Nanopore Stochastic Sensing Based on Non-covalent Interactions

Abstract

A variety of species could be detected by using nanopores engineered with various recognition sites based upon non-covalent interactions, including electrostatic, aromatic, and hydrophobic interactions. The existence of these engineered non-covalent bonding sites was supported by the single-channel recording technique. The advantage of the non-covalent interaction-based sensing strategy was that the recognition site of the engineered nanopore was not specific for a particular molecule but instead selective for a class of species (e.g., cationic, anionic, aromatic, and hydrophobic). Since different species produce current modulations with quite different signatures represented by amplitude, residence time, and even characteristic voltage-dependence curve, the non-covalent interaction-based nanopore sensor could not only differentiate individual molecules in the same category but also enable differentiation between species with similar structures or molecular weights. Hence, our developed non-covalent interaction-based nanopore sensing strategy may find useful application in the detection of molecules of medical and/or environmental importance.

Figure 1.

a) Molecular graphics representation of the wild-type protein homoheptameric pore, showing the mutation site (in red) in this work; and b) schematic representation of the principle of the non-covalent interaction-based nanopore sensing strategy.

Figure 2.

Typical single-channel recording trace segments, demonstrating the non-covalent interactions observed in stochastic studies of the translocation of various peptide molecules through pores of wild-type and mutant alpha-hemolysin proteins. All the experiments were performed in an electrolyte solution containing 1 M NaCl and 10 mM Tris•HCl (pH 7.5), with α-HL protein pores added in the cis compartment of the chamber. In the series of experiments with cationic peptide R-K-R-A-R-K-E (20 μM), which was added in the trans chamber compartment, the applied potential was +40 mV (cis at ground); in the experiments with anionic peptide D-D-D-D-D-D (which was added in the cis compartment of the chamber), a positive +30 mV (cis at ground) voltage was applied. In the traces of (M113R)₇ and (M113K)₇, the final concentration of D-D-D-D-D-D was 1.41 μM, while 30 μM D-D-D-D-D-D was added in other protein pores; in the case of hydrophobic peptide cyclo(pro-gly)₃ (21.5 μM), which was added in the trans chamber compartment, the voltage was +50 mV (cis at ground); and with aromatic peptide Y-F-F, (which was added in the trans comparment of the chamber), the experiments were performed at −25 mV (cis at ground). In the traces of (M113F)₇, (M113W)₇, and (M113Y)₇, the final concentration of Y-F-F was 1 μM, while 10 μM Y-F-F was added in the traces of other protein pores.

Figure 3.

Single-channel recordings of anionic peptides D-D-D-D-D-D in α-hemolysin mutant protein homoheptamer pores (M113RT145R)₇, (M113RT145R)₇, and (M113R)₇, supporting the electrostatic interaction nature, and also suggesting that the sensitivity of the non-covalent interaction-based nanopore sensor could be improved significantly by introducing multiple functional groups to the nanopore. The experiments were performed at +30 mV (cis at ground) in an electrolyte solution containing 1 M NaCl and 10 mM Tris•HCl (pH 7.5). Both the α-HL protein and anionic peptide D-D-D-D-D-D (1.4 μM) were added in the cis compartment of the sensing chamber.

Figure 4.

Simultaneous detection of multiple analytes. (Left) Typical single-channel recording trace segments, showing different binding behaviors of α-hemolysin mutant protein homoheptamer (M113K)₇ pore with anionic peptides D-D-D-D-D-D (D6) and D-D-D-D-D (D5). The experiments were performed at +30 mV (cis at ground) in an electrolyte solution containing 1 M NaCl and 10 mM Tris•HCl (pH 7.5). Both the α-HL protein and anionic peptides D6 and/or D5 were added in the cis chamber compartment. (Right) Typical single-channel recording trace segments, showing different binding behaviors of α-hemolysin mutant protein homoheptamer (M113Y)₇ pore with peptides F-Y-F, endomorphin-1 (Y-P-W-F), and Y-Y-Y-Y-Y-Y (Y6). The experiments were performed at −25 mV (cis at ground) in an electrolyte solution containing 1 M NaCl and 10 mM Tris•HCl (pH 7.5). The α-HL protein was added in the cis compartment, while peptides Y-F-F, endomorphin-1, and/or Y6 were added in the trans compartment of the chamber device.

Figure 5.

a) Single-channel recordings and b) all points histograms, showing different binding behaviors of α-hemolysin mutant protein homoheptamer (M113Y)₇ pore with peptides endomorphin-1 (Y-P-W-F) and endomorphin-2 (Y-P-F-F). The experiments were performed at +50 mV (cis at ground) in an electrolyte solution containing 1 M NaCl and 10 mM Tris•HCl (pH 7.5). The α-HL protein was added in the cis chamber compartment, while peptides endomorphin-1, and/or endomorphin-2 were added in the trans compartment of the sensing chamber.