Biosensor Development with a Glutamate Receptor Ion-Channel Reconstituted in a Lipid Bilayer

Abstract

A glutamate receptor ion channel (RIC) protein, isolated and purified from rat brains, was reconstituted into artificial bilayer lipid membranes. This RIC protein was found to serve as a recognition site for a sensitive detection of L-glutamate. Two types of RIC sensors were tested. When the RIC was reconstituted as a single protein in a patchclamp membrane configuration, digital "off/ on" signals were obtained for L-glutamate. With multiple proteins as a multi-channel sensor, the integrated do signals were related to the glutamate concentration. The results from these two configurations are further discussed in terms of signal amplification and concentration dependence of the sensor.

An Evaluation of Signal Amplification by the Ion Channel Sebsir Based on a Glutamate Receptor Ion Channel Protein

“…Since ca. 2.1 x 10s ions are estimated to permeate through the channel in each of the channel-open periods (vide supra) [7], the molecular response ratio obtained above would reflect the integrated amplification due to ca. 10 channel-openings per second.…”

“…The remarkable detection limits of these ion channel sensors are evidently based on the intense signalamplifying ability of the G1uR ion channel protein, as supported by an estimation in our previous study that the average number of ions permeated through the channel during each of the channel-open period is ca. 2.1 x 105 [7]. This number can be regarded as a signal amplification factor.…”

“…We have recently reported a series of fundamental studies aimed at a new class of sensors that would be designated as "ion channel sensors" [2][3][4][5][6][7][8]. In the studies based on artificial systems, the "molecular response ratio" has been calculated as an indication of signal amplification [2][3][4]6].…”

Methods of Analysis for Chemicals that Promote/Disrupt Cellular Signaling

“…[7][8][9][10][11] A membrane-protein-based ion-channel sensor for glutamate and active (uphill) transport sensor for glucose were first reported later in 1990 and 1993, respectively. [12][13][14][15] Coulometric biosensors using glutamate receptor (GluR) ion channel protein as a signal-amplifying sensory element that exploits the glutamate-triggered Na + ion current through bilayer lipid membranes have been fabricated. 13 The formation of stable planar bilayer lipid membranes was achieved by applying the folding method across a small circular aperture bored through a thin polyimide film.…”

A Glass Capillary Ultramicroelectrode with an Electrokinetic Sampling Ability

“…Glass capillary micropipets have also been employed for microiontophoresis to inject exogenous substances into specific regions in biological systems. 9 Taking an advantage of the very small tip size, pulled glass capillaries have been employed as microholders for constructing electrochemical microsensors, such as carbon fiber microelectrodes, 10,11 bilayer lipid membrane microsensors [12][13][14] and oxygen microsensors, 15 and also in achieving enhanced and variable mass-transfer rates at a hydrodynamic ultramicroelectrode. 16 The use of glass capillaries with a conical or cylindrical tip (1.5 -2 µm) as a diffusional microburet for microtitration [17][18][19] and quantitative reagent delivery for fine chemical manipulations 20,21 has also been reported.…”