Studies On Single-molecule Chemistry Of Gold Compounds Based On Biomimetic Nanopore

Bioinorganic chemistry is an interdisciplinary subject between biology andinorganic chemistry,which mainly studies the chemical interactions between biological ligands and inorganic elements.However,there are significant static and dynamic heterogeneities in the real world chemical systems,and it is difficult for researchers to reveal the intrinsic molecular mechanism of chemical reactions in an ensemble fashion.Since all chemical reactions can be decomposed into a series of single-molecule reactions,which only involve the process of bond breaking and formation of an individual molecule.Thus,studying single-molecule chemical reactions,such as observing the formation and breaking of single bonds,calculating single-molecule reaction kinetics,and capturing short-lived intermediates,can help us understand the intrinsic molecular mechanisms of complex chemical reactions.Nanopore technology is a rapidly developing single-molecule researchmethod in recent years,which has the advantages of label-free,rapid detection and high resolution.According to the properties of nanopore materials,they can be divided into three types:biological nanopore,solid nanopore and composite nanopore.Among them,biological nanopore is based on protein expression and purification,so it has atomic precision,stable and reproducible nanostructures and interfacial chemistry.Moreover,compared with other types of nanopores,biological nanopore has a higher resolution,not only to distinguish single bases of nucleic acids,but also enough to sense as small as single metal ions.Therefore,biological nanopore is the only means of current nanopore research on single-molecule chemistry,and can be constructed as nanoreactors that interact with inorganic elements to dynamically monitor the process of single-molecule bio-inorganic chemical reactions in real time.Studies of single-molecule chemistry in biological nanopores can be traced back to 1997 years.The Hagan Bayley group at Oxford University in the United Kingdom designed and constructed engineered hemolysin(?-hemolysin,?-HL)nanopores,and observed the coordination interactions between metal ions such as Zn²⁺,Co²⁺with amino acid residues at the level of single-molecule.However,due to the wide aperture of the?-HL channel(?1.4 nm)and the cylindrical recognition site,all observed signals exhibited weak blocking signal fluctuations(1-3 pA).To date,almost all single-molecule reactors based on biological nanopores are constructed from?-HL nanopores.Recently,our group unexpectedly found that tetrachloroaurate ions([AuCl₄]^-)can coordinate with methionine residues(M113)in the wild-type?-HL(WT?-HL)nanopore through the reversible Au(?)-sulfide coordination.However,as mentioned above,the signal of[AuCl₄]^-ions in?-HL is small and exhibits a variety of irregular fluctuations,which may be related to the non-specific binding of tetrachloroaurate(?)with other amino acids such as lysine(K147 or K131)distributed over its long cylindrical restriction.Inspired by the nanopore sequencing technology,we introduced methionines at the recognition site of the conical-shaped Mycobacterium smegmatis porin A(MspA)nanopore,and observed amplified[AuCl₄]^-signals,thus we successfully constructed an engineered MspA single-molecule reactor.Subsequently,we further studied a series of single-molecule chemical reactions based on gold compounds in the MspA nanopore.The main research content of this paper includes the following four parts:1.Studies on coordination chemistry of Au(?)and methionine in an engineered conical-shape MspA nanoporeBased on the known sulfur-gold(S-Au)coordination chemistry,we firstobserved the single-molecule chemical reaction between[AuCl₄]^-and methionine(M113)in a WT?-HL nanopore.Then,a Met?Gly mutation(M113G)was introduced to WT?-HL by pore engineering.No[AuCl₄]^-binding events were detected by?-HL M113G,which is evidence of the coordination interaction between Au(?)and methionine(M113)in the WT?-HL.By site directed mutagenesis in M2 MspA,we introduced methionine residues at the narrowest point 91 of MspA.Compared with the barrel configuration of?-HL,the constriction of MspA is narrower,so that it has a higher resolution and is less susceptible to interference from surrounding amino acids.Therefore,the amplification of the[AuCl₄]^-current blockade and the improvement of the signal-to-noise ratio were observed in the experiments.At+200 mV,the blockade depth of[AuCl₄]^-can reach about?55 pA,which is the largest single ion binding event reported in nanopore measurement.These phenomena indicate that MspA may be a superior template engineered as a nanoreactor,which can detect chemical intermediates or kinetics in single-molecule.2.Single-molecule chemistry of Au(?)and methionine in an engineered conical-shape MspA nanoporeAu(?)and Au(?)are both gold compounds.We have demonstrated that Au(?)can coordinate with methionine residues in the MspA nanopore at the single molecule leve.However,there is no related single-molecule chemistry research to Au(?)and methionine.In this experiment,we studied the interactions between dichloroaurate(?)([Au Cl2]^-)and methionine residues in an engineered MspA nanopore and systematically compared the binding differences of two valences Au(?)and Au(?)from the terms of reaction kinetics,blockade depth,and the number of gold ions bound in the pore.Single-molecule data suggest the[Au Cl2]^-is able to form stable complexes with methionine residues,and shows a higher affinity for thioether than[AuCl₄]^-.Moreover,due to the size difference,more gold(?)ions capable of binding simultaneously to the nanopore.In addition,we also proved that even under acidic conditions,no redox reaction occurs between Au(?)and methionine.The investigation of the[AuCl2]^-binding to an engineered MspA nanopore brings insights in understanding the coordinated gold(?)ion in protein receptors and the single-molecule action difference between two valences of gold ions.3.Biothiols sensing based on gold ion embedded MspA nanoporeFor protein nanopore prepared by biological expression,it is composed of 20conventional amino acids,so it is difficult to possess any chemical properties other than amino acids.However,when a single gold ion is binding with methionine,it can temporarily stay in the channel that imparts chemical properties to the MspA nanopore,thereby forming a composite biological channel embedded with gold ion.As we know,gold can not only coordinate with thioethers,but also form stronger interactions with thiol-contained molecules.Therefore,the gold ion embedded in the pore can serve as a gold atom bridge,one end of which is connected to the thioether on the nanopore,and the another end is connected to the thiol-contained molecules,so as to realize the detection of various biothiols in the solution.In the experiment,we constructed a single-molecule chemical reaction system based on MspA nanopore embedded with gold ion,and realized the detection of three most common biothiols L-cysteine(Cysteine,Cys),L-homocysteine(Homocysteine,Hcy)and L-Glutathione(Glutathione,GSH).The sensing mechanism is simple,fast and lable free.It may be engineered into a portable sensor chip for clinical detection.4.Studies on the embedding of gold clusters in an engineered conical-shape MspA nanoporeAlthough gold ions can be temporarily embedded in the MspA nanopore to achieve the sensing of biothiols,it is currently difficult to fix them for a long time.Compared with gold ions,gold clusters with a size of 1-2 nm are more stable and controllable.It may be possible to embed single gold cluster that with unique physical and chemical properties into the MspA nanopore.In the experiments,we initially tested two thiol ligand-modified gold clusters,and observed the translocate characteristics of a single gold nanocluster under voltage.Single-molecule experiments indicate that the negatively charged[Au₂₃(SC₆H₁₃)₁₆]^-gold nanocluster can stay in the channel stably and realize the embedding of gold cluster in an engineered conical-shape MspA nanopore,which is because the cluster size is larger than the narrowest recognition site of the MspA nanopore.Based on the embedding of single gold cluster,we are expected to further study the interaction of gold nanoclusters with other analytes or observe metal-catalyzed chemical reactions at the single molecule level.