A Novel Method Study For Detection Of Cysteine And RNase A Based On Silver Nanoclusters

How to realize rapid and sensitive detection of biomolecules,which closely related to diseases,has always been a problem in biochemical analysis.Recently,the emergence of new nanomaterials with unique physicochemical properties has made it possible to construct new biomolecular detection systems.Among of them,silver nanoclusters(AgNCs)synthesized with DNA as a template showed advantages including strong photoluminescence,easy synthesis,ultra-small size,low toxicity and good biocompatibility.Thus,it can be widely used in the biosensing and molecular imaging.Based on the basement of previous study,we further synthesized a series of DNA-AgNCs materials,which can emmit strong fluorescence signal,by using single-stranded DNA as the template.After performing the characterization of the system,it was used to detect the expression levels of two biomolecules,cysteine(Cys)and ribonuclease A(RNase A),which are tightly related to many physilocial processes of life.The contents are as follows:1.Detection of Cys based on DNA-AgNCs fluorescence probeIn this chapter,a simple Cys detection method was developed using the principle that the interaction between Ag~+and Cys thiol groups can cause fluorescence quenching of DNA-AgNCs.The data of quenching experiments showed the optimal reaction condition of 30°C and pH 6.0,which can obtain maximal fluorescence quenching efficiency of DNA-AgNCs more than 99%.Under ideal experimental conditions,the linear range of this method for Cys detection is 0.1-100 nM with a detection limit of 0.05 nM.Meanwhile,the method showed high anti-interference ability and selectivity.Finally,the method was used for detection of Cys/mercapto compound levels in clinical serum samples.The results showed that the linear range of Cys detection is 1 nM-100 nM in serum with the detection limit of 0.5 nM.Using standard addition assay,it was found that the recovery of Cys was 101.3%to 104.2%with relative standard deviations(RSD)less than 10%.These results clearly demonstrate the high accuracy and good anti-interference ability of the new method for Cys analysis under complex conditions.According to our point,this method combining with high-throughput techniques is expected to be widely used in the diagnosis,prognosis,and evaluation of treatment efficiency associated with Cys.2.An ultrasensitive fluorescence system for detection of RNase A and drug screening based on DNA-AgNCs probeBased on the previous chapter,we further constructed a fluorescent signal self-quenching AgNCs nanoprobe using molecular engineering technology.By monitoring the effect of RNase A on the fluorescence signal of self-quenching AgNCs probe,a sensitive and specific platform for RNase A analysis was established.The results show that the optimal ratio and time for hybridization between RNA and DNA in the assay system is 2.5 and 60 min,respectively.The optimal conditions for RNase A assay are 37°C,30 min at pH 6.6.Under these optimal conditions,RNase A concentration showed a good linear relationship with fluorescence change in the range from 0.2 pg/?L to 10 pg/?L with a detection limit of 0.098 pg/?L.The results of specificity and stability assay show that the method has good anti-interference ability and high selectivity.The detection result using standard addition method showed that the recovery rate of RNase A in the serum was about 98.47%to 104.35%with a coefficient of variation less than 10%.These results clearly demonstrate the high accuracy and good anti-interference ability of the new method for RNase A assay under complex environment.In addition,the method was used to screen the effectors of RNase A from natural compounds.Among of the 15 investigated compounds,7natural compounds with active and 1 with inhibitory effect on this enzyme were screened.In summary,these results indicate that this high sensitivity and low cost method for RNase A assay has broad prospects in the fields of activity detection,drug screening and related functional evaluation.