J. Pack. Sci. Tech. Vol. 12 No. 6 (2003)

一般論文

Application of a contact dehydrating sheet to the
preparation of an immobilized-enzyme
membrane for biosensor
Sachie OGAWA, Naoko HAMADA-SATO* , Takeshi KOBAYASHI,
Chiaki IMADA and Etsuo WATANABE
Some enzymes were immobilized in an inner layer of a filter paper using the osmotic actionof a contact dehydrating sheet (CDS). CDS was examined to determine if the efficiency andrapidity of an enzyme reaction could be improved, thereby making better of the preparation of animmobilized-enzyme membrane. The activity of the membrane, in which an enzyme exists in its
layer, was compared with that of a conventional enzyme adsorption membrane. pH was
examinedmodulated for the enzyme to obtain a higher migration rate into the membrane. The
membrane in which layer an enzyme exists and the enzyme adsorption membrane were placedon the oxygen electrode. The glucose sensor, the D-amino acid sensor and the sarcosine sensor
were constructed. The use of CDS successfully increased enzyme migration into the membraneand enhanced the signal intensity per unit of enzyme. From this point of view, there is a verystrong possibility that biosensors utilizing an immobilized-enzyme membrane prepared with CDS
can be developed. This is the first report on the application of CDS to bioengineering fields
except for food wrapping.
Keywords : Immobilized-enzyme membrane, contact dehydrating sheet (CDS), enzyme
accumulation membrane, enzyme adsorption membrane.

1.

Introduction
A contact deh yd rating sheet (C DS) was invented as a materi al fo r remo ving water fromfoods such as fish and meat by osmotic action without heating, freeze-drying and related
processes. CDS consists of two pieces of polyvinyl alcoholate film co mp osed of ed ible sugarplaced between two layers of a semi-permeable membrane made of polyvinyl alcohol 1 ) ～ 3 ) . The
film has many pores that enable the permeation of small molecules such as water andtri meth ylamine but retains relatively larg e mo lecules such as proteins and enzymes 4 ) . To use
CDS, for instance, a piece of meat should be wrapped completely allowing full contact between
CDS and the entire surface of the meat (for a pre-determined period of storage at 0~5℃ ) .
Even at such low temperatures, CDS can selectively remove water from food through its semip e r m e a b l em e m b r a n e .

___________________________________________________________________________________________"Corresponding author. E-mail : hsnaoko@s.kaiyodai.ac.jp
Laboratory of Applied Microbiology, Graduate School of Marine Science and Technology, Tokyo University of Marine
Science and Technology, 5-7, Konan 4, Minato-ku, Toky o 108-8477, JAPAN- 3 5 1 -Application of a contact dehydrating sheet to the preparation of an immobilized-cnzyme niembrare for biosensor

When compared to conventional chemical analytical techniques, biosensor has a numberof advantages that include the direct and accurate detection of components in samples, rapidityof signaling, convenience in operation, and high biospecificity. One disadvantage of biosensors
is their lack of stability. The progress in enzyme immobilization technology has assisted infurther improvement to obtain a stable enzyme activity 5 ) 6 ) . Conventionally, an enzyme isimmobilized on a membrane. In order for an enzyme to function as a receptor, chemical
modification methods are used, for example, 1) covalent bonding, 2) crosslinking, 3) coveringwith polymers, and 4) adsorption 7 ) . However, an enzyme is damaged by chemical agentsduring covalent bonding or crosslinking. Furthermore, the disadvantages of these methods
include the need for a large amount of enzyme and their high cost.
In this work, we aimed to develop a novel method of immobilizing enzymes inside amembrane using CDS, which has been used so far in only field of food industries as wrapping
materials. Conventionally an enzyme is immobilized randomly in a membrane. The
immobilized enzyme system using CDS is developed to improve the efficiency and rapidity ofan enzyme reaction. Furthermore, the CDS modification is considered to causes minimal
damage to the enzyme. It is also expected that the advantage of the new method lies in its
simple preparation and low consumption of enzymes.

2. Materials and Methods

2.1.

Materials
Glucose, D-alanine, sarcosine and glucose oxidase (GO) (E.C.I.1.3.4 from Aspergillussp.) (pI 4.2) were obtained from Wako Co. (Osaka, Japan). D-amino acid oxidase (AO)
(E.C.1.4.2.2 from porcine kidney) (pis 4.0-7.0) and sarcosine oxidase (SO) (E.C.I.5.3.1 from
Arthrobacter sp.)(pl 4.9) were purchased from Funakoshi Co. (Tokyo, Japan) and Toyobo Co.
(N.Y., U.S.A.), respectively. All the materials were used as received.

2.2. Preparation of enzyme solution
GO and SO (0.15 U each) were dissolved in 6 μ l of 0.1 M phosphate buffer at pHs 5.8
and 7.0, respectively. AO (0.15U) was also dissolved in 6 μ l of 0.1 M Tris-HCl buffer at pHs
7.0and8.2.

2.3. Preparation of enzyme adsorption membrane (AM) and membrane in which an enzyme
exists in its layer (LM)The enzyme adsorption membrane (AM) was prepared using a filter membrane from- 3 5 2 -

J.Pack.Sci. Tech- Vol. 12 No.6 (2003)

ADVANTEC Co. (No. 51, depth 0.17 mm, Tokyo, Japan), which was cut into 5×5 mm pieces.
Each piece was dipped in 6 μl of the above enzyme solution. To prepare the membrane inwhich layer an enzyme exists, the above AM was placed on CDS (Showa Denko Plastic
Products Co., Ltd, Tokyo, Japan) overnight in a refrigerator at 4°C. The longer themembrane remains on the surface of the CDS, the further the enzyme migrates into themembrane, which should facilitate efficient enzyme reaction.

2.4. Enzyme electrode preparation
The oxygen electrode employed in this study was a Clark-type electrode consisting of aplatinum cathode, a lead anode, an alkaline electrolyte (KOH) and an oxygen-permeable
Teflon membrane. The enzyme membranes were placed on the Teflon membrane of theoxygen electrode and covered with a dialysis membrane. The LM was carefully placed on the
Teflon membrane of the oxygen electrode, with the enzyme layer touching the cathode.

2.5. Apparatus for sensor system
The measurement system consisted of an enzyme electrode, a flowcell, an electrometer(Hokuto Denko, Co., Tokyo, Japan, HE-104), a recorder (Rikadenki, Co., Tokyo, Japan, U1), an airpump (Nihon Suisoukougyou Co., Tokyo, Japan, a4000), a peristaltic pump
(Rikadenki, Co., Tokyo, Japan, MP-3N), and a buffer tank. The enzyme electrode was fixedin the flowcell, and measurement using this system was continuously performed (Fig. 1).

Fig. 1. Schematic diagra m of the enzy me s e n so r s ys t e m a n d o x yg e n ele c t ro d e(1) Pt electrode, (2) Immobilized-enzyme membrane, (3) Dialysis membrane, (4) Flow cell.

- 3 5 3 -Application of a contact dehydrating sheet to the preparation of an immobilized-enzyme membrane for biosensor

2.6. Assay procedures
A phosphate-buffered solution was transferred continuously through the sensor by theperistaltic pump. The dissolved oxygen in the buffer was always saturated by aeration. Since
the concentration of dissolved oxygen was proportional to the current decrease, 1 kΩ resistancewas connected between the enzyme electrode and the recorder in parallel, and current wascalculated based on the recorded potential difference. After the output current became steady,a substrate solution was injected into the flow line and current decrease was recorded. The
assay was performed with 0.1M phosphate buffer (pH 7.0) for glucose sensor8) and sarcosine
sensor9). Tris-HCl buffer (0.1M, pH 8.2) was used as D-amino acid sensorl0). The principle of
the above enzyme reactions is shown in Fig. 2. This study was performed at a flow rate of 0.1
ml/min. The temperature was controlled at 30"C during the enzyme reaction. A 50 μl aliquotof the substrate solution was injected into the enzyme sensor system.

Fig. 2. Principle of enzyme reaction, a) Glucose sensor, b) D-amino acid sensor, c) Sarcosine sensor

2.7. Inspection of LM activity
After the enzyme was immobilized in the layer, the membrane was separated into twopieces lengthwise. The side facing CDS was named the lower membrane and the other side of
the membrane that did not contact with CDS, was named the upper membrane. These
membranes were respectively placed on the oxygen electrode and then the enzyme electrodewas placed in the sensor system following the above procedure. A substrate solution was
injected to obtain a response. When the lower membrane had a larger response than the upper
membrane, it was considered that the LM was established. To gain a response curve, the chart
speed was adjusted to either 3 cm/h or 10 cm/h.

- 3 5 4 -

J.Pack.Sci. Tech. Vol. 12 No.6 (2003)

3. Results and Discussion

3.1. Preparation of LM
Fig. 3 shows the schematic diagram of the preparation of the LM. Firstly, 5×5 mm of
the AM was placed on CDS and then the interface was attached overall. The water migrated
inside CDS from the filter membrane due to osmotic pressure. The enzyme can not passthrough the semipermeable membrane, which was accumulated at the bottom of the filter
membrane.
As shown in Fig.4, it was considered that most of the enzyme accumulated in the lower
membrane since there was a larger response detected in the lower membrane than in the upper
membrane. The LM was prepared in this manner.
The optimal pH for the reaction of glucose with GO to produce gluconolactone is 7.0

Fig. 3. Schematic diagram of enzyme accumulated on one side of intrafilter membrane using CDS.
● H2O. ○ Enzyme (Oxidase).

Fig. 4. Relationship between enzyme activities of the inner and outer of membranes determinedusing the sensors. (■ , ●) Lower membrane. (□,○) Upper membrane.
a) Glucose sensor (pH 5.8, pH 7.0). b) D-alanine sensor(pH 7.2, pH 8.2). c) S a rc os ine sensor(pH 5.8, pH 7.0).

- 3 5 5 -Application of a contact dehydrating sheet to the preparation of an immobilized-enzymc membrane for biosensor

with the isoelectric point (pI) observed at pH 5.6 11). The pHs 7.0 and 5.8 were examined using
the CDS-immobilized- enzyme system. O2 consumption was higher near the pi than near the
pH optimum when examining the lower portion of the membrane, as shown in Fig. 4. pH
slightly higher than the pI was used to ensure the solubility of the enzyme but also to decrease
the net charge on the protein. At pH 7.0, the enzyme will have a larger net negative charge.
This elevated net charge probably slowed enzyme migration into the membrane due to theelectrostatic properties of the membrane. In other words, at a higher net charge (pH 7.0), theinteraction between the enzyme and cellulose increases, which leads to slowing migration. On
the other hand, enzyme migration near the pi should be faster due to minimized electrostaticand hydrogen bonding interactions of GO with the functional hydroxyl and ether groups of thecellulose membrane. Therefore, it appears that two factors control biosensor efficiency : 1) pH
optimum of the enzyme and 2) migration rate.
In the upper membrane, the sensor had a stronger signal at the pH optimum than at thepI (Fig. 4). This result is opposite to that observed in the lower membrane but is still
expainable. A greater amount of enzyme should reside in the upper membrane at pH 7.0 (slowmigration) that at pH 5.8 (fast migration). This is further evidence that an enzyme migrates
into the membrane interior more efficiently at pH 5.8 than at pH 7.0. Thus, GO accumulated
in the lower membrane more efficiently at pH 5.8 than at pH 7.0, which also increased theobtainable signal per unit of enzyme. pH 5.8 lies in the range of stable pHs for GO since thisenzyme is active between pHs 4.0 and 7.013). The order of efficacy was as follows: pH 5.8(lower membrane)>pH 7.0 (lower membrane)>pH 7.0 (upper membrane)> pH 5.8 (upper
membrane).
It was likewise shown that when examining D-amino acid oxidase, a stronger signal was
obtained in the lower membrane compared with the upper membrane at pHs 7.2 and 8.2 (Fig.
4b). Although the optimal pH of this enzyme is 8.2, the LM membrane functioned better when
the pH was adjusted to 7.2 (Fig. 4b). pH 7.2 is closer to the pi of the enzyme than pH 8.2.
Sarcosine oxidase was also investigated. Sarcosine oxidase accumulated in the lower membraneat pHs 5.8 and 7.0 (Fig. 4c). A higher signal was obtained at pH 5.8, which is closer to the piof this protein than pH 7.0. The optimum pH of this enzyme is between 7.5 and 8.5, and the
pH at which sarcosine oxidase is stable between 6.5 and 9.0. Thus, the pH 5.8 is far fromboth the optimum pH and pH range at which this enzyme is stable. However, the LM was
prepared in better condition more efficient at pH 5.8 than at pH 7.0, which implies theimportance of approaching the pI when an enzyme migrates into the membrane using CDS.
When the immobilized- enzyme-membrane was separated into the upper and lower
membranes using a pair of tongs, it was considered to increase errors over the depth of the- 3 5 6 -

J.Pack.Sci. Tech. Vol. 12 No.6 (2003)

membranes. However, the larger response was continuously observed at the lower membrane.
Thus, enzymes were considered to accumulate on a close limit at the bottom of the lowerm e m b r a n e .
The above results suggest that (1) the enzyme LM is obtained as enzymes accumulated
on a close limit at the bottom of the membrane by CDS and (2) the optimal pHs of the enzyme
solution for enzyme LM preparation are 5.8 for the glucose sensor, 7.2 for the D-amino acidsensor and 5.8 for the sarcosine sensor.

3.2. Differences between AM and LM of enzyme activity
Fig. 5 shows the schematic diagram of both LM and AM placed on the tip of oxygen
electrode. Once substrate get into the enzyme immobilization membrane, the oxidation ofsubstrate by enzyme causes the oxygen consumption. Simultaneously, the consumption ofo x yg en a r ou n d th e me mb r a n e r es u l t s t h e cu r r e n t d ec r e a s e s . T h at i s , t h e su b s t r a t e
concentration is calculated from the amount oxygen consumed. When substrate diffuses into
the enzyme immobilization membrane, the diffusion velocity (DV) is shown as the follow
f o r m u l a .

Width of filter membrane(x) : 5 mm

Length of filter membrane(y) : 5 mm

Thickness of filter membrane(z) : 0.17 mm

Diffusion Time(t)

The number of substrates(N)

Reaction Volume(V) : X, Y, Z

Namely, the diffusion velocity is proportional to the number of substrates per an unit
volume of the enzyme membrane. On the other hand, the frequency in a collision between
enzymes and substrate was far higher in LM than AM. Because, in the case of AM, enzymes
were considered to be immobilized randomly and the reaction distance z1 was 0.17 mm, whichequals to the thickness of filter membrane as shown in Fig. 5.
In the case of LM, enzymes were considered to assembled at the bottom (most interior)of the membrane close to the tip of oxygen electrode and the reaction distance z 2 wasconsidered to be reach almost to zero. Therefore, the volume of the field which substrate
reacts with enzyme was also considered to reach almost to zero. From the results mentioned
above, it was concluded that the enzyme reaction advanced more rapidly in the case of LM
compared to AM.

- 3 5 7 -Application of a contact dehydrating sheet to the preparation of an immobilized-enzyme membrane for biosensor

Fig. 5. Diagram of substrate diffusion through filter membrane.

3.3. Calibration curve of enzyme sensor
Fig. 6 presents the calibration curve for each of the three sensors. For the D-alanine
sensor, as shown in Fig. 6-a, a linear relationship between the concentration of D-alanine andthe current decrease was obtained for 0.5～2.0 mM D-alanine. The correlation coefficient (R2)were 0.995 on the enzyme LM and 0.964 on the AM. The slope (x = 0.424) of the calibration
curve of the enzyme LM was about 1.5 times larger than the slope (x = 0.288) of the calibration
curve of the AM. This indicated that an approximately 50% increase in biosensor efficiency
was obtained using the CDS technology. Similarly, using the glucose and sarcosine sensors
shown in Fig. 6-b and Fig. 6-c, respectively, linear relationships between the concentrations of
glucose and sarcosine and their current decreases were obtained for glucose and sarcosine in therange of 0.5-4.5 mM and 0.05-3.50 mM, respectively. The R2 value for the enzyme LM
were 0.999 and 0.977 for the glucose sensor and the sarcosine sensor, respectively. The R2

Fig. 6. Calibration curve for enzyme sensors.
●LM (with CDS). ○AM (without CDS),a) D-alanine sensor, b) Glucose sensor, c) Sarcosine sensor.

- 3 5 8 -

J.Pack.Sci. Tech. Vol. 12 No.6 (2003)

value for the AM were 0.990 and 0.985, respectively. Sensors signal was improved by 10% and
20% in the sarcosine and glucose sensor systems, respectively, when using the CDS.

Conclusion
A novel method for preparing immobilized enzyme membrane of biosensors using CDS
was proposed. By use of CDS, enzyme was migrated into the membrane interior and it waspossible to prepare for enzyme accumulation membrane. Enzyme migration rate was optimized
by a change of pH in enzyme solution. Enzyme accumulation membrane was examined todetermine if it increase accuracy of biosensor signal compared with traditional enzyme
adsorption membrane. Improvement of signals by use of CDS was observed among threedifferent type of enzyme membranes, therefore it was expected that CDS successfully increased
migration of enzyme into the membrane interior and enhanced signal intensity per unit ofenzyme in order to obtain improvement in biosensor efficiency.

A c k n o wl e d g e m e n t s
We would like to thank Mr. K. Saito and Ms. K. Takashima for valuable suggestions.
We thank Showa Denko Plastic Products Co., Ltd for donating CDS.

References
1) Orita, S.. Nihonshokuhinkagakukougakukaishi, 44(12), 86-87 (1997).
2) Takuno, M.: Kagaku, 46(4), 264 (1991).
3) Satou. A.: Shinsozai, 8, 77-78 (1991).
4) Kikuchi, K.: Kagakugijutushi MOL., 5, 1-6 (1987).
5) Skladal, P.: Electroanalysis, 9, 737 (1997).
6) Yabuki, S.: Denki Kagaku, 9, 912-917 (1998).
7) Karube, I.: Biosensor. Kyouritusyuppan, 19 (1986).
8) Jung-Ho, K., Tai-Jin, K., Dong-Hee, R. and Bong-Soo, N.: Food Science andBiotechnology, 7(1), 28-34 (1998).
9) Zeller. H. D., Hille R. and Jornes. H. S. : Biochemistry, 28, 5145-5134 (1989).
10) Chibrongodze,H., Hayashi,K. and Toko, K. : Sensors and Materials, 13(2), 99-106 (2001).
11) Swoboda, B.E.P. and Massey, V. J.BioI.Chem., 240, 2209 (1965).
12) John, S. W. and Dorothy, C. W. : Source book of Enzyme, 112-113 (1997).

- 3 5 9 -