Synthesis Of Metal Clusters And Their Applications In Biological Analysis

As a new type of small-size nanomaterials,metal nanoclusters usually contain few to hundreds of metal atoms.Comparing with the conventional nanomaterials,the nanoclusters possess the different physicochemical properties,such as their precise molecular composition,good fluorescence properties and enzyme-like catalytic activity.The prepared nanoclusters using the biomolecular as a template usually occupy good biocompatibility,fluorescence tunability and catalytic activity.At the same time,the prepared nanoclusters could maintain the biological activity of biomolecules.Therefore,it has attracted extensive attention in the field of bioanalysis.In this research,combined with previous reported work in our lab,two different fluorescent signals Au nanoclusters were prepared with the same ploypeptide,respectively.The prepared Au nanoclusters was further modified with a KL Peptide which specific targeting the HER-2 receptor protein in order to selective inhibition of HER-2 high expression breast cancer cell lines.For another research,copper and platinum clusters with fluorescent signal and bacterial inhibitory activity were prepared on the basis of copper and platinum elements,using egg white protein as a template by optimising experimental conditions,and their biological effects were initially investigated..The main contents of the experiment are as follows:(1)Development of an integrated probe for the diagnosis and treatment of HER-2 overexpressed breast cancerHER-2 receptor protein is an important factor in the prognosis of gastric and breast cancers,and is overexpressed in 20-30% of patients with malignant breast cancer.Therefore,the development of novel anti-tumour drugs that specifically inhibit HER-2 receptors in tumours with high HER-2 receptor expression is important for tumour prevention,control and treatment.Based on two Sv-Au NCs with different fluorescence emission signals synthesized using the Sv peptides reported by our group previously as templates,the KL6 peptide with the function of targeting epidermal growth factor receptor 2(HER-2)was covalently bound to the prepared Sv-Au NCs cluster material by a bio-covalent coupling method.The size and morphology of the materials were characterized by DLS and HRTEM,the molecular composition of the materials was characterized by MALDI-TOF-MS,the enzyme-like catalytic activity of the materials was investigated by TMB catalysis,and the biosafety of the materials was initially evaluated by CCK-8 cell viability test and hemolysis test.The targeted property of the materials was investigated by the constructed SPR biosensor.Finally,we employed the different tumor cell models,SK-Br-3,MDA-MB-231,MAC-145 and MCF-7,with different expression level of Her-2 receptor protein to investigate the inhibition effect to tumor.The experimental results showed that the KL6-Sv-Au NCs nanoclusters drugs can maintain the reductive sulfhydryl group of some KL6 peptides,and the enzyme-like catalytic activity of the gold cluster materials.The prepared KL6-Sv-Au NCs cluster also showed good selectivity for the SK-Br-3 cell line which with high expression of the HER-2 receptor.Cell viability and hemolysis test assays showed the prepared possessed good biosafety of the prepared clusters.The results of the tumour cell inhibition assay showed that the gold cluster probes modified by the KL6 peptide with different fluorescent signals specifically inhibited HER-2 overexpressing SK-Br-3 cells,while showing good biosafety against other tumour cells and normal cells.This work provides new options and ideas for the development of safe and highly available drugs for the treatment of cancers with HER-2 receptor overexpression.(2)Preparing of metal nanoclusters with egg white protein and their bioeffectTraditional biological template molecules to synthesize metal nanocluster usually focus on functional proteins and functional polypeptides.Commercial functional proteins and peptides have the disadvantage of being expensive and limited in source.Egg whites as an important protein resource occupy the advantage of being cheap and easy to obtain.Therefore,it is good choice to explore the preparation of different metal cluster probes using the egg whites protein.Herein,copper clusters with blue fluorescence emission(?ex = 330 nm,?em = 410 nm)and platinum clusters with green fluorescence emission(?ex = 380 nm,?em = 450 nm)were synthesised by optimising the experimental conditions using the egg whites of commercially available eggs.The size and morphology of the material were characterized by DLS and HRTEM,and the material was characterized by fluorescence spectroscopy,ultraviolet spectroscopy,and infrared spectroscopy.The optimal enzymatic activity of the material was investigated by optimising the conditions for TMB-catalyzed colour development experiments.The anti-tumour effect of the platinum clusters was initially evaluated by a CCK-8 cell viability test,the enzyme-like catalytic activity of the copper clusters was further investigated by a DABcatalyzed chromogenic assay,and the in vitro antibacterial effect of the clusters was also investigated.The HRTEM data showed good dispersion and uniformity of size for both metal probes,with the Pt cluster probes measuring 3.22 ± 0.20 nm and the Cucluster probes measuring 1.98 ± 0.24 nm.The results of the characterisation by fluorescence tracing,infrared spectroscopy and ultraviolet spectroscopy show that the synthesised metal probes have good stability.The experimental results showed that LHRH,a functional peptide targeting lung cancer A549 cells,inhibited A549 tumour cells by binding to EW-Pt NCs through biocovalent coupling,and that platinum clusters at concentrations up to 200 ?M led to apoptosis of nearly half of the cells.Copper clusters have good enzyme-like catalytic activity,enabling DABcatalyzed chromogenic imaging of cells.In vitro inhibition assays showed that both EW-Pt NCs and EW-CuNCs inhibited S.aureus and E.coli,and the inhibition effect of EW-CuNCs was better than that of EW-Pt NCs.It provides new options and new ideas for the development of non-toxic,low-cost and highly effective anti-tumour and anti-bacterial drugs.