gramicidin-a and 6-carboxyfluorescein

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 10

Topics: Antibodies, Monoclonal; Biosensing Techniques; Fluoresceins; Gramicidin; Haptens; Ion Channels; Limit of Detection; Lipid Bilayers; Nanopores

Using fluorescent, anionic dyes such as carboxyfluorescein as model solutes, it is shown that the forces allowing such solutes to be retained within sealed lipid vesicles, against a large concentration gradient, can be primarily electrostatic in nature. At temperatures distant from that of the ordered-fluid lipid phase transition a small number of the anionic dye molecules trapped within lipid vesicles are capable of traversing the lipid bilayer and establishing an electrical diffusion potential across the membrane. Further solute movement can then only occur with the concomitant permeation of ions which restore electrical balance. A significant flux of dye can be triggered by (a) increasing the permeability of the membrane to ions (for example by the addition of ionophores such as gramicidin, or by allowing the lipid to approach a phase transition) or by (b) adding lipophilic counterions such as tetraphenylborate or dinitrophenol to the system.

Topics: Anilino Naphthalenesulfonates; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane Permeability; Fluoresceins; Fluorescent Dyes; Gramicidin; Lipid Bilayers; Membrane Potentials; Phosphatidylcholines