Detection Of Biomolecules And Metal Ions By Aptamer DNA-templated Fluorescence Silver Nanoclusters

In this study,we have designed a variety of ssDNA templates,and synthesized the corresponding fluorescent DNA-Ag NCs.The DNA templates designed contained two components,Ag NC-nucleation segment with C-rich sequence at the 5' and/or 3' terminuses and an aptamer segment for combining with corresponding target.Upon the optimization of the C-rich sequence,we have selected two DNA-Ag NCs that can high sensitively detect ATP,further investigation were performed to determine the preliminary mechanism of interaction between ATP and DNA-Ag NCs.Meanwhile,the proposed method also could detect the formation of G-quadruplex by embedding the G-rich sequence at the aptamer segment.The main results are as follows:?1?If aptamer segment is the ATP aptamer,thus the DNA-Ag NCs could be used for detecting of ATP,and further applied to monitor the activity of ADA.The detection principle is as follows: when ATP was introduced,the combination of ATP and aptamer segment induced the change of the microenvironment of DNA templates,which ultimately lead to the change of the fluorescence intensity of Ag NCs.We studied a total of fourteen Ag NCs prepared by different ssDNA templates,and found that they showed different sensitivity to ATP.BT3T3-and BT3T3?R?-Ag NCs could detect ATP with the limits of detection of 0.44 and 0.65 mM based on fluorescence turn-on assay.Moreover,ATP could protect the fluorescence intensity of BT3T3?R?-templated Ag NCs from quenching for at least 9 h,thus we further investigated the feasibility of the assay for ADA on the basis of the BT3T3?R?-Ag NCs/ATP complex sensor system.Both BT3T3-and BT3T3?R?-Ag NCs were chosen to make particular studies on the interaction mechanism with ATP by UV-vis,TEM,fluorescence and TCSPC methods.The results showed that when DNA-Ag NCs presented a strong fluorescence,the addition of ATP failed to cause a large change of fluorescence intensity;on the contrary,after Ag NCs was kept for 24 h and emitted a weak fluorescence,adding ATP was able to result in the large fluorescence enhanced of 43 and 33 times for BT3T3-and BT3T3?R?-Ag NCs,respectively.The possible mechanism was also suggested that ATP binding to aptamer segment of template induced the change of the DNA secondary structure,which made the aggregated Ag NPs disperse into Ag NCs with an average diameter of about 2 nm that were responsible for the large fluorescence increase.?2?If aptamer segment is G-rich DNAs,thus the DNA-Ag NCs could be used for detecting the formation of the G-quadruplex DNAs.The detection principle is as follows: if the G-rich DNA can fold into G-quadruplex,thus the two darkish DNA-Ag NCs located 3' and 5' terminuses will approximate,which will lead to enhance the fluorescence signal.Upon addition of the complementary DNA of G-rich DNA?C-DNA?,the G-quadruplex structure translated into double helix structure and the two DNA-Ag NCs are separated,then the fluorescence signal is quenched.This novel approach presented a universal feasibility for variety of G-quadruplex structures whereas the chair-type structure of TBA gave the largest sensitivity among them,that is,the assay partially depended on the structure of G-quadruplex DNAs,the closer the 5' and 3' terminuses was,the higher the sensitivity was.In addition,the ionic strength,DNA sequences and the stability of G-quadruplex structures also affect the sensitivity of assay.?3?Above studies found that BT5A5-Ag NCs presented a strong and stable fluorescence,although it had no significant change after addition of ATP,it had good selectivity for Cu²⁺ and Hg²⁺ among other metal ions and the limit of detection was found to be 5.82 and 4.88 nM respectively,which met the detection requirement for drinking water of the U.S.Environmental Protection Agency.More importantly,the detection system has also been successfully applied to the detection of Cu²⁺ and Hg²⁺ in the real water samples and showed a potential application value.