Construction Of The Sensing Interface In Nanopore Confined Space And Its Applications In Single-molecule Analysis

In the past two decades,nanopore technology,as an important member of single-molecule detection technology,has attracted considerable attention due to its intrinsic advantages such as label-free,amplification-free,high sensitivity,and real-time identification.As the first discovered and used membrane nanopores,biological nanopores are particularly appealing due to their atomic-precision structural reproducibility,easy to be produced in large quantities,and pore sizes on a similar scale as many biologically important analyte molecules.In 1996,Kasianowicz and his colleagues took the lead in detecting individual polynucleotide molecules using natural alpha hemolysin(?-HL)nanopore,paving the way for single-molecule analysis in nanopores.The nature of common biological nanopores is protein,which makes it easy for us to engineer them,such as site-directed mutagenesis or incorporation of specific adaptors,and then make well-defined local changes to the nanopores,so that the pores can better achieve certain specific applications.Based on native or engineered biological nanopores,researchers have realized single-molecule measurement of various significant analytes such as metal ions,nucleic acids,and proteins.Although having achieved many remarkable results,there are still some issues that need to be solved for the single-molecule analysis based on this unique nanopore confined space.For example,the research of metal ions sensing in the nanopores still focuses on the ions'detection,but it remains unclear what the overall law of ions sensing in nanopores is.The translocation speed of nucleic acids through nanopores is generally very fast,so it is necessary to develop simple and feasible strategy to slow down the passage of nucleic acids.The ineffective sequencing time in nanopore DNA sequencing accounts for the main proportion and the detection throughput remains to be improved.Aiming at these problems mentioned aboved,this dissertation inverstigated the overall law of ion sensing in the nanopore confined space,developed a simple and general strategy to retard nucleic acids translocation,and proposed a new measurement scheme for nanopore DNA sequencing.The main research contents are included in the following three parts:1.Single molecule observation of hard–soft-acid–base(HSAB)interaction in engineered MspA nanoporesMost early works of metal ion sensing in biological nanopores were carried out in?-HL nanopores,and the sensing signal generated was usually very small(1?3 p A).The MspA nanopore is conical structure with a wide vestibule and sharp constriction,and it has been proved to own higher sensitivity and spatial resolution than?-HL from its excellent performance in nanopore DNA sequencing.However,other analytes were poorly investigated in MspA nanopores except nucleic acids.Our group pioneered to use MspA nanopores to sense HAu Cl₄ and observed the largest single-ion sensing signal(?55 p A),which indicated that MspA nanopore is also a suitable nanoreactor for ion sensing.However,most works of ions sensing in nanopores are still restricted to the detection of ions,the overall law of ion sensing at the single-molecule level is unclear in this nanopore confined space.In this work,we engineered the most sensitive position91 of MspA nanopores to be different amino acids,constructing the functional sensing interface in nanopore confined space,and then studied the coordination interaction between three representative amino acids(aspartic acid,histidine and cysteine)and a variety of divalent metal ions at the single-molecule level.By comparing the coordination kinetic parameters,we found that the ion sensing in the nanopore confined space followed the principle of HSAB theory,that is“hard acids prefer to coordinate to hard bases and soft acids to soft bases”.In addition,we could adjust the coordination strength of metal ions and amino acid residues at the single-molecule level by fine-tuning the p H.The above results show that MspA nanopores are very suitable for detecting various extremely small analytes,such as monoatomic and polyatomic ions,small molecules and chemical reaction intermediates.And the HSAB theory at the single-molecule level will be of benefit to the subsequent nanopore design for ion sensing.2.Calcium ion flux regulates nucleic acids sensing in biological nanoporesNucleic acids sensing in biological nanopores is the most studied field.However,the speed of nucleic acid translocation through nanopores is usually so fast(1?10?s/base)that only burr signals can be obtained.As a result,much translocation details are missed.The commonly used method to slow down nucleic acid speed is to construct double-stranded DNA(ds DNA),and then utilize ds DNA unzipping during its translocation to slow down the speed and obtain specific sensing signals.The preliminary chain construction of this method is complicated,and nucleic acids with different sequences need to be designed different complementary chains,leading to its poor generality.Therefore,there is an urgent demand to develop a simpler and general method to slow down nucleic acids translocation.Inspired by retarded nanopore translocation of micro RNA in the fluorenscence nanopores sensing,we systematically investigated this phenomenon in the electrophysiological single-channel recording.We found that the introduced calcium ion flux played an important role in the retarded nucleic acids translocation.The probable reason was the strong interaction between calcium ion and nucleic acids backbone.By asymmetrically placing KCl/Ca Cl₂buffer in the measuring chamber,the translocation speed of nucleic acids(DNA/RNA)was slowed down and the capture rate was also improved simultaneously.Finally,by means of the deceleration effect,we could easily distinguish three RNA homopolymers directly.This deceleration method of changing the buffer in principle is applicable to a wide variety of nanopores and nucleic acid analytes.That is,the deceleration stragedy we proposed is universal while simple and easy to operate.3.An immediate response strategy of nanopore DNA sequencingIn 2012,Jens H.Gundlach group demonstrated the nanopore DNA sequencing using a mutant MspA nanopore and phi29 DNA polymerase.However,during the actual sequencing process,since the samples are obtained randomly,more than 90%of the time is spent waiting for the samples to enter nanopores,and afterwards most of the signals obtained are translocation signals of the DNA library rather than sequencing signals,so valuable samples and measurement time are wasted.At the same time,the signal acquisition mode based on ionic current limit the detection throughput due to the complexity of integrated circuits.Fluorescence nanopore technique has the advantage of high throughput,but the drawback of background fluorescence has restricted its effective measuring time to only about 15 minutes,which requires us to shorten detection time.If immediate response DNA sequencing can be achieved once the voltage is applied,combining with the high-throughput advantage of fluorescence nanopore technique,the performance of nanopore DNA sequencing can be improved greatly in both sides of detection time and detection throughput.For this purpose,we developed a new DNA sequencing strategy.By coupling one strand of DNA of the sequencing library to the MspA nanopore,we constructed the sequencing library at the pore entrance.Combined with the deceleration function of phi29 DNA polymerase,we ultimately realized the envision of reporting DNA sequencing signals immediately once a voltage was applied.