The Establishment Of Metal Nanoclusters Biosensors And Its Applications In Bio-enzyme Detection

Recent years,metal nanoclusters as a new type of light-emitting nano-materials has attracted more and more attention from the researchers due to its ultra-small size(usually composed of several to ten atoms and the size is smaller than2 nm)and unique physical and chemical properties.So far,metal nanoclusters had been used in biological analysis,biological imaging,environmental monitoring,industrial catalysis and electronic devices,etc.In this thesis,we used metal nanoclusters as a biosensor to detection enzyme molecules.The main contents are as follows:The first chapter,we studyed the background of the fluorescent metal nanoclusters and introduced the results and significance of this thesis.The second chapter,we developed a simple,ultra-sensitive and label-free fluorescent probe for trypsin and its inhibitor based on DNA hosted Cu nanoclusters(Cu NCs).With the addition of protamine,the fluorescence intensity(FL)of Cu NCs could be enhanced obviously through forming protamine/DNA complexes between Cu NCs and protamine.The fluorescence intensity of Cu NCs would decrease in the present of trypsin due to the protamine hydrolysis,which allows the sensitive detection of trypsin.And this system was further used for trypsin inhibitor detection.Compared with the previous reports,this method does not need complex DNA sequence design,fluorescence label and has a much lower detection limit.The third chapter,a label-free,highly sensitive and simple assay for one step detection of protein kinase(PKA)activity and inhibition that avoids the fluorescent dye process has been established.The detection was based on thefluorescence(FL)quenching of peptide-Ag nanoclusters(Ag NCs)caused by antibody modified Au nanoparteicles(anti-Au NPs)via fluorescence resonance energy transfer(FRET).With PKA and adenosine 5'-triphosphate(ATP)introduced,the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation,and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs.According to the fluorescence quenching of Ag NCs,the activity of protein kinase can be facilely monitored with high sensitivity.We further explored the inhibitory effect of H-89 for protein kinase activity.The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells,which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.The fourth chapter,we established a simple and sensitive fluorescence probe to detect protein kinase(PKA)activity based on DNA-Cu NCs.This method was based on the strong interaction between adenosine triphosphate(ATP)aptamer and ATP.When ATP was added,fluorescent Cu NCs could not be formed due to the lack of effective substrate and the fluorescence of Cu NCs decreased.However,when PKA was added,the fluorescence of Cu NCs recovered because that ATP had been translated into ADP by PKA and ADP can not be bond with ATP aptamer.We can effective monitored the activity of protein kinase according to the variation of fluorescence signal.We also used this method to detect protein kinase inhibitor H-89.In addition,this method was also used to explore the activity of drug-induced PKA in Hela cells,which makes the sensor had a great application value in biochemistry and targeted kinase drug discovery research.