Application Of Nanopore Single-Channel Recording In Metal Ions Detection

Water is a key resource in sustainable development. Developing new technologies for effective monitoring and prevention of water pollutants is not only vital for human health, but also a critical issue of cutting edge science. Among the various types of pollution, heavy metal ions have attracted the most attention, due to their resistance to microbial degradation and long-term accumulation in human body. And even trace amounts of heavy metal ions would have severe toxic effects.This thesis intends to introduce a novel nanopore-based single molecule sensing technique. This technique has undergone rapid development in the last20years and is now extensively used in DNA sequencing. Yet, is application in the detection of aqueous heavy metal ions was rarely reported.In this thesis, we designed a properly structured G-rich single stranded DNA (ssDNA) to selectively detect Pb2+and Ba2+based on a nanopore single-channel recording system, in which the random coiled ssDNA was transformed to a G-quadruplex structure by Pb2+and Ba2+and thus altering the translocation profile and generating characteristic signals in the recorded current. After optimization, the detection limit for Pb2+and Ba2+were both reduced to0.8nM. Selectivity study shows that there was no interference from other metal ions tested. And this was confirmed by surveying with a matrix sample containing Pb2+and Ba2+and all other cations, indicating the potential of this strategy for simultaneous detection of Pb2+and Ba2+in the absence of any masking agents. We also varied the molar ratio of DNA1to Pb2+and Ba2+to study the separation ability of this system on co-existing Pb2+and Ba2+, finding that semi-quantification could be realized. In chapter4, we synthesized a ligand which was able to coordinate with rare earth (RE) metal ions, o-carboxylbenzaldehyde-2-pyrroleformylhydrazone. And it holds the potential to be combined into the nanopore system for RE metal ion sensing.Details of the projects are given below: 1. Build suitable nanopore single-channel recording detection system for Pb2+and Ba2+. We designed4DNA strands that could be stabilized by Pb2+on the basis of the classic sequence of TBA and PS2.M. After screening with the hemolysin nanopore platform, the most effective strand DNA1,(A)3oGTGGGTAGGGCGGGTTGG(A)3o, was obtained, which was used for the following experiments to detect Pb2+and Ba2+. Through trial and error,(CH3)4NC1(recording buffer) and+180mV/+200mV (recording voltage) were finally used to construct the nanopore system for the detection of Pb2+and Ba2+.2. We tested the performance of our system to detect Pb+and Ba+, individually. Circular dichroism (CD) patterns shows that both Pb2+and Ba2+could induce the G-quadruplex formation, of which the former possessed stronger stabilizing ability. This discrepancy was verified by nanopore recording, in which translocation of Pb2+-DNA1generated much longer durations than its counterpart Ba+-DNA1and could be readily identified in the2D event-density plots. Both Pb2+-DNA1and Ba+-DNA1have a new distinctive cluster associated with the dissociation and translocation of G-quadruplex structure. From the histogram of dwell time, the most probable duration time of Pb+-DNA1and Ba+-DNA1in nanopore was determined to be100ms and10～20ms, respectively. Later on, we set out to study the sensitivity and selectivity of this strategy for the detection of individual Pb2+and Ba+. No interference from the other12relevant metal ions (K+, Na+, NH4+, Ca+, Mg+, Li+Zn2+, Cd2+, Cu2+, Cr3+, Fe3+, Hg2+) was observed, and the detection limit was lowered to0.8nM for both ions after the introduction of asymmetrical salt concentration.3. We then moved on to study the possibility of simultaneous detection of Pb2+and Ba2+. The co-existence of interfering ions did not bring in any problem to the sensing system, as verified in the matrix experiment which was performed in the presence of all metal ions. Next, we studied if this strategy was able to determine the respective concentration of Pb2+and Ba2+Gaussion curve was used to fit the histogram patterns of Pb2+-DNA1, Ba2+-DNA1and Pb2++Ba2+-DNA1, and the areas under the curve, denoted as Apb, and ABa, was corresponded to the concentration of Pb+and Ba+, respectively. As the molar ratio of DNA1:Pb2+:Ba+was varied from2:1:1to1:1:1and1:2:2, the corresponding ratio of ABa to Apb (ABa/Apb) was calculated to be0.52,0.35,3, indicating the potential of this method for simultaneous detection of Pb2+and Ba2+. Interestingly, when the amount of DNA1was fixed, Apb/ABa was declined with the increase of the concentration of Pb2+and Ba2+while maintaining the relative concentration of Pb2+and Ba2+equal.4. We synthesized a complex-structured ligand, o-carboxylbenzaldehyde-2-pyrroleformylhydrazone, and characterized it with1H NMR and13C NMR. Through elemental analysis and FTIR, the as-synthesized ligand was determined to be capable of coordinating with the thirteen RE metal ions tested, including La3+, Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Yb3+, Y3+. The formed complex shared a general formula, RE(C13H10N3O3)(C13H11N303)·2H2O, indicating that one RE ion was coordinated with two water molecules and two ligand molecules, in which one of the ligands had sacrificed a hydrogen atom. This complexation would increase the hydrodynamic size of ligands, thus holding the potential for being combined into the nanopore system and used in RE metal ion detection.In summary, we have developed a new method to sensitively and selectively detect trace amount of Pb2+and Ba2+in solution using a nanopore-based single-channel recording system. Moreover, semi-quantitative information could be obtained by this method for simultaneous detection of Pb2+and Ba2+. This thesis has broadened the applications of nanopore sensing technique and would provide some guidelines for the development of novel metal ions sensors.