Anal. Chem. I W S , 85, 363-369
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Na+/o-Glucose Cotransporter Based Bilayer Lipid Membrane Sensor for D-Glucose Naomi Sugao, Masao Sugawara, Hirotsugu Minami, Masayuki Uta> and Yoshio Umezawa*J Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060, Japan
A new type of amperometrlc blosensor for glucose was fabrlcated udng a Na+/r+glucosa cotranrporteras the dgnaltransducing sensory element that exploits the ~-glucosetriggered Na+ Ion current through bilayer llpld membranes (BLMs). The planar BLM was formed by the folding method across a small aperture of a thin Teflon fllm. The Na+/b glucose cotramporter,Isolatedand pumedfromsmall Intestinal brush border membrane of guinea pigs, was embedded Into BLMs through proteollporromes. The number of the protein molecules thus IncorporatedIn the present sendngmembrane was estlmated to be ca. lo7. The sensor response was measured as an lonlc current through the BLM arklng from cotransportedNa+ Ion flux under a constant applled potential and was only Induced by wlucose above lo4 M, but not by the other monocurccharklesexcept for walactose. The effect of appiled potentlala, Na+ and K+ Ion concentrations, and the additkn of a competitlve Inhlbitor, phlorlzln, were scrutinized to charactorlze the sensor output. The results were brlefly dkcussed In terms of the potential use of the Na+/wlucobe cotranrporter as a sensory element for pglucose.
INTRODUCTION For biosensor developments, few membrane proteins have been utilized until now, although they have been known to display important roles in various modes of transmembrane signalling such as ion-channel and active transport phenomena.' The membrane proteins that have been exploited as potential sensory elements include nicotinic acetylcholine receptor (nAChR),2-' auxin-receptor ATPase? H+/lactose cotransporter) and maltose binding protein.10 However, in most cases, only the receptor function rather than the function of transmembrane signalling was utilized. In our series of studies toward the development of ion channel sensors,based t Present address: Department of Environmental Technology,Kitami Institute of Technology, Kitami 090, Japan. t Permanent address: Department of Chemistry, Faculty of Science, The University of Tokyo, Hongo, Tokyo 113, Japan. (1)Alberta, B.; Bray, D.; Lewis, J.; Raff, M.; Roberta, K.; Watson, J. D. Molecular Biology of the Cell; Garland Publishing, Inc.: New York, 1991;Chapter 6. (2)Dalziel, A. W.; Georger, J.; Price, R. R.; Singh, A.; Yager, P. Membrane Proteins, Proceedings of the 1986 Membrane Protein Symposium; Goheen, S . C., Ed.; Bio-Rad Laboratory: Richmond, CA, 1987; pp 643-673. (3)Gotoh, M.; Tamiya, E.; Momoi, M.; Kagawa, Y.; Karube, I. Anal. Lett. 1987,20,857-870. (4)Eldefrawi, M. E.; Sherby, S.M.; Andreou, A. G.; Mansour, N. A.; Annau, Z.; Blum, N. A.; Valdes, J. J. Anal. Lett. 1988,21, 1665-1680. (5)Taylor, R. F.; Marenchic, I. G.; Cook, E. J. Anal. Chirn. Acta 1988, 213,131-138. (6)Rogers, K. R.; Valdes, J. J.; Eldefrawi, M. E. Anal. Biochem. 1989, 182,353-359. (7)Rogers, K. R.;Valdes, J. J.; Eldefrawi, M. E. Biosens. Bioelectron. 1991,6,1-8. (8)Thompson, M.; Krull, U. J.; Venis, M. A. Biochem. Biophys. Res. Commun. 1983,110,300-304. (9)Kiefer, H.; Klee, B.; John E.; Stierhof, Y.-D.; Jiihnig, F. Biosens. Bioelectron. 1991,6,233-237. (10)Zhou, L. Q.;Cass, A. E. Biosens. Bioelectron. 1991,6,445-450.
0003-2700/93/0365-0363$04.00/0
on artificial as well as biological ~ystems,ll-~9 a purified glutamate receptor ion channel protein, one of the most important membrane proteins, was utilized as a signalamplifying sensory element: the sensor exploited the glutamate-triggeredNa+ion-channelcurrent through bilayer lipid membranes (BLMs), yielding remarkable sensitivity. In the present study, we exploited another kind of membraneprotein called Na+/D-glucosecotransporter for the sensingof glucosein aqueous solution. Thisapproach is based on a principle defined as "active or uphill transport", which is of course different from that of conventionalglucose enzyme electrodes. Active transport of D-glucose across the cell membrane is the process that enables accumulation of a small concentration of extracellular glucose into the cell against ita concentration gradient. This process is known to be displayed by a transport protein called Na+/D-glucosecotransporter.20.21The driving force for D-glucose to be pumped is provided as an electrochemical Na+ gradient. The Na+/D-glucosecotranaporter is specifically activated by Na+ ions that populate rich in the extracellular side, and the energy conversion proceeds in a coupled transport: a Na+ flux, following its electrochemical gradient, is coupled to a D-glucose flux against its concentration gradient with a coupling stoichiometry of either Na+: D-glucose = 1:l or 2:1.21 Na+ ions that enter the cell together with D-glucose are pumped out by Na+, K+-ATPase, so that the Na+ gradient is virtually infiiitely maintained. In the present Na+/PglucosecotransporterembeddedBLM sensor (Figure 11, the electrochemical Na+ gradient Aji, the drivingforce for the activetransport of Pglucuse, was provided
as
where f i ~ ~ + hand f i ~ ~ + care k the electrochemicalpotentials of Na+ ions in the trans and cis sides, respectively, a ~ the~ + activity of Na+ ions in the side d e f i e d by the superscript, AE the applied electricpotential difference across the membrane, (11)Umezawa, Y.; Sugawara, M.; Kataoka, M.; Odashima, K. In ZonSelective Electrodes, 5; Pungor, E., Ed.; Akadbmiai Kiad6 (Pergamon Press): Budapest (Oxford), 1989;Vol. 5, pp 211-234. (12)Odashima, K.; Umezawa, Y. In Biosensor Technology; Buck, R. P., Hatfield, W. E., Umaiia, M., Bowden, E. F., Us.; Marcel Dekker: New York. 1990: ChaDter 6. (13)Odkhima, K.;Sugawara, M.; Umezawa, Y. Trends Anal. Chem. 1991. 10. ~ .. - 207-215. (14)Sugawara,M.;Kojima, K.; Sazawa,H.; Umezawa, Y.Anal. Chern. 1987.59, 2842-2846. (15)Sugawara, M.;Kataoka, M.; Odashima, K.; Umezawa, Y. Thin Solid Films 1989,180, 129-133. (16)Nagase, S.; Kataoka, M.; Naganawa, R.; Komatsu, R.; Odashima, K.; Umezawa, Y. Anal. Chem. 1990,62,1252-1259. (17)Sugawara, M.; Sazawa, H.; Umezawa, Y. Lamgmuir 1992,8,6O$b
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(18)Uto, M.; Michaelis, E. K.; Hu, I. F.; Umezawa, Y.; Kuwana, T. Anal. Chem. 1990,6,221-225. (19)Minami, H.; Sugawara, M.; Odashima, K.; Umezawa, Y.; Uto, M.; Michaelis, E. K.; Kuwana, T. Anal. Chem. 1991,63,2787-2795. (20)Crane, R. K. Physiol. Rev. 1960,40,78S825. (21)Kimmich, G. A. J. Membr. Biol. 1990,114, 1-27. 0 1993 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
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0 D-Glucose
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m 0 Na+
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Flguro 1. Schematlc diagrams representlng the principle of a Na+/ DglucosecotransporterembeddedBLM sensor. (A) A modelstructve of the proposed sensor. (B) The driving force (electrochemical Na+ gradient) for energizing the Na+/Dgiucose cotransporter system Is provided by a Na+ concentration gradlent directed from the trans to the CISsides plus an electrlc potential applied negative to the cis side relative to the trans side. The Dglucose sample solution was injected to the trans side solution.
(m
and R, T, and F have their usual significance. It can be seen from eq 1that the larger the Na+ concentration gradient or the applied potential is, the larger the rate of D-glucose transport becomes. In the present sensing system,the cotransported Na+ ions across the BLM was measured as Na+ ion currents under a constant applied potential. The magnitude of this current was found to be a new analytical measure of the amount of D-glucose in solution.
EXPERIMENTAL SECTION Materials. L-a-Phosphatidylcholine (PC; from frozen egg yolk; 100 mg/mL hexane solution) was purchased from Sigma Chemical Co. (St. Louis, MO). Cholesterol was from Wako Pure Chemicals Co. (Osaka, Japan) and recrystallized twice from methanol. D-Glucose, L-glucose, D-mannose, and D-fructose (Wako Pure Chemicals Co.) were all of analytical grade. D-Galactose (Sigmagrade; containing
