A New Method For Detecting Protease Activity Using DNA-AgNCs As A Fluorescent Probe

Protease plays a crucial role in a wide variety of physiological or pathological processes.Many methods have been developed to detect the activity of individual protease,including fluorimetry.Traditional fluorescence techniques are sensentive and accurate.However,they require laborious labeling in most cases.Therefore,it is necessary to establish a new,simple,homogeneous and label-free method for detection of protease activity.Silver nanoclusters offer several features that make them become a new alternative fluorophore for detection,including sub-nanometer size,high quantum yield,light stability,and biological compatibility.In this paper,two fluorescent bioanalytical methods have been developed for the detection of chymotrypsin and protein disulfide isomerase activity by using silver nanoclusters,respectively.The applications of the developed methods were validated by the inhibition and recovery test.Our proposed methods are simple,label-free,and high sensitive to detect protease.The main contents of this paper as follows:In chapter one,the characteristics and the applications of the new silver nanoclusters were firstly introduced.Then,the types of proteases and their importance in physiological or pathological processes were summarized.Finally,the advantages and disadvantages of the traditional fluorescence methods were discussed and the aim and ideas of the study were proposed.In chapter two,a new fluorescent biosensing method for assay of chymotrypsin activity was developed using DNA-AgNCs and graphene oxide(GO).A peptide probe was also designed using chymotrypsin-cleavable amino acid sequence and a cysteine terminus.The peptide probe formed Ag-S bond to DNA-AgNCs to enhance the fluorescence of DNA-AgNCs.After the addition of GO,the peptide was adsorbed to the negative GO surface and the fluorescence of DNA-AgNCs was quenched by FRET.The peptide was then degraded into amino acid fragments upon addition of chymotrypsin;these fragments were released from the GO surface,and the FRET was terminated.The developed label-free method features lower cost and higher sensitivity to chymotrypsin activity assay compared with conventional fluorescence analysis.The method can be used to analyze chymotrypsin(as low as 3 ng/mL)across a dynamic range of 0.0-50.0 ng/mL.The proposed biosensing strategy can also be extended to other proteases by using different peptide substrates.In chapter three,a method for the detection of protein disulfide isomerase(PDI)based on the turn-on to turn-off of DNA-AgNCs has been developed.The detection mechanism is based on two facts:(1).the transformation of sulfhydryl(-SH)to disulfide bonds(S-S)is reversibly catalyzed by PDI;(2).The fluorescence response of DNA-AgNCs to-SH and S-S are different.The developed method was lable-free,and unnecessary to involve coenzyme(NADPH)and reduction agent DDT.DNA-AgNCs can bond to GSH and the fluorescence of DNA-AgNCs is enhanced because the formation of Ag-S bond.In the presence of PDI,leading to the quenching of enhanced fluorescence of DNA-AgNCs because the GSH was transformed to GSSG.The method can be applied to detect PDI as low as 0.07 nM in the linear range of 1-50 nM.Compared with the conventional methods,this method is simple and sensitive,needs shorter reaction time.The potential application of the method for inhibition of rutin was also demonstrated,the IC50 was 14.02 nM.This method can be expected to be used for the early detection of clinical PDI and the screening of antithrombotic drugs.