New Methods For The Preparation Of Gold Nanoclusters/Nanoparticles And Their Applications In Bacterial Detection

Gold nanomaterials,the earliest reported nanomaterials,have the advantages of simple preparation,good biocompatibility,easy surface modification and stable property,and have been widely used in biomedical analysis and imaging.Gold nanoclusters(Au NCs)are new luminescent materials in recently years.Gold nanoclusters have molecule-like properties,good biocompatibility and strong fluorescence.Thus,they will be of wide applications.However,the quantum yields of most gold nanoclusters are very low.We prepared one kind of gold nanoclusters with high quantum yield and a kind of vancomycin-modified gold nanoparticles by new methods,and established new colorimetric bacterial assays.The main contributions are as follows:1.Syntheses of strongly luminescent,highly heat-stable gold nanoclustersWe prepared a kind of gold nanoclusters(Trp-c AMP-Au NCs)with the quantum yield as high as 59.6% by hydrothermal reaction with adenosine cyclic monophosphate(c AMP)as template molecule,tryptophan(Trp)as function ligand and chloroauric acid in a weak acid medium.The fluorescence of Trp-c AMP-Au NCs has no change under 120? temperature.The quantum yields of most gold nanoclusters reported so far are less than 6%,and these nanoclusters lost their fluorescence at high temperature.Our method provides a new idea for the preparation of gold nanoclusters with intense luminescence.These highly heat-stable gold nanoclusters can be applied to the fluorescence labeling of DNA polymerase chain reaction(PCR),promoting the application of metal nanoclusters in the high temperature reaction.2.Investigation on the mechanisms of fluorescence enhancement and heat-resistance of gold nanoclustersFor explaining these phenomena,we used adenosine monophosphate(AMP),adenosine diphosphate(ADP)and adenosine triphosphate(ATP)as analog of template molecule to replace c AMP,used tryptophan-like tryptamine to replace tryptophan,respectively.It was found that the phenomenon of fluorescence enhancement is still observed,indicating that the template molecule and tryptophan were not the main factors for the fluorescence enhancement of gold nanoclusters,and that tryptophan's contribution is little to the fluorescence enhancement of gold nanoclusters through its energy transfer,which is only a secondary factor.Under the condition of no hydrothermal reaction,the fluorescence cannot be enhanced.The hydrothermal condition is an important factor for the strong luminescence of these gold nanoclusters.The results show that the gold atoms rearrangement caused by the hydrothermal reaction and thus the formation of a more compact structure of gold nanoclusters result in the intense luminescence.This is the first case of hydrothermally-induced emission enhancement of Au NCs.Our finding offers a new way for the fabrication of strongly luminescent metal nanoclusters for wide applications.On the basis of the finding that tryptophan molecules have a good protective effect on gold nanoclusters,we investigated the mechanism of heat resistance of the gold nanoclusters.Tryptophan was replaced by tryptamine,indole,3-indoleacetic acid,3-methylindole and indigo.The results show that the high temperature resistance of the gold nanoclusters is a result of tryptophan's high thermal decomposition temperature and its strong interaction with gold atoms.The highly heat-stable Trp-c AMP-Au NCs is promising for the labeling in high temperature reaction like PCRs.3.The interaction of bovine serum albumin stabilized gold nanoclusters as fluorescent labels with different bacterial cellsBacterial detection is of great significance in the field of public health,and the detection of bacteria by using fluorescent dyes/materials in combination with fluorescent microscopes is the most commonly used detection method.The gold nanoclusters modified by the protein and the polypeptide as the stabilizer have good biocompatibility,and have good application potential in the field of bioanalysis.We used bovine serum albumin as a protective agent to prepare the fluorescent gold nanoclusters(BSA-Au NCs),and to interact with different species of bacteria(S.aureus,M.luteus,B.subtilis,E coli,P.vulgaris,and P aeruginosa).The absorption capacity of various bacterial cells on the BSA-Au NCs was evaluated,and the factors affecting the absorption of fluorescent gold nanoclusters by microbial cells were also analyzed.This research laid a foundation for developing fluorescent gold nanoparticles for the identification of bacteria.4.Colorimetric assay of Gram-positive bacteria by using vancomycin-modified gold nanoparticlesVancomycin-modified gold nanoparticles(Van-Au NPs)were synthesized by a simple one-step reaction with vancomycin(van)and chloroauric acid.In this reaction,vancomycin is both a reducing agent and a protective agent for gold nanoparticles.Although many papers for the preparation of gold nanoparticles modified with small molecule have been reported and the antibiotic vancomycin is widely used,however the direct modification of gold nanoparticles with vancomycin via a one-step reaction has never been successfully achieved.We tried various reaction conditions,and the prepared Van-Au NPs have very high activity,can be specifically combined with the Gram-positive bacteria,and can distinguish the Gram-positive bacteria and the Gram-negative bacteria by their color change in the solution.On this basis,a visual colorimetric assay using Van-Au NPs and silver staining reagent was established for Gram-positive bacteria.The detection limits of bare eyes for Gram-positive S.aureus,M.luteus,and B.subtilis are 1×10~9,1×10~9,and 1×10~9 cells/m L,respectively.This assay is rapid and easy,without the use of any microscopic devices,and is promising to be used as a rapid method for the identification and quantification of bacteria in clinics,environment,and fieldwork.