Profiling single-molecule reaction kinetics under nanopore confinement

Abstract

The study of a single-molecule reaction under nanoconfinement is beneficial for understanding the reactive intermediates and reaction pathways. However, the kinetics model of the single-molecule reaction under confinement remains elusive. Herein we engineered an aerolysin nanopore reactor to elaborate the single-molecule reaction kinetics under nanoconfinement. By identifying the bond-forming and non-bond-forming events directly, a four-state kinetics model is proposed for the first time. Our results demonstrated that the single-molecule reaction kinetics inside a nanopore depends on the frequency of individual reactants captured and the fraction of effective collision inside the nanopore confined space. This insight will guide the design of confined nanopore reactors for resolving the single-molecule chemistry, and shed light on the mechanistic understanding of dynamic covalent chemistry inside confined systems such as supramolecular cages, coordination cages, and micelles.

Fig. 1. (a) A schematic illustration of the single-molecule reaction between R and a K238C AeL nanopore (P); (b) the current trace model for the reaction event and non-reaction event. The kinetics model describes the four states: (1) capture of R into the nanopore, (2) energizing of a cysteine residue for [P⋯R]^‡ by the collision of R with the K238C site, (3) formation of a covalent bond between R and K238C, (4) release of R after deactivation. The non-reacted reactant escapes from K238C AeL either from the cis side or trans side. k₁, k₂ and k₃ represent the kinetics constant of each step. [R] refers to the concentration of the reactant. t_I-R stands for the time interval between adjacent reaction events. t_I-NR refers to the time interval between adjacent non-reaction events. t_D-R denotes the time duration of reaction events. t_D-NR denotes the time duration of non-reaction events.

Fig. 2. Kinetics evaluation of R₁ reacting with the K238C AeL nanopore. (a) The reaction rate (f_PR = 1/t_I-R) and fraction of effective collision (ECF) at different R₁ concentrations. (b) Reaction rate (f_PR) and ECF under different voltages from +60 mV to +110 mV. (c) The relationship between the ECF and non-reaction event duration (t_D-NR). The voltage dependent results of t_D-NR and t_I-NR are shown in Fig. S5a and b, respectively. (d) The collision threshold energy (ε₀) under different voltages from +60 mV to +110 mV. All data were acquired at 20.0 ± 2.0 °C in 1.0 M KCl, 10.0 mM Tris, and 1.0 mM EDTA solution buffered at pH 8.0 in the presence of 50.0 μM R₁.

Fig. 3. The single-molecule reaction of R₂ (a), R₃ (b), R₄ (c), and R₅ (d) with a K238C AeL nanopore. Left: the ionic current trace at +60 mV. The red and blue symbols represent the reaction events, and non-reaction events, respectively. Right: voltage dependent of the reaction rate (f_PR = 1/t_I-R) and ECF. The current fluctuation of the reaction events may be attributed to the conformational changes of the peptide or the possible intermediates. All data were acquired at 20.0 ± 2.0 °C in 1.0 M KCl, 10.0 mM Tris, and 1.0 mM EDTA solution buffered at pH 8.0 in the presence of 50.0 μM reactant. The short bumping events have been excluded.