.The studies in mechanism showed that the fluorescence enhancement of AuNCs@Cyt induced by COS was attributed to the AIEE property.In addition,the method can be applied to the quantitative detection of ALP in diluted human serum with a good recovery.In this study,a novel nano-substrate was developed for enzyme activity detection,which expanded the biological function and application of MNCs.Thirdly,guanosine monophosphate-protected gold and platinum nanoclusters(Au-Pt NCs@GMP)have been prepared by doping platinum atoms,which show a high catalytic activity for the oxidation of substrates 3,3?,5,5?-tetramethylbenzidine(TMB)and 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS).The initial maximum velocity(V_max)for TMB was253.8×10^-8 M s^-1,which was the highest values among the reported oxidase-like catalyst.The in-depth mechanistic studies by ¹H and ³¹P NMR spectra,transmission electron microscope(TEM),X-ray photoelectron spectroscopy(XPS)of the optimized Au-Pt NCs@GMP reveal them to be of ultrasmall size with a unique ligand structure,especially the synergistic effect between gold and platinum endow the product with a high catalytic performance.Therefore,this study provides a novel nano-catalyst with high oxidase-like activity,and proves that the doping of platinum atoms can improve the catalytic activity of gold nanoclusters through synergistic effect.Fourthly,the catalytic activity of AuNCs@CMP was further investigated.In the presence of H₂O₂,the V_max for TMB catalyzed by AuNCs@CMP was calculated to be 174.83×10^-8 M?s^-1.In addition,the AuNCs@CMP can catalyze the production of reactive oxygen species(ROS)in the presence of H₂O₂ in vitro.In addition,both the tumor cells of He La and SW480 can activate and promote AuNCs@CMP,leading to the generation of ROS while the normal cells 293T produce a small amount of ROS.Further studies of MTT and Flow cytometry displayed that the AuNCs@CMP showed higher cell apoptosis in He La and SW480 cells.The in-depth studies in mechanism reveal that the weak acidic environment and abundant of H₂O₂ in tumor cells are the fundamental to stimulate AuNCs@CMP to produce plenty of ROS.Therefore,this study explores the high peroxidase-like activity of MNCs,which catalyze the production of large amounts of ROS and kill the cancer cells in the tumor microenvironment(TME).