A New enzymatic receptor to be used in a biosensor

Journal of Membrane Science, 49 (1990) 95-102 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

A NEW ENZYMATIC BIOSENSOR*

RECEPTOR

95

TO BE USED IN A

EIJI OHASHI Central Research

Laboratory,

Nippon

Suisan Kaisha Ltd., 559-6, Kitano-cho,

Hachioji,

Tokyo 192 (Japan)

EIICHI TAMIYA and ISA0 KARUBE Research Center for Advanced Science Meguro-ku, Tokyo 153 (Japan)

and Technology,

University

of Tokyo, 4-6-l

Komaba,

(Received October 12,1987; accepted in revised form August 18, 1989)

Summary An artificial lipid membrane which incorporated an enzyme was prepared and used for the molecular recognition element of a sensor. This membrane constituted alanine aminotransferase and an acetylcellulose membrane containing phospholipid and cholesterol. The membrane exhibited specific response to the substrate, resulting in a change in membrane potential. In the experiment, this enzyme was used as an L-alanine receptor, since the enzyme does not show catalytic activity under restricted conditions. When L-alanine was applied to the membrane, surface charge density increased with application time. Membrane potential appeared to be shifted mainly by the change of surface charge density caused by formation of an enzyme-alanine complex.

Introduction

Biochemical sensors using immobilized enzymes or micro-organisms, socalled enzyme sensors or microbial sensors, have been developed [l-3]. These sensors are very useful in clinical analysis and food process control because of their excellent sensitivity and selectivity [4,5]. In contrast, few papers have been presented on taste sensors, for which the food industry has a great requirement. Relatively little is understood about the biochemical basis of the taste reception system [ 6-81, but it has been proposed that biological taste reception is initiated by the specific adsorption of taste substances onto the specific site of taste cell membranes, not by their transportation. In the case of sweet-tasting substances or some amino acids, the specific receptor proteins are incorporated in the taste cell membrane [9,10]. When a *Paper presented at ICOM ‘87, Tokyo, Japan, June 8-12,1987.

0376-7388/90/$03.50

0 1990 Elsevier Science Publishers B.V.

96

taste substance binds to a specific receptor, a change in membrane potential is induced due to the conformational change in the receptor or change in the surface charge density. In this report, a new sensing system, which was a model of the taste reception system, was constructed using an artificial planar lipid bilayer which incorporated an enzyme that could act as a receptor element. Enzymes were used instead of actual receptor proteins because the actual receptor proteins are difficult to obtain and are typically very unstable, while enzymes are easily available and possess greater stability. Therefore the enzyme was used under restricted conditions in which enzyme reaction did not occur. Experimental Materials The enzyme used was alanine aminotransferase obtained from Boehringer Mannheim Yamanouchi (Tokyo), which acted as a receptor of L-alanine. This enzyme catalyzes reaction ( 1) : L-Ala+2-oxoglutarate-+pyruvate+~-Glu

(I)

In our measurement system no 2-oxoglutarate was present; therefore the enzyme cannot catalyze the above reaction even if there are many L-alanine molecules in the system. However, the affinity to L-alanine remains, and thus the enzyme functions only as a receptor. Phosphatidylcholine and phosphatidylserine were purchased from Sigma Chemical Co. These reagents were used without further purifications. Cholesterol and L-alanine were purchased from Nakarai Chemical Ltd. (Kyoto) and Wako Pure Chemical Industries Ltd. (Osaka), respectively. Other reagents used were of analytical grade. Preparation of planar enzyme-lipid membrane The planar lipid membrane was made as follows. A Millipore membrane filter, which was used as a support, was immersed in a chloroform solution of phospholipid and cholesterol for 15 min, then the membrane was taken out of the solution and dried under a nitrogen gas stream for 2 hr. This procedure enabled the filter pores to till with lipid. Liposomes incorporating enzyme were made as follows. Enzyme was added to a multilamellar liposome suspension prepared by vortex mixing 100 mM KC1 solution and lipid thin films. This solution was then sonicated at 40 W for five 30 set periods. The Millipore membrane filter was immersed in this liposome suspension, 30 mM CaCl, was added, and the system was then left overnight at room temperature. The planar lipid membrane in the Millipore membrane filter and the liposomes became fused, and enzyme was thus introduced into the planar lipid membrane.

97

Fig. 1. Schematic diagram of the membrane potential measuring system. 1: Recorder; 2: electrometer; 3: Ag/AgCl electrode; 4: H-type cell; 5: silicone rubber; 6: lipid membrane; 7~3: salt solutions C(l), C(2); 9: stirring bar.

Measurement of membrane potential In order to measure the membrane potential, the system shown in Fig. 1 was used. The Millipore membrane filter was put between silicone packing pieces and positioned at the center of a glass cell of the so-called H-type, and aliquots of various salt solutions were added. The mean diameter of this membrane was 12 mm. The electrical potential of the membrane was measured using Ag-AgCl electrodes connected to an electrometer (Hokuto Denko Co., HA lOl), and was displayed on a recorder. One electrode was earthed as shown. The measurements were carried out at room temperature while the solutions in the cells were stirred. Results and discussion Electrical potential of enzyme-lipid membrane Figure 2 shows the relationship between salt concentration C( 1) and membrane potential (A@). Lipid membranes were prepared from phosphatidylcholine (PC) and cholesterol (Chol) or PC, phosphatidylserine (PS) and Chol. The Millipore membrane filter (pore size 0.025 pm, type VS) was immersed in a chloroform solution of PC and Chol or PC, PS and Chol, and the enzymeliposome was prepared from the same lipid composition. Membrane potential was measured using KC1 solution under the constant concentration ratio condition. In the case where two different concentrations of salt solution, C ( 1) and C( 2), come into contact with a membrane, the membrane potential is expressed as a sum of surface potential and diffusion potential [ 111. The depen-

Fig. 2. Potential response of lipid membrane with or without enzyme. Membrane was prepared from chloroform solution oflipids PC:PS:Chol=l:O:l (A,A); PC:PS:Chol=4:1:5 (0,O). Total lipid concentration was 200mg/ml. ( l ,A ), Including alanine aminotransferase.

dence of the membrane potential (A$) on electrolyte concentration is approximated by the following eqn. (2 ) when uni-univalent electrolyte solutions are separated by a charged membrane, and when the electrolyte concentration is much higher than the surface charge density of the membrane: gTA$=

(2t-1)ln

y+

2(Y--1) Y

t(l-t&-

C(l)

(2)

where y indicates salt concentration ratio, R, T and F have their usual meaning and t and 8 indicate the transportation number and surface charge density, respectively [ 121. Then, when the membrane potential is measured at various concentrations of salt solution under conditions of constant y, T and 0, the membrane potential is directly proportional to the reciprocal of the salt concentration. In other

99

TABLE 1 Characterization

of Millipore-lipid

membrane”

Lipid composition

Transportation number

Surface charge density ( X 10e3 es/l)

PC, PC, PC, PC,

0.522 0.506 0.518 0.518

3.26 4.30 26.7 34.7

Chol Chol+ enzyme PS, Chol PS, Chol+enzyme

“Membrane

potential

was measured using KC1 solution at y= 2.

words, transportation number and surface charge density can be calculated from these data. In Fig. 2, the reciprocal of KC1 concentration l/C( 1) is shown on the horizontal axis and the membrane potential on the vertical axis. Electrical potential was measured under the constant condition y= 2. In this plot, the membrane potential changed almost linearly at high concentration. Phosphatidylserine, which is an acidic phospholipid, gives the membrane a negative charge. Therefore the difference between the slopes of the lines shown in Fig. 2 reflects the difference in charge density. On the other hand, the incorporation of enzyme did not exert a significant effect on charge density. The surface charge density and transportation number were calculated from the slope and intercept of the above-mentioned data using eqn. (2). Surface charge density was determined to be 4.30 x 10e3 eq/l for the membrane incorporating enzyme, PC and Chol. These data are summarized in Table 1. In every case, transportation numbers showed almost the same values but the charge densities were very different. Potential change associated with the adsorption of L-alanine A 100 mM L-alanine solution in phosphate buffer, pH 7.4, was applied to the membrane for 30 min at room temperature. After incubation, the membrane was washed lightly with alanine-free phosphate buffer and membrane potential was measured according to the same procedure. Figure 3 shows the change in membrane potential before and after applying L-alanine solution. The membrane potential decreased at every salt concentration. This change was considered to be a result of the formation of an alanine-enzyme complex. On the other hand, a decrease of surface charge density was observed after washing the L-alanine-binding membrane. A membrane that had been in contact with L-alanine solution for 60 min indicated 5.00 x 10V3eq/l surface charge density. L-Alanine solution in the cell was then replaced by alanine-free phos-

Fig. 3. Potential response of enzyme-lipid membrane to L-alanine. Lipid membrane was prepared from PC, Chol in the same manner as described in Fig. 2. Before (0 ) and after (0 ) applying 100 mM L-alanine solution for 30 min.

-yyY-s0

/”

z

/O

0.520

$ :

/-4._._&-_~--______-_n____

n

30

60 Time

(

min

Fig. 4. Change in surface charge density membrane.

I

90

) (0 ) and transportation

Lipid membrane prepared from PC and Chol.

number

(A ) of enzyme--lipid

101

phate buffer. After 30 min washing the surface charge density reverted to 4.30 x lop3 eq/l, which was the original level. Figure 4 shows the change in surface charge density and transportation number plotted against time of application of alanine solution. After each measurement, the membrane was washed using alanine-free phosphate buffer for 30 min, and it was confirmed that the surface charge density was dependent on the application time but transportation number was almost constant. These data suggest that the observed change may be due partly to rearrangement in the enzyme structure during binding to alanine, consequently effecting a change in the membrane properties, or to the negative charge of the adsorbed alanine molecules. Selectivity of enzyme-lipid membrane The selectivity of the alanine aminotransferase-incorporating lipid membrane was demonstrated by applying the homologous amino acid glycine. A plot of the change in surface charge density against concentrations of alanine and glycine is shown in Fig. 5. No response was observed when glycine was applied to the system up to 100 mM. Thus the change in surface charge density may be caused by the specific adsorption of L-alanine onto the membranebound enzyme and the difference between the alanine and glycine responses may be reflected by the selectivity of enzyme itself. Generally, water-soluble proteins change their conformation in a hydrophobic environment. In the case of this experiment, it is not clear whether the conformation of enzyme is changed

3 .Y 2 $

4.6

;

4.4

2 " : h L s

4.2

100

50 Substrate

concentration

( mM)

Fig. 5. Difference between response to L-alanine (0 ) and glycine (0 ) of enzyme-lipidmembrane.

102

not; however, incorporation did not exert a significant change in the affinity for L-alanine.

or

Conclusions A planar lipid membrane incorporating alanine aminotransferase was used to detect L-alanine. The enzyme functioned as a receptor, not as a catalyst. The membrane was made of phospholipid, cholesterol and enzyme, with a Millipore membrane filter used as a support. A change in membrane potential was induced by the specific adsorption of alanine, resulting in a change of surface charge density.

References 1 2 3 4 5 6 7 8 9 10 11

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I. Karube, E. Tamiya, J.M. Disks and M. Gotoh, A microsensor for urea based on an ionselective field effect transistor, Anal. Chim. Acta, 185 (1986) 195. I. Karube and E. Tamiya, Biosensors for environmental control, Pure Appl. Chem., 59 (1987 ) 545. I. Kubo, I. Karube and T. Moriizumi, Micro ammonia sensor, Anal. Lett., 19 (1986) 697. R. Stevanato, L. Avigiliano, A. Finazzi-Agro and A. Rigo, Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase, Anal. Biochem., 149 (1985) 537. M.N. Rahni, G.G. Guilbault and N. Olivera, Immobilized enzyme electrode for the determination of oxalate in urine, Anal. Chem., 58 (1986) 523. K. Yoshii and K. Kurihara, Role of cations in olfactory reception, Brain Res., 274 (1983) 239. K. Torii and R.H. Cagan, Biochemical studies of taste sensation, Biochim. Biophys. Acta, 627 (1980) 313. J.E. Segall, A. Ishihara and H.C. Berg, Chemotactic signaling in filamentous cells of E. cob, J. Bacterial., 161 (1985) 51. K. Yoshii and K. Kurihara, Ion dependence of the eel taste response to amino acids, Brain Res., 280 (1983) 63. A.F. Russo and D.E. Koshland, Jr., Separation of signal transduction and adaptation functions of the aspartate receptor in bacterial sensing, Science, 220 (1983) 1016. M. Aizawa, S. Kato and S. Suzuki, Immunoresponsive membrane. I. Membrane potential change associated with an immunochemical reaction between membrane-bound antigen and free antibody, J. Membrane Sci., 2 (1977) 125. M. Nakagaki and K. Miyata, Membrane potential and permeability coefficient of cellulose membrane, Yakugaku Zasshi, 93 (1973) 1105.