Changes in the nanopore ionic current during entry of a target molecule underlie the sensing capability and dominate the intensity and extent of applications of the nanopore approach. The volume exclusion model has been proposed and corrected to describe the nanopore current blockage. However, increasing evidence shows nonconformity with this model, suggesting that the ionic current within a nanopore should be entirely reconsidered. Here, we revisit the origin of nanopore current blockage from a theoretical perspective and propose that the noncovalent interactions between a nanopore and a target molecule affect the conductance of the solution inside the nanopore, leading to enhanced current blockage. Moreover, by considering the example of an aerolysin nanopore discriminating the cytosine DNA and methylcytosine DNA that differ by a single methyl group, we completely demonstrate, by nanopore experiments and molecular dynamics simulations, the essential nature of this noncovalent interaction for discrimination. Our conductance model suggests multiplicative effects of both volume exclusion and noncovalent interaction on the current blockage and provides a new strategy to achieve volume difference sensing at the atomic level with highly specific current events, which would promote the nanopore protein sequencing and its applications in real-life systems.

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Detection of DNA methylation in real human serum is of great importance to push the development of clinical research and early diagnosis of human diseases. Herein, taking advantage of stable pore structure of aerolysin in a harsh environment, we distinguish methylated cytosine from cytosine using aerolysin nanopore in human serum. Since wild-type (WT) aerolysin enables high sensitivity detection of DNA, the subtle difference between methylated cytosine and cytosine could be measured directly without any specific designs. Methylated cytosine induced a population of I/I = 0.53 while cytosine was focused on I/I = 0.56. The dwell time of methylated cytosine (5.3 ± 0.1 ms) was much longer than that of cytosine (3.9 ± 0.1 ms), which improves the accuracy for the discrimination of the two oligomers. Moreover, the pore-membrane system could remain stable for more than 2 h and achieve the detection of methylated cytosine with zero-background signal in the presence of serum. Additionally, event frequency of methylated cytosine is in correspondence with the relative concentration and facilitate the quantification of methylation.

Aerolysin has been used as a biological nanopore for studying peptides, proteins, and oligosaccharides in the past two decades. Here, we report that wild-type aerolysin could be utilized for polynucleotide analysis. Driven a short polynucleotide of four nucleotides length through aerolysin occludes nearly 50% amplitude of the open pore current. Furthermore, the result of total internal reflection fluorescence measurement provides direct evidence for the driven translocation of single polynucleotide through aerolysin.

Nanopore techniques offer the possibility to study biomolecules at the single-molecule level in a low-cost, label-free and high-throughput manner. By analyzing the level, duration and frequency of ionic current blockades, information regarding the structural conformation, mass, length and concentration of single molecules can be obtained in physiological conditions. Aerolysin monomers assemble into small pores that provide a confined space for effective electrochemical control of a single molecule interacting with the pore, which significantly improves the temporal resolution of this technique. In comparison with other reported protein nanopores, aerolysin maintains its functional stability in a wide range of pH conditions, which allows for the direct discrimination of oligonucleotides between 2 and 10 nt in length and the monitoring of the stepwise cleavage of oligonucleotides by exonuclease I (Exo I) in real time. This protocol describes the process of activating proaerolysin using immobilized trypsin to obtain the aerolysin monomer, the construction of a lipid membrane and the insertion of an individual aerolysin nanopore into this membrane. A step-by-step description is provided of how to perform single-oligonucleotide analyses and how to process the acquired data. The total time required for this protocol is ∼3 d.