Enzyme-immobilized Langmuir-Blodgett film for a biosensor

463

ENZYME-IMMOBILIZED LANGMUIR-BLODGETT FILM FOR A BIOSENSOR* M. S R I Y U D T H S A K , H.

YAMAGISHIAND T. MORIIZUMI

International Cooperation Center for Science and Technology, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152 (Japan)

(ReceivedJuly 27, 1987;acceptedSeptember14, 1987)

A lipid-protein monolayer for a biosensor was prepared utilizing a LangmuirBlodgett technique. The enzyme glucose oxidase was used as the protein. Three types of lipid were chosen to change the surface charge of the polar group. The enzyme was immobilized on the lipid monolayer by adsorption from the subphase solution onto the lipid monolayer on the air/water interface. It was found that the lipid-enzyme interaction was dominated by electrostatic forces, and the characteristics of the film can be controlled by expansion and recompression of the adsorbed monolayer. Finally, a glucose sensor was fabricated by depositing the film onto a hydrogen peroxide electrode.

1. INTRODUCTION It is known that a lipid monolayer has a surface charge on a hydrophilic group and the total charge on a protein, i.e. the enzyme, can be controlled by the pH of the solution. This means that under suitable conditions it is possible to adsorb protein molecules on lipid layers and prepare protein-immobilized monolayers for biosensor applications. We used this method to fabricate enzyme-immobilized biosensors 1. 2. MATERIALAND FILM PREPARATION Glucose oxidase (GOD) (13.3 U m g - 1) purchased from Tokyo Kasei Chemical was used without any purification. Three kinds of lipids, namely, arachidic acid (C2o), arachidic acid methyl ester (C2oMe) and stearyltrimethylammonium chloride (C18N) were used to form monolayers on the subphase. Since ClaN itself cannot form a monolayer, it was mixed with C2oMe in a molar ratio of 1:4 (abbreviated as CIaNo.2). All lipids were dissolved in chloroform at a concentration of 5 mM. The subphase was 1 m M H E P E S buffer o f p H 7.0, which was adjusted with NaOH. Paper presented at the Third International Conference on Langmuir-Blodgett Films, G6ttingen, F.R.G., July 26-31, 1987. *

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464

M. SRIYUDTHSAK,H. YAMAGISHI,T. MORIIZUMI

The enzyme-immobilized lipid monolayer was prepared by using Fromherz's method 2. The Langmuir-Blodgett (LB) trough is a multicompartment one. The lipid solution was spread, left for 5 min and then compressed to a surface pressure of 15-20 mN m-1. The enzyme solution, dissolved in the same buffer at a concentration of 2 mg ml- 1, was injected into another compartment. Then the monolayer was moved to the enzyme compartment. There, enzyme molecules were adsorbed onto the lipid film for 60 min. Again, the enzyme-adsorbed lipid monolayer was moved away from the enzyme compartment to a dipping compartment. Transfer was achieved by the classical dipping method at a speed of 5 mm min-1. At first, the enzyme-lipid monolayer was transferred at a surface pressure of 20 mN m - 1 to a solid substrate. Then the remaining monolayer was expanded to a pressure of 10 mN m - 1 and transferred to another substrate. Finally, the monolayer was recompressed again to the pressure of 2 0 m N m -1 and transferred to the third substrate. In all cases, only two layers were deposited. Three types of solid substrates, a slide glass plate, a quartz substrate and a glass plate with hydrogen peroxide electrodes 3, were used to investigate the properties of the deposited films. To prepare hydrophobic surfaces, all substrates were dipped in 0.2~ n-octadecyltrichlorosilane toluene solution for 30 min and then rinsed with fresh toluene. 3. EXPERIMENTALDETAILSAND RESULTS

3.1. Enzyme activity Slide glass plates were used to investigate the activities of the enzyme-lipid monolayers. The substrates were put into glucose, indigo-carmine (DH2) and copper sulphate with histamine (as a catalyst) solutions. Hydrogen peroxide, produced from the decomposition of glucose by GOD, oxidizes the DH 2 (eqns. (1) and (2)). The activities of the film were estimated by the change in optical absorbance of DH2 at 613nm. The comparison of the activities for the different lipid monolayers, prepared under the different conditions mentioned above, is shown in Fig. 1. C 6 H 1 2 0 6 + H 2 0 + 0 2 GOD ~H 2 0 2 + C 6 H 1 2 0 7

(1)

H202+DH2

(2)

catalyst , 2 H 2 0 + D

It is found, by comparing the G O D LB layers with the different types of lipids, that the C 18No.2 monolayer showed the highest activity. Among the layers with the same type of lipid, the recompressed film had the highest activity except for that deposited at 10 mN m - 1. 3.2. Relative protein amount To evaluate the amount of enzyme in the film, an o-phthalaldehyde was used to examine the relative number of amino groups. A quartz substrate was used in this case. A complex of o-phthalaldehyde and the amino groups in G O D was formed by the reaction shown in eqn. (3). The observation of fluorescence emission (excitation, 340 nm; emission, 455 nm) from the complex enables us to compare the relative amount of the enzyme molecules in the film. The results are shown in Fig. 2.

465

ENZYME-IMMOBILIZED LB FILM FOR A BIOSENSOR

0.15 O 1

; 0.10

2

~ o.os

3

~C20--

C20Me~

- - C18 NO.2~

Fig. 1. Comparison of the activities of the enzyme-immobilized LB films.

150

1

.~100

~ so

3

2

C20Me~

~C20--

~ C1BN0.2~

Fig. 2. C o m p a r i s o n of the relative amounts of protein in the enzyme-immobilized LB films: 1, 20 m N m - 1 after adsorbing GOD; 2, 10 m N m - ~ after expansion from 20 m N m - 1; 3, 20 m N m - 1 after recompression from 10 naN m - 1.

SCH2CH2OH

G ?o

+ R - - N H 2 + HSCH2CH2OH -* ~

N--R

(3)

Here, the trend is similar to that found for the activity measurements. The best film can be obtained from the C18N0.2 monolayer which was expanded once and recompressed. 3.3. Glucose sensor

The enzyme-adsorbed LB film was deposited onto a hydrogen peroxide electrode to construct a glucose sensor. The hydrogen peroxide electrode was fabricated on a slide glass by evaporating 2000/~, of gold over about 200/~ of chromium. Comparisons of the sensor sensitivities (expressed as H202 molar

466

M. SRIYUDTHSAK, H. YAMAGISHI, T. MORIIZUMI

quantities) are shown in Fig. 3. They show the same trend as the results of the above two experiments. Figure 4 shows the output of the sensor with the GOD-CtsN0. 2 film when the deposition surface pressure was changed. The measurement was performed in 0.01 M phosphate buffer of pH7.2 at 22_+2°C. It indicates that deposition at a higher pressure gives rise to increased sensor outputs. It is foundthat the glucose concentration can be measured up to 1000 mg dl-1 without saturation of the output.

C20Me

C20

I00

C18N0.2

~80

~0

!

20

:

•

I

. ,, . I , I. !

.

200 400 600

.

.

.

.

,

200

400 600 Gtucose [mg/d[]

,

200

,

,

,

400 600

Fig. 3. Comparison of the sensitivities of the glucose sensors (the "100" on the ordinate scale corresponds to the output current in H202 solution with a concentration of 300 ttM): A , transfer at 20 dyn era- 1 after adsorbing G O D ; O, transfer at 20 dyn c m - ~ after recompression.

0.150

D

I [~aA] 0.10-

U 0

0.05o o

a

t~

a

0.00

2~o

56o

Glucose

[ mg/dl}

7~o

;doo

Fig. 4. Sensitivities of the glucose sensor when the deposition surface pressure was changed (the lipid is ClsNo.2): I , 3 0 m N m-1 before expansion; A, 3 0 m N m - 1 after expansion; Fq, 35 m N m -1 after expansion; O, 40 m N m - 1 after expansion. The adsorptions were performed at 5.2 m N m - ~.

4. DISCUSSION It is found that the best film can be obtained by the adsorption of the enzyme onto the C~8No.2 monolayers and by the deposition after expansion and recompression of the adsorbed film. The results suggest that an electrostatic interaction

467

ENZYME-IMMOBILIZED LB FILM FOR A BIOSENSOR

between the lipid monolayer and enzyme molecules dominates the adsorption process. This can be understood by considering the charging conditions of the lipid monolayer and the total charge on the enzyme. It is known that the isoelectric point of GOD is about 4.2. Therefore, at pH 7.0, GOD has a negative total charge. At neutral pH, the surface charge of arachidic acid, arachidic acid methyl ester and the mixed ammonium salt is thought to be negative, neutral and positive respectively. Consequently, the best film can be obtained from the ammonium salt film. It is interesting that treatment of the adsorbed film by expansion and recompression, changes the properties of the film. We suggest that the structure of the molecular assembly of the film varies during the treatment. The effect of deposition pressure on sensor characteristics shown in Fig. 4 can be understood by looking at a pressure-area isotherm. Figure 5 shows the isotherm curves of the three types of lipid monolayers before and after the enzyme GOD adsorption. The area per molecule is not much changed above a surface pressure of

5O

50

"--40

40

Z

E 30

30

20

20 2

10

1

0

I

2o Aree

(a)

10 I

30

molecule

per

i

i

40 [~21

i

i

t,J

I

10

50

(b)

I

20 310 40 5JO Areo per molecule [ A 2]

i

2 5C "-4C

\

Z

E 30 20 I(; 0

(c)

I

I

I

I0 20 30 Area per molecule

I

40 [~,2]

I

50

Fig. 5. Pressure-areaisothermoflipidmonolayers before and after adsorbingenzymeOOD:(a)curve I, C~o; curve 2, C2o after adsorbing G O D ; (b) curve 1, C2oMe; curve 2, C2oMe after adsorbing G O D , (c) curve 1, C t sNo.2; curve 2, C 1sNo.2 after adsorbing GOD.

468

M. SRIYUDTHSAK, H. YAMAGISHI, T. MORIIZUMI

20 mN m - 1 before adsorption takes place. However, alter tlae enzyme was adsorbed, the area per molecule increased in the. sequence C2o, C20Me and CIaNo.2. We assumed the model of the enzyme-lipid molecular structure shown in Fig. 6. The increase in area of pressure-area curves is considered to arise from adsorption and permeation of enzyme molecules into the lipid monolayer (Fig. 6(b)). When the monolayer was expanded, a reorganization of the lipid-enzyme molecules occurred (Fig. 6(c)). Therefore, when this monolayer is recompressed again, the characteristics of the film must change. This can be seen as a big change in the pressure-area isotherm. Before the enzyme was adsorbed, the liquid-solid phase transition was seen clearly. However, after the adsorption the phase transition points disappeared and the area per molecule became larger. When the deposition pressure was raised, a considerable decrease in the area per molecule occurred, the packing of the enzymelipid molecules on the subphase goes up and the density of the transferred enzyme molecules increases, giving rise to sensor sensitivity enhancement. (a)

....

jd2

d2.1/.

....

Fig. 6. The presumed model of the lipid-enzyme molecule: (a) lipid monolayer over enzyme solution, (b) adsorption of enzyme molecules onto monolayer, (c) expansion of the adsorbed monolayer leads to a reorganization of the enzyme molecules and (d) a close-packed monolayer can be obtained when recompressing the monolayer aaain. 5. CONCLUSION

Selecting the lipid materials to change the surface charge, the number of adsorbed enzyme molecules can be enhanced and the properties of the film can be improved. Furthermore, by expansion and recompression of these films, the monolayers reorganized themselves. Thus, better characteristics from these monolayers may be obtained. The present study showed the feasibility of using the enzyme-immobilized LB films as sensing membranes for biosensors.

ENZYME-IMMOBILIZED LB FILM FOR A BIOSENSOR

REFERENCES 1 T. Moriizumi, Thin Solid Films, 160 (1988) 413. 2 P. Fromherz, Biochim. Biophys. Acta, 225 (1971) 382. 3 I. Takatsu and T. Moriizumi, Sensors and Actuators, 11 (1987) 309.

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