Binding of a protein at a ligand-modified ion channel can lead to closure or distortion of the channel, resulting in a digital on/off response of the ion flux. This principle could be applied as a strategy for sensor design using artificial membrane ligands or receptors. Important details of modeling and a technique of realization of the new molecular electronic sensor device are presented. The response of the sensor induced by analyte binding depends on the analyte's size and ability to close or distort the ion channel. Testing different ion channels in a typical single molecule-binding event modifies the membrane current by about 0.2 to 20 pA. The gated channel-based sensor is set up by using elements from different fields. It consists of a stable transmembrane channel (for example, a chemically engineered 6.3 helix peptide antibiotic) with a ligand covalently bound at the peptide channel entrance, an electrochemical sensor chip with a photostructurized hydrophobic polymer frame, a hydrophilic ion-conducting membrane support, a lipid membrane incorporating the engineered ion channel, and a sensitive membrane current amplifier or a sensitive fluorescence monitor. Detection of channel opening or closure can either be obtained directly by monitoring membrane conductivity or by monitoring a transient change in pH or ion concentration within the membrane compartment. This transient change can be induced by various means, and its decay is directly correlated to the ion permeability of the membrane. Nonspecific binding at a lipid membrane does not interfere with the ion flux through ion channels. This is a basic advantage of this principle compared to that of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or hybridization of labeled DNA/RNA probes. Furthermore, because nonspecific binding events are mainly transient, specific binding events directly at the ion channel can be separated from them by evaluating the current peak time scale. Theoretically, the sensitivity of such a device can approach one molecule. Under practical conditions, the sensitivity is limited by the diffusion process of the analyte to the receptor ligand but not by the molecular electronic device. The new type of biosensor combines sensor design and technology from immunoassays and biorecognition assays with automated analysis systems and knowledge from the field of nanotechnology. Based on the detection principle, miniaturized portable sensor chips with either electronic or optical detection are under construction. These may have capabilities exceeding those of existing analytical instruments (for example, ELISA tests, medical analyzer, and high-pressure liquid chromatography [HPLC].
Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies
