Capone Ricardo, Blake Steven, Restrepo Marcela Rincon, Yang Jerry, Mayer Michael
Department of Chemical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, Michigan 48109-2099, USA.
J Am Chem Soc. 2007 Aug 8;129(31):9737-45. doi: 10.1021/ja0711819. Epub 2007 Jul 11.
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.
在单分子水平上检测化学过程是灵敏分析测定的最终目标。我们最近报道了通过监测短杆菌肽A（gA）衍生物与溶液中的分析物反应时单离子通道电导的变化来检测单个分子上化学修饰的可能性。这些基于肽的纳米传感器检测gA衍生物电荷的反应诱导变化，这些衍生物被设计为在其C末端附近带有特定官能团。在此，我们讨论五个关键设计参数以优化此类化学调制离子通道传感器的性能。为了实现一个有效的传感器来测量连接到gA孔C末端的基团电荷的变化，应满足以下条件：（1）电荷变化应尽可能靠近孔的入口发生；（2）反应前后的电荷在操作pH范围内应明确界定；（3）记录缓冲液的离子强度应尽可能低，同时保持可检测的离子流通过孔；（4）施加的跨膜电压应尽可能高，同时保持膜的稳定；（5）支撑膜中的脂质应为两性离子型或与gA衍生物带不同电荷。我们表明，在高跨膜电位（>100 mV）和低记录缓冲液离子强度（≤0.10 M）的条件下，区分gA的两种不同电荷衍生物的主要要求是孔入口处的电荷变化；此处报道的异二聚体gA孔的电导不取决于孔出口处电荷的差异。我们基于脂质双层膜附近阳离子浓度的电荷诱导局部变化对此不对称特性提供了一个简单解释。基于电荷的离子通道传感器在超灵敏功能检测方面具有巨大潜力，因为每个单独传感元件的单个化学修饰可导致通道电导的易于检测的变化。
