A Novel Synthesis Strategy For Silver Nanoclusters Based On The Enzymatic Polymerization And Its Application As A Versatile Biosening System

Silver nanoclusters encapsulated by DNA (DNA-AgNCs) have attractedextensive attention in the fields of biosensing and bio-imaging in the recent years.With the advantages of tunable fluorescence emission, high quantum yield, goodphotostability low biological toxicity, and biocompatibility, DNA-AgNCs have beenwidely applied in the design of biosensing systems and bio-imaging. As a uniqueDNA polymerase, terminal deoxynucleotidyl transferase (TdT) has been widely usedin the nuclease labeling because of its template-free property. In the present study, wehave demonstrated a new proof-of-concept of the synthesis of AgNCs based on DNAtemplate generated by DNA polymerization via TdT. And this synthesis strategy canbe applied to detection of nuclease activity and protein.1. We have firstly demonstrated a novel synthesis strategy for silver nanoclustersbased on DNA polymerization by TdT. A cytosine-rich (C-rich) DNA sequence waspolymerized by TdT, and it was further used as template to encapsulate thefluorescent DNA-AgNCs. Some factors that may infuence the synthesis ofDNA-AgNCs were also investigated. In additon, the DNA-AgNCs encapsulated by thepolymerization product (DNA-P-Cn) were compared with those encapsulated by thesynthetic C-rich DNA. Furthermore, transmission electron microscope (TEM) andX-ray energy dispersive spectrometer (EDS) were employed to characterize theDNA-AgNCs. We infered that the enhanced fluorescence intensity of DNA-AgNCsencapsulated by DNA-P-Cncould be accounted for the two reasons:(1) Longer DNAsequence could provide better protection for AgNCs and remove the polar solventform the surface of the AgNCs, leading to a fluorescence enhancement.(2) Thefluorescence resonance energy transfer (FRET) effect probably occurs and leads to anenhanced fluorescence emission. Finally, the photostability turned out to be quitesatisfactory.2. Based on this novel DNA-AgNCs synthesis strategy, a―turn-on‖and label-freemethod for TdT activity detection was developed with a very low limit of detection(LOD) which is about200times lower than the previous reports. In addition, thisdetection method has a very good reproducibility and recovery. Finally, three commoninhibitors of TdT were investigated.3. Based on the TdT polumerization and synthesis of DNA-AgNCs, a―turn-on‖ method for nuclease detection was developed with universality. EcoRI and ExoIII astypical examples of endonuclease and exonuclease, respectively, were chosen as thetarget analytes. The results show that the detection system has very good sensitivitybecause there is no pre-existing C-rich sequence without cleavage of nuclease andpolymerization of TdT. Excellent selectivity was also obtained because of thesubstrate specificity of nuclease. This detection system can be extended to detectionof other DNA-related enzymes just with some change of the substrate sequences.4. Based on the thrombin aptamer and nicking enzyme Nb.BbvCI, theDNA-AgNCs synthesis strategy was applied to the amplified―turn-on‖detection ofthrombin. The detection of trace thrombin was achieved after rational design of twoDNA probes–an aptameric probe and a amplification probe. Because of the substratespecificity of aptamer, this detection method processes very good selectivity. Thisstrategy can be well extended to the detection of other analytes after some changes onthe aptamer sequence.