This study aims to understand nanopore technology from the standpoint of capillary electrophoresis separation. The nanopore electrochemical measurements could be regarded as "single molecule electrophoresis". Similar to the case of capillary electrophoresis, the single target molecules migrate inside a nanopore under an external electric field. The recognition ability of the nanopore mainly depends on the charge, shape, and size of the target molecules under the electric force. The confined space of an Aerolysin nanopore matches the size of single biomolecule, while the amino acid residues along the inner wall of the nanopore facilitate electrokinetic regulation inside the nanopore. Under the applied voltage, each molecule enters the nanopore, generating the characteristic migration velocity and trajectory. Therefore, statistical analysis of the current amplitude, duration, frequency, and shape of the electrochemical signals would help differentiate and identify a single analyte from the mixture. Herein, we used an Aerolysin nanopore for identifying the oligonucleotides of 5'-CAA-3' (CA2), 5'-CAAA-3' (CA3), and 5'-CAAAA-3' (CA4), which differ in length only by one nucleotide, as the model system to demonstrate single-molecule electrophoresis. The diameter of the Aerolysin nanopore is around 1 nm, and the pore length is approximately 10 nm. Under an applied voltage of 80 mV, the nanopore experiences a high electric field strength of about 80 kV/cm. The phosphate groups of the nucleotides carry negative charges in an electrolyte buffer solution of 1.0 mol/L KCl, at pH 8. Therefore, CA2, CA3, and CA4 carry 2, 3, and 4 negative charges, respectively. During nanopore sensing, CA2, CA3, and CA4 are subjected to electrophoretic forces and thus move inside the nanopore. Because the Aerolysin nanopore is anion selective, the direction of electroosmotic flow through the nanopore is consistent with the anion flow direction. Under the combined effects of the electrophoretic force and electroosmotic flow, CA2, CA3, CA4 will transverse through the Aerolysin nanopore at different migration velocities. Note that the oligonucleotide shows strong electrostatic interaction with the two sensitive regions of Aerolysin, which comprises polar amino acids around R220 and K238. The strong interaction between the sensitive region of Aerolysin and the analyte would further modulate the translocation of oligonucleotides. Therefore, each oligonucleotide follows a different migration trajectory as it individually transverses through the nanopore. The migration speed and migration trajectory are recorded as ionic blockages in nanopore electrochemistry. The scatter plots of the blockage current and blockage duration of the mixed sample of CA2, CA3, and CA4 show three characteristic distributions assigned to each type of oligonucleotide. Since the net charge increases with increasing length of the oligonucleotide, CA3 and CA4 experience a stronger electrophoretic force than does CA2 inside the nanopore, leading to higher migration velocity. Therefore, the blockage duration of CA3 and CA4 is 5 times longer than that of CA2. By Gaussian fitting, the fitted blockage currents of CA2, CA3, and CA4 are 20.7, 15.7, and 12.7 pA, respectively. Similar to our previous results, the blockage current increases with the chain length when the oligonucleotides comprise not more than 14 nucleotides. Therefore, nanopore-based single-molecule electrophoresis allows for the electrochemical identification of CA2, CA3, and CA4 that differ in a length by only one nucleotide. Understanding the "single-molecule electrophoresis" concept would promote the application of electrochemically confined effects in single-molecule electrophoresis separation. The combination of single-molecule electrophoresis with a microfluidic system and a nanopore array is expected to aid the separation and identification of single molecules.
Keywords: electrochemically confined effects; nanopore; single-molecule electrophoresis.