| In recent years,metal pollution in industrial waste water has attracted considerable attention due to its potential hazards to human safety and environmental stability.It is well known that a small amount of metal has a positive effect on life activities.However,excessive metal ions may lead to a variety of diseases.Therefore,rapid detecting,real-time monitoring and effective removing the metal ions in the environment,food and human body are of great significance.Among the various techniques for removing metal ions,the chelating method is the most versatile and effective approach.Therefore,it is very important to study the reaction process,mechanism of metal ions and metal ion chelating agents.However,the results of traditional research methods are based on the average effect of a large number of molecular reactions and the formation and dissociation of metal-chelator complexes is largely unexplored at the single-molecule level.In recent years,the nanopore detection technique has played an important role in the study of single molecule reactions by combining biotechnology with nanopore single-molecule technology through recording current changes in individual ion channels.In the past two decades,the nanopore detection technology with the merits such as low cost,simple operation,rapid detection,good selectivity,high efficiency and sensitivity,has become a powerful tool for detecting and analysing at the single-molecule level,widely used in the field of DNA sequencing,isomers distinguishing,reaction kinetic process monitoring,covalent and noncovalent bonding interactions and the detection of nucleic acids,polymers,organic molecules and metals,etc..In this work,based on the engineered carboxymethyl-β-cyclodextrin(CMβCD),a new method for the detection of chelating with metal ions at a single-molecule level is presented and a variety of divalent metal ions and small organic molecules were detected.The main research contents are as follows:1.Nanopore single·molecule platform for investigating metal-chelator interactionsIn this experiment,Cu2+and CMβCD are selected as a model system and we will illustrate the details with single-molecule interaction between Cu2+ and CMβCD with an engineered a-HL(M113N)7 pore.The experimental results show that the bioavailable CMpCD as a natural derivative possesses higher affinity for Cu2+.The formation constant Kf of Cu2+ and CMβCD increases 14,417-fold in the nanopore than that in the bulk solution.There are four binding modes in the single molecules interaction of Cu2+ and CMβCD,which were observed by the reversible current blocking signals.In addition,the thermodynamic and kinetic processes of the interaction between Cu2+ and CMβCD were investigated by changing the concentration of Cu2+,pH,transmembrane voltage,and temperature.The experimental results showed that it is favored to form stable Cu2+-CMβCD complexes at weak acidic conditions.With the increasing of the voltage and temperature,the binding frequency of CMpCD with Cu2+ could increase,while the stability of Cu2+-CMPCD complexes Would decrease.This work provides a new platform for studying the interaction of metal ion and metal ion chelator and the method of rapid and efficient detection of metal ions.Moreover,bioavailable CMpCD is expected to be used in the treatment of metal ion poisoning,which has both important theory meaning and real application value.2.Simultaneous multiplexed analysis of metal ions using a protein nanopore equipped with a funtionalized molecular adaptorSingle molecule detection of divalent metal ions was realized by using CMβCD as the adapter.According to different kinetic constants between diverse divalent metal ions and CMβCD,the interaction between divalent metal ions and CMBCD was studied at the single-molecule level.In addition,according to the difference between the current blocking rates △I2/I0 and/or the residence time τoff produced by the different metal-CMpCD complexes,the distinction between some divalent metal ions as well as the simultaneous detection of several different divalent metal ions(eg.,Cd2+,Hg2+ and Cu2+)was achieved.Experimental results showed that the effect of CMpCD on each component was not affected by the presence of other ions in the mixture compared with the individual detection.This work takes advantage of the nanoscopic dimensions of the sensor element not ouly provides a new method for the sensitive and rapid detection of metal ions,but also plays an important role in the effective removal of metal ions.3.Study on simultaneous detection and interaction with of metal ions and organic molecules based on functionalized molecular adaptersIn this experiment,the simultaneous detection of 1-AdNH2·HCl and Cu2+ was conducted based on the characteristics of that 1-AdNH2·HCl and Cu2+ through different sites of action with CMpCD.The experimental results showed that the △I/I0 and τoff of each component in the mixed system is consistent with that in the individual detection.However,when metal ions and organic molecules interact with CMpCD at the same time,the effect of each other will decrease the residence time τoff.The mechanism of the simultaneous interaction of CMpCD with metal ions and organic molecules have been studied at the single-molecule level by the current blocking signals.This work not only provides a platform for studying the effects of metal ions and organic molecules with CMβCD,but also realizes efficient detection of metal ions and organic molecules coexisting contaminants.In addition,the effective removal of metal ions and organic molecules coexisting with pollutants is of great significance to create a green and healthy environment. |
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