Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity,Analytical Chemistry

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Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-01-16 00:00:00 , DOI: 10.1021/acs.analchem.7b02961
Young Hun Kim , Leibniz Hang , Jessica L. Cifelli , David Sept ₁ , Michael Mayer ₂ , Jerry Yang

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Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 10³ times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.

中文翻译：

基于频率的Gramicidin A纳米孔分析可检测具有皮摩尔灵敏度的小分子

检测低浓度小分子的方法可用于广泛的分析问题，包括临床测定方法的开发，复杂生物系统的研究以及生物战剂的检测。本文介绍了一种半合成离子通道平台，该平台能够以皮摩尔灵敏度检测小分子分析物。我们的方法利用了由短杆菌肽A（gA）纳米孔形成的离子通道的瞬态性质以及观察到的单通道事件的频率，该事件是双层膜中可逆地二聚的游离gA分子浓度的函数。我们最初使用蛋白质（此处为单克隆抗体）隔离C的离子通道活性。-末端修饰的gA衍生物。将小分子分析物引入电记录介质后，它会竞争性地结合蛋白质并释放gA衍生物，从而恢复其单离子通道活性。我们发现，监测gA通道事件的频率可以检测溶液中小分子的皮摩尔浓度。在某种程度上，由于基于频率的分析的数字开/关性质，该方法为10 ³比测量通过gA通道的宏观膜离子通量作为检测的基础灵敏两倍。因此，这种新颖的方法极大地提高了小分子分析物基于纳米孔的传感器的检测限度，该限度有可能整合到可以弥补当前已建立的测定方法的小型化和低成本装置中。

更新日期：2018-01-16

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