Synthesis Of Peptide-Metal Nanoclusters For Bioanalysis Research

Metal nanoclusters as a new class of metal nanoclusters have attracted great attention in recent years for their unique and intriguing physical and chemical properties. However, strong reducing agents which would disrupt the biological function of models were required in these synthesis processes and further complex chemical steps are needed to purify and functionalize the clusters for their later biologic applications. Therefore. it’s necessary to find out a simple method to synthesize metal nanoclusters without any reducing agents. Protein kinase which catalyzes protein phosphorylation and has a crucial role in treatment of diseases including cancer. Therefore, the establishment of high sensitivity and simple operation detection method of protein kinase not only provide the basis for the early diagnosis of cancers, but also helps to treat cancer-related phosphorylation of discovery and screening of new drugs. In this thesis, we synthesized metal nanoclusters by a one-step peptide method without any strong reducing agents, and designed a series of routes to detect the protein kinase which would help us find out a simple and sensitive method. The main work of my graduation thesis will be written in details in the sections below.1. A label-free, sensitive and simple method to detect protein kinase based on the selective aggregation of phosphorylated peptide-gold nanoclusters(peptide-Au NCs) triggered by Zr4+ ion coordination is developed. The Au NCs were synthesized by peptide without any strong reducing agents, which prevent peptides from being disrupted. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide-Au NCs and the concentration of casein kinase II(CK2) in the range of 0.08 to 2.0 U mL-1 with a detection limit of 0.027 U m L-1(3σ) was obtained. The feasibility of this Au NCs-based sensor was further demonstrated by the assessment of kinase inhibition by ellagic acid, 5,6-dichlorobenzimidazole-l-β-D-ribofuranoside, emodin, and quercetin in human serum. As expected, the IC50 value(inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.045 μmol/L. With more sophisticated design of the peptide substrate sequences, the detection of other enzymes will be realized. With characteristics of homogeneous, facile, universal, label-free, and applicable for kinase assay, the proposed sensor provides potential application in kinase-related biochemical fundamental research and inhibitor screening.2. Due to the remarkable fluorescent property of peptide-Ag NCs, it can be used to detect the phosphorylation degree of protein. Firstly, the phosphorylation reaction was occurred in the presence of CK2. Because of the phosphorylation of peptide, shearing action to peptides caused by carboxypeptidase Y was stopped. Secondly, one–step peptide synthesis method was used to synthesize the Ag NCs with the peptide being template and protective agent in the absence of strong reducing agents. However, peptide cannot be used to synthesize Ag NCs without CK2 due to the shearing action of carboxypeptidase Y. The phosphorylation degree of peptides could be calculated through the difference of fluorescence intensity. Although this method realizes the detection of protein kinase, some environmental factors such as temperature and stirring speed may have effect on the final results and the fluorescence intensity of peptide-Ag NCs is much lower than peptide-Au NCs。Therefore,there is more study have been taken.3. Because of the shortcoming of above work, we ameliorated the protein kinase detection assay. Peptide-Au NCs were regarded as fluorescence probes to establish a novel and sensitive mehod for PKA fluorescence “turn on” detection via the fluorescence quenching of peptide-Au NCs caused by carboxypeptidase Y. A novel, green and simple one-step biomineralization method was employed to synthesize peptide-Au NCs. The peptide-Au NCs were sheared by carboxypeptidase Y which contribute to the fluorescence quenching. However, after the phosphorylation of peptide-Au NCs, the fluorescence of peptide-Au NCs still remained due to the inhibition to carboxypeptidase Y caused by phosphate group. Through the difference of fluorescence intensity, we can detect the protein kinase in a high sensitive way. This detection method we designed has some advantages which can avoid some complex procedures may have potential applications in disease diagnosis.4. A novel and sensitive ratiometric fluorescent assay for monitoring kinase activity based on carboxypeptidase Y digestion by using Au nanoclusters(Au NCs) and CdSe/ZnS QDs@SiO2 as the dual-emission nanoparticles is developed. This approach can not only sensitively monitor the kinase activity with affording built-in correction which avoids environmental interferences, but also realize visual detection. Under optimal conditions, a linear relationship between the PL intensity ratio(I415/I630) of CdSe/ZnS QDs@SiO2@Au NCs and protein kinase A(PKA) concentration in a range of 0.01 to 40 U m L-1 with a detection limit of 0.004 U mL-1(3σ) is obtained. The feasibility of this CdSe/ZnS QDs@SiO2@Au NCs-based ratiometric sensor has been further demonstrated by assessment of kinase inhibition by ellagic acid in human serum. The IC50 value(inhibitor concentration producing 50% inhibition) for ellagic acid is estimated to be 0.10 μmol/L. The detection of other enzymes can also be realized with precise design of the peptide substrate sequences. This approach can not only sensitively monitor the kinase activity with affording built-in correction which avoids environmental interferences, but also realize visual detection. The present assay is universal and visual for kinase assay and promises potential application in kinase-related biochemical fundamental research and inhibitor screening.5. The double fluorescent probe was built with two peptide biomineralized metal nanoclusters which can realize the purpose of detecting PKA and CK2 at the same time. We have synthesized blue-light-emitting peptide-Cu NCs and red-light-emitting peptide-Au NCs by using protein kinase PKA and CK2 as template. Fluorescence of these two metal nanoclusters will enhance when these two protein kinases were added. This lable-free, sensitive and simple method which employed different metal nanoclusters can be used to detect several protein kinases simultaneously and have potential applications in diagnosis of diseases including cancer.