Coulometric biosensors using glutamate receptor (GluR) ion channel protein as a signal-amplifying sensory element that exploit the glutamate-triggered Na+ ion current through bilayer lipid membranes have been fabricated. 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. The multichannel type sensing membranes, formed across an aperture of ca. 120 microns diameter, contained more than 10 GluR proteins and showed L-glutamate-triggered response as a composite of individual single-channel currents. The single-channel type sensing membranes, formed across an aperture of ca. 20 microns diameter, contained a sufficiently small number of GluR proteins so that the response was observed as a series of single-channel pulse currents. Dependence of the integrated channel current on the glutamate concentration was examined. A sharp concentration dependence of up to ca. 1.5 x 10(-7) M and 3 x 10(-6) M for the multichannel and single-channel type sensors, respectively, was observed. A high selectivity for L-glutamate compared with D-glutamate for inducing the channel current was observed. A detection limit as low as ca. 3 x 10(-8) M was attained for the multichannel type sensor. This remarkable sensitivity is discussed in terms of the potential use of GluR ion channel protein for a new type of sensing system.

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.

A new type of amperometric blosensor for glucose was fabricated using a Na+/D-glucose cotransporter as the signal-transducing sensory element that exploits the D-glucose-triggered Na+ ion current through bilayer lipid membranes (BLMs). The planar BLM was formed by the folding method across a small aperture of a thin Teflon film. The Na+/D-glucose cotransporter, isolated and purified from small intestinal brush border membrane of guinea pigs, was embedded into BLMs through proteoliposomes. The number of the protein molecules thus incorporated in the present sensing membrane was estimated to be ca. 10(7). The sensor response was measured as an ionic current through the BLM arising from cotransported Na+ ion flux under a constant applied potential and was only induced by D-glucose above 10(-9) M, but not by the other monosaccharides except for D-galactose. The effect of applied potentials, Na+ and K+ ion concentrations, and the addition of a competitive inhibitor, phlorizin, were scrutinized to characterize the sensor output. The results were briefly discussed in terms of the potential use of the Na+/D-glucose cotransporter as a sensory element for D-glucose.

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Lipid membranes made of lecithin and cholesterol were formed by self-assembly in a small aperture on an agar support. The membranes exhibited an average electric resistance of 135 Gfl and a capacitance of 0.43 µF/cm2. Gramicidin, known to form a channel in uni-lamellar lipid bilayers, reduced the electric resistance to a Mf level, thus showing the membranes to be of a uni-lamellar bilayer type. The membrane stability was investigated against perturbation with electric potentials and against mechanical agitation in the contacted aqueous solution. About 80% of the membrane preparations remained intact after applying electric potentials of between +1500 mV and -1500 mV. A similar percentage of the membranes stayed intact under 100 rpm magnet stirring in a 30 ml vessel. Membranes containing valinomycin responded to K+ ions with changes in both the membrane conductance and the membrane potential.