Designing Nanosensors Based on Charged Derivatives of Gramicidin A

Abstract

Detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. We recently reported the possibility to detect chemical modifications on individual molecules by monitoring a change in the single ion channel conductance of derivatives of gramicidin A (gA) upon reaction with analytes in solution. These peptide-based nanosensors detect reaction-induced changes in the charge of gA derivatives that were engineered to carry specific functional groups near their C-terminus.1 Here, we discuss five key design parameters to optimize the performance of such chemomodulated ion channel sensors. In order to realize an effective sensor that measures changes in charge of groups attached to the C-terminus of a gA pore, the following conditions should be fulfilled: (1) the change in charge should occur as close to the entrance of the pore as possible; (2) the charge before and after reaction should be well-defined within the operational pH range; (3) the ionic strength of the recording buffer should be as low as possible while maintaining a detectable flow of ions through the pore; (4) the applied transmembrane voltage should be as high as possible while maintaining a stable membrane; (5) the lipids in the supporting membrane should either be zwitterionic or charged differently than the derivative of gA. We show that under the condition of high applied transmembrane potential (>100 mV) and low ionic strength of the recording buffer (< or =0.10 M), a change in charge at the entrance of the pore is the dominant requirement to distinguish between two differently charged derivatives of gA; the conductance of the heterodimeric gA pore reported here does not depend on a difference in charge at the exit of the pore. We provide a simple explanation for this asymmetric characteristic based on charge-induced local changes in the concentration of cations near the lipid bilayer membrane. Charge-based ion channel sensors offer tremendous potential for ultrasensitive functional detection since a single chemical modification of each individual sensing element can lead to readily detectable changes in channel conductance.

Chemically Reactive Derivatives of Gramicidin A for Developing Ion Channel-Based Nanoprobes

“…ably amplify a signal by monitoring a flux of ions through a single pore; (2) its current levels are distinct and quantized, which facilitate the measurement of changes in conductance on a single pore level; (3) its C-terminus is conducive to derivatization without compromising function (2,3,(9)(10)(11)(12)(44)(45)(46)(47)(48); (4) its availability in gram quantities from commercial sources makes it possible to generate tailored derivatives on practical scales; (5) its self-incorporation into lipid bilayers makes its use straightforward as opposed to most ion channel proteins that require incorporation into bilayers through proteoliposome fusion (49)(50)(51)(52); (6) its electrophysiological characteristics are well explored, which makes it an excellent model for fundamental biophysical studies of pores in membranes; (7) its size is nanoscale, which is conducive to miniaturized device designs and may enable parallel assays developed in a high density format; and (8) its activity is localized in a membrane, a property useful for applications where control of location is important. Figure 1A shows the sequence and helical structure of gA. Gramicidin A reversibly dimerizes in a bilayer ( Figure 1B), forming an N-terminus to N-terminus dimeric structure held together by a network of hydrogen bonds (53)(54)(55).…”

Truncated β-amyloid peptide channels provide an alternative mechanism for Alzheimer’s Disease and Down syndrome

“…Images were acquired with a Multimode AFM, using a Nanoscope IVA controller (Veeco). Single-channel conductance measurements were carried out using painted membranes as published previously (8,13,14,(46)(47)(48)(49) (SI Materials and Methods). Cell (APP-deficient mouse fibroblast cell line) calcium imaging (Fluo-4 NW) was carried out for p3 and N9 (under normal and reduced extracellular calcium levels, as well as with and without preincubation with zinc chloride), p3-F19P mutant, and scrambled p3 peptides under normal extracellular calcium level.…”

Membrane protein-based biosensors

“…Accordingly, synthetic pores have been reported in recent studies to overcome the limited variations of the pores based on the pore-forming toxins. A peptide nanopore, gA, was partially modified as a semi-synthetic pore, and it revealed the significant influence of the C-and N-termini on the pore properties [75][76][77]. Using the DNA origami technology, DNA was assembled into sophisticated nanopores with a gating mechanism, functionally resembling ligand-gated ion channels [78][79][80][81].…”