单分子电泳:纳米孔道电化学的新认识

该文旨在从电泳分离技术的角度认识纳米孔道电化学单分子分析技术,这种技术可以作为"单分子电泳"来理解和研究。纳米孔道电化学单分子分析技术与电泳的本质都是采用外加电场使待测分子产生电迁移。待测分子性质不同,且与介质材料孔道外露基团相互作用不同,使得分子移动速度具有差异,据此实现分离识别。气单胞菌溶素(Aerolysin)纳米孔道,由于其孔径与待测分子尺寸相匹配,其孔道内壁可以看作是由氨基酸组成的具有调控单个分子电迁移能力的特异性孔道界面。每一个氨基酸残基都相当于一个探测单元,在电场力的作用下,待测分子逐一进入孔道时与每一个探测单元相互作用方式、程度与时长不同,从而形成了单个待测分子特征的迁移速度和迁移运动轨迹。在纳米孔道实验中,每秒可以有上千个待测分子穿过孔道,产生特征阻断电流信号。通过对这些信号的阻断电流、阻断时间、阻断频率、信号特征等进行统计分析,可以从"单分子电泳"水平对单个待测物实现高通量的分辨和识别。该文以Aerolysin纳米孔道分辨仅有一个核苷酸差异的寡聚核苷酸(5′-CAA-3′、5′-CAAA-3′、5′-CAAAA-3′)为例,详细阐述了纳米孔道"单分子电泳"的单核苷酸...

Single-molecule electrophoresis: renewed understanding of nanopore electrochemistry

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.