- Email: [email protected]
Preparations of Langmuir-Blodgett films of enzyme-lipid complexes: A glucose sensor membrane
Thin Solid Films, 180 (1989) 65-72
65
P R E P A R A T I O N S O F L A N G M U I R - B L O D G E T T FILMS OF E N Z Y M E - L I P I D COMPLEXES: A G L U C O S E SENSOR M E M B R A N E * Y. OKAHATA, T. TSURUTA, K. IJIRO AND K. ARIGA
Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152 (Japan) (Received April 25, 1989; accepted May 16, 1989)
A stable monolayer of water-soluble enzyme (glucose oxidase (GOD)) could be prepared by spreading an organic solution of lipid-coated G O D on a water subphase. Langrnuir-Blodgett (LB) films of the G O D - l i p i d monolayers could be deposited on a platinum electrode and these acted as a glucose-sensing ultrathin membrane with a high sensitivity and a short response time. The complexing of enzyme with lipid molecules in advance is a useful technique for producing a waterinsoluble monolayer and LB films of water-soluble enzymes. This technique will become a new tool not only for preparing sensor membrane but also for assembling water-soluble proteins on biological electrical components, so-called "biochips".
1. INTRODUCTION Recently it has been of interest to use monolayer and Langmuir-Blodgett (LB) films of enzymes and antibodies as biosensors or biomolecular switches because of their high selectivity for substrates and antigens respectively 1'2. However, it seems to be difficult to prepare stable monolayers of water-soluble proteins on a water subphase because of their solubility in the subphase and their easy denaturation at the air-water interface a. Recently we developed a new lipid-coated enzyme which could be prepared as a precipitate by mixing aqueous solutions of enzyme and synthetic dialkyl amphiphiles, and the obtained enzyme-lipid complex was soluble only in organic solvents without being denatured (Fig. 1). For example, the lipaselipid complex has been found to function vigorously as a catalyst for esterification in a highly enantioselective manner in homogeneous and non-aqueous isooctane 4' 5. In this paper, we report that the enzyme-lipid complex forms a stable monolayer on a water subphase and LB films can be transferred onto a substrate (platinum electrode). When glucose oxidase (GOD) is employed as an enzyme, the GOD-immobilized LB films (2-10 layers) on platinum electrodes act as a sensitive and ultrathin glucose sensor membrane. * Enzyme-lipidcomplex4. For part 3 of this series(a preliminaryreport), see Y. Okahata, T. Tsuruta, K.. ljiro and K. Ariga, Langmuir, 4 (1988) 1373.
0040-6090/89/$3.50
© ElsevierSequoia/Printedin The N e t h e r l a n d s
Y. OKAHATAet al.
66
Synthetic
~
dialky._.~l amphiphile...._..ffi~s
CHa(CH2)ls'OC~-NHCO'CHz'N? -CH3
J/[/~\
CH3(CH2)ls-O~ ---/ O 2C1sgluN*
Br"
CH3
CH3(CHzhr'O-~ CH3(CH2)17-O--~ CH 2ClsOH CH3(CH2)17"O-~ ~-O.CH=.COO" Na ÷ CH3(CH2)17-O-~ 2CleCOO-
Fig. 1. A schematicillustration of the GOD-lipid complex. 2. EXPERIMENTAL PROCEDURES
2.1. Formation of the glucose oxidase-lipid complex Preparations of the monolayer-forming amphiphiles dihexadecyl N-(trimethylammoniomethylcarbonyl)glutamate bromide (2C16gluN+) 6, 1,3-dioctadecyl-2glycerol (2CtsOH) 7 and 1,3-dioctadecylglycero-2-oxymethylcarboxylic acid (2C 18COOH) 7 have been reported elsewhere, A buffer solution (25 ml, 0.01 M acetate, p H 5.6) of 25-50 mg of G O D (157 000 unit g - 1; Amano Pharmaceutical Co., Japan) and an aqueous dispersion of 100 mg of cationic 2C16gluN ÷, anionic 2 C 1 8 C O O - or non-ionic 2CI s O H amphiphiles was mixed, and the precipitates obtained after incubation at 4°C for 1 day were iyophilized. The characterizations of the pale yellow powder obtained are summarized in Table I. The protein content in the complex was determined from elemental analysis (carbon, hydrogen and nitrogen) and UV absorption of aromatic residues of proteins at 270 nm in chloroform. TABLE I PREPARATIONAND CHARACTERIZATIONOF GLUCOSEOXIDASE--LIPIDCOMPLEXES Lipid
Cationic 2C16gluN+ Non-ionic 2C~sOH Anionic 2C1sCOO-
GOD content (wt.%) b
Preparation a Amount of lipid (mg)
GOD (mg)
Yield(rag(%))
100 100 100
25 50 50
105 (84) 45 (30) 9 (6)
24 3.0 > 0.1
a 25 ml of aqueous solutions of GOD and lipid moleculesweremixed and the precipitate was lyophilized. bObtained from the elemental analysisand the UV absorption at 270 nm,
2.2. Langmuir-Blodgett films of the glucose oxidase-lipid complex A benzene or chloroform solution (1.0 ml rag-1) of the G O D - l i p i d complex was spread on Milli-Q water (Millipore Co.) at 20 °C in a Teflon-coated trough (475mm × 1 5 0 m m ) with a microprocessor-controlled Teflon barrier (San-Esu
67
GLUCOSE-SENSING ENZYME-LIPID LB FILMS
Keisoku Co., Fukuoka, Japan) 7' 8. The monolayer was continuously compressed at a speed of 180mm2s -1 and surface x-area A isotherms at 20°C were stored automatically in a microcomputer (NEC, PC 9801 model). The substrate (platinum electrode or quartz crystal microbalance (QCM)) was lowered and raised at a speed of 5-100mmmin -1 through the monolayer on the subphase at 4 0 m N m -1 Transfer ratios were confirmed from the frequency decrease of the QCM (8 mm x 8 mm, AT cut, 9 MHz) which constitutes a very sensitive mass measuring device because the resonance frequency change AF (Hz) on deposition of a given mass Am (g) on the QCM electrode is as followsS-~°: Am = (1.27 _+0.01) × 10 -9 AF
(1)
The LB films of GOD-lipid complex were transferred onto a platinum electrode as well as onto a QCM plate.
2.3. Detection of glucose The platinum electrode deposited with LB films of GOD-lipid complexes was placed in stirred 0.01 M acetate buffer solution (50 ml, pH 5.6) at 25 °C together with an Ag[AgCI counterelectrode. The potential of +0.6 V vs. AglAgCI was applied to the GOD/Pt electrode in order to detect H202 produced by the enzymic oxidation of glucose: OOD glucose + 02 , gluconic acid (2) gluconic acid + H 2 0 2
on electrode) 2H+
+ 02 "[-2e-
The electric current was recorded in response to the addition of glucose (10- 6-10- 3 M) into the buffer solution. 3.
RESULTS AND DISCUSSION
3.1. Characterization of the glucose oxidase-lipid complex Table I shows the yield and the GOD content of the complex prepared from GOD and cationic 2Cx6gluN ÷, anionic 2ClaCOO- , or non-ionic 2C18OH amphiphiles. The cationic 2Ca6gluN + amphiphiles gave the complex containing 24 wt.% proteins in a high yield of 84%. The pale yellow powder obtained was soluble in organic solvents such as benzene, chloroform and isooctane but insoluble in aqueous solutions. The complex was calculated to involve 150-250 lipid molecules per GOD molecule, which is roughly consistent with the number (ca. 200) of lipid molecules required to cover the surface of GOD as a lipid monolayer. Considering that the GOD-lipid complex is soluble only in organic solvents, we assume that cationic head groups of 2C16gluN ÷ bind to the negatively charged surface of GOD proteins with electrostatic interactions and that lipophilic dialkyl tails of lipids solubilize the complex in organic solvents as schematically illustrated in Fig. 1. When the non-ionic dialkyl 2C18OH amphiphiles were employed instead of the cationic 2C 16gluN+, the complex was obtained in the relatively low yield of 30% and the content of proteins was only 3 wt.%, probably because of the lack of an
68
Y. OKAHATA et
al.
electrostatic interaction between the hydrophilic head groups of the lipids and the enzyme surface. In the case of anionic 2C~8COO- amphiphiles, the formation of a complex with G O D was hardly observed because of the electrostatic repulsion.
3.2. Langmuir-Blodgett films of the glucose oxidase-lipid complex rc-A isotherms of the G O D - l i p i d complexes and the pure lipid molecules are shown in Fig. 2. The monolayer of the 2C 16gluN + 24~oGOD complex exhibited two steep rises in the curve corresponding to the expanded liquid phase and the condensed solid phase, which was stable up to 50 mN m - 1 and very similar to that of the pure 2C16gluN + amphiphiles (Fig. 2(a)). In the case of the monolayer of the 2C~ sOH-3~oGOD complex, the limiting area per molecules at zero surface pressure was 0.52 nm z which was larger than that of the pure lipid molecules. The monolayers of the GOD-lipid complex were stable without undergoing denaturation for at least 6 h on the air-water interface.
60 luN+/ GOD-24% ~-: 20 2C~sgluN ~ , ~ . ~ . _
(a)
~
.
6O
z,O
20
(b)
.
0. . . .
°o~z
,80H
" , ~ 2 C , 8 O H / GOD-3%
.I
0.4 a6 o,s 1.o tz t4 ts A / nmZrnolec:~
Fig. 2. Surface pressure ~-area A isotherms of GOD--lipid complexes and pure lipid molecules at 20 °C.
Only two layers of the monolayer of the 2C16gluN+-24?/oGOD complex could be transferred (Y type; transfer ratio, 1.02+0.01) when a platinum electrode (1 cm x 1 cm x 0.1 cm) was lowered and raised through the monolayer at a surface pressure of 40 mN m - 1 and at a dipping speed of 100 mm m i n - 1. On the contrary, more than 30 layers (Y type) of the monolayer of the 2 C 1 8 O H - 3 ~ G O D complex could be transferred with a good transfer ratio (1.01 _+0.02) at a surface pressure of 40 mN m - 1 and a dipping speed of 5 mm m i n - 1. The true transferred amount was determined from the frequency decrease (mass increase) of the Q C M plate as the substrate according to eqn. (1)s: the masses of two layers of 2C16gluN+-24~GOD and ten layers of 2 C 1 8 O H - 3 ~ G O D were calculated to be 201 ng (48 ng of G O D in the complex) and 1320 ng (41 ng of G O D in the complex) respectively. The obtained masses of LB films on the Q C M plate agreed well with the theoretical value calculated from the area per molecule on the subphase and the area (23.8 mm 2) of the QCM electrode.
3.3. Electric current response to glucose Typical amperometric responses of the platinum electrode (1 c m × 1 cm)
69
GLUCOSE-SENSING ENZYME-LIPID LB FILMS
deposited with two layers of the 2C16gluN+-24~oGOD LB film (GOD, 203 ng) to the addition of glucose are shown in Fig. 3. A steady state current increase of 0.05 ___0.01 IxA was observed within 5 s of each addition of 70 IxM glucose. Figure 4 shows good linear correlations between the current and the glucose concentration in the range 0-1.5 mM glucose. Although the slope for the 2C16gluN+-24~oGOD LB films (two layers; GOD, 203 ng) was larger than that of2C~sOH-3~GOD LB films (two layers; GOD, 27 ng) the current per microgram of GOD and for a millimolar glucose concentration was calculated to be 3.2-3.6 I~A for both LB films. Thus the GOD seems not to be denatured during complexation with lipids and the reactivity in the LB film is independent of head group charges or structures of lipid molecules.
i
I 0
10
20 30 time/sec
Fig. 3. Electric current response of the 2Ct6gluN+-24%GOD LB films (two layers) on a platinum electrode to the addition of 70 IxM glucose (marked by the arrows) in aqueous solution (pH 5.6, 0.01 M acetate) at 25 °C.
1.2'
,o ~=08
2CIBgtu~
0.6
d 0.4 0.2 0.5
1.0 [Glucose]/mM
I. 5
Area per molecule/nrn 2 0.95 Q8 0.58 0.46
O.E
OA
~O.E
0.3 0
E
~-o.~
0.2 -~
;o.~
0.t
°O
~b
2'0
3'0
4b
0
Surface pressure / mN.m-~
Fig. 4. Linear correlation between the electric current of two layers of 2C16gluN+-24~GOD or 2G8OH-3%GOD LB films on a platinum electrode and the concentration of glucose in solution at 25 °C. Fig. 5. Effect of surface pressure during the transfer of the 2C16gluN +-24%GOD LB films (two layers) on the electric current response to 1 mM glucose.
Figure 5 shows the effects of the surface pressure during the transfer of the 2C16gluN+-24~oGOD LB films (two layers) on the current responding to 1 mM glucose. The amount of LB films transferred (GOD content) was confirmed to increase with increasing surface pressure from the frequency measurements of the QCM plate as a substrate 8. On the contrary, the electric current exhibited a large increase at low surface pressures although the current increased gradually with increasing surface pressure (GOD amount) in the region of high surface pressures. At low surface pressures, the molecular packing of LB films may be loose and glucose
70
v. OKAHATAet al.
molecules may easily penetrate into the LB films. As a result, the current response to glucose was relatively large at the low surface pressure although the G O D content on the electrode was low. Figure 6 shows the effect of the number of layers of the 2C18OH-3~oGOD LB films on the electric current response to glucose and H202. The current response to the addition of 1 mM glucose increased with increasing number of layers (GOD content) below 10 layers. However, the current decreased gradually for more than 20 layers, which can be explained by the barrier effect of thick LB films for the penetration of glucose molecules into LB films. The current response to the addition of H202 reflects the penetration behaviour of H202 molecules into LB films, since H202 molecules are reduced directly on the electrode without GOD. The current for H202 decreased substantially for more than ten layers of LB films. This indicates that the penetration of glucose or H202 molecules is disturbed by the GOD-lipid LB films more than ten layers thick. Amount of GOD/IJg
0.1
E
0.2
0.3
0.4
0.5
% q~
0.4
80o
0.3
600
0.2
400
< "~
0.1
200
"6
M
0
10
20
30
U
Number of layers
Fig. 6. Effect of the number of layers of 2C1 s O H - 3 % G O D LB films on the electric current response to the addition of glucose and H202 (1 mM) into aqueous solution at 25 °C.
The barrier effect of LB films is clearly shown in the current response to glucose for the double-decker-type LB films in Fig. 7. When some layers of the pure 2C16gluN ÷ LB films were deposited over two layers of the 2C16gluN÷-24~GOD LB films on the electrode, the current for glucose decreased linearly with increasing number of layers of the covered pure lipid LB films (see Fig. 7(a)). This indicates that the upper pure lipid LB films act as a barrier for the penetration of glucose to G O D molecules buried in the lower LB films. When two layers of 2C16gluN+-24~GOD were deposited over some layers of the pure 2C16gluN + LB films on the electrode, the current for glucose decreased also with increasing number of layers of pure lipid LB films (see Fig. 7(b)). In this case, the permeability to the electrode of H202 produced by the reaction of G O D with glucose is reduced with increasing number of layers of pure lipid LB films underneath the complex films. Figure 7 indicates that glucose and H202 molecules cannot penetrate through pure lipid LB films more than six layers thick. The GOD-immobilized LB films on the platinum electrode were stable, in that enzymes were not solubilized into aqueous solutions, and they exhibited a good reproducibility (more than 30 times) of the amperometric response even after 3 months. When the complex-coated electrode was prepared without the use of the LB
GLUCOSE-SENSING ENZYME-LIPID LB FILMS
71
1.0 0.8
~,,.,-
~o 0A, •2 (a) ~
G~u¢ose pa
,
1.0
L 0.4 ~
B,a~
layers
0.2
(b)
CO
2
4
6
8
10
Number of layers
Fig. 7. Barrier effect of double-decker-type LB films formed from 2Ct6gluN+-24%GOD LB films (two layers) and pure lipid molecules on the current response to 1 mM glucose at 25 °C.
method, the amperometric response was not reproducible and diminished with time, probably because of leaching out of the complex (or enzyme) from the electrode. 4. CONCLUSION The GOD-immobilized LB film has the following features as a sensor membrane compared with currently available GOD-immobilized thick polymer films11-15: (i) a short response time, (ii) a small amount of enzyme required for preparations, and (iii) a simple ultrathin membrane. We can also prepare easily double- or triple-decker-type LB films in each layer of which different enzymes are incorporated. The complexing of enzyme with lipids in advance is a useful technique not only for producing a good sensor membrane but also for assembling other water-soluble proteins on biological electrical components, so-called "biochips". REFERENCES 1 G.G. Guilbault, Analytical Uses oflmmobilized Enzymes, Dekker, New York, 1984. 2 T.M.S. Chang, Biomedical Application oflmmobilized Enzymes and Proteins, Plenum, New York, 1977. 3 T. Ishii and M. Muramatsu, Bull. Chem. Soc. Jpn., 44 (1971) 679. 4 Y. Okahata and K. Ijiro, J. Chem. Soc., Chem. Commun., (1988) 1392. 5 Y. Okahata, Y. Fujimoto and K. Ijiro, Tetrahedron Lett., 29 (1988) 5133. 6 Y. Okahata, S. Hachiya and T. Seki, J. Polym. Sci., Polym. Lett. Edn., 22 (1984) 595. 7 Y. Okahata, K. Ariga, H. Nakahara and K. Fukuda, J. Chem. Soc.. Chem. Commun., (1986) 1069. K. Ariga and Y. Okahata, J. Am. Chem. Soc., 111 (1989) 5618. 8 Y. Okahata and K. Ariga, Jr. Chem. Soc., Chem. Commun., (1987) 1535. 9 G. Sauerbrey, Z. Phys., 155 (1959) 206. 10 Y. Okahata, H. Ebato and K. Taguchi, J. Chem. Soc., Chem. Commun., (1987) 1363. Y. Okahata and O. Shimizu, Langmuir, 3 (1987) 1171. Y. Okahata, H. Ebato and X. Ye, J. Chem. Soc., Chem. Commun., (1988) 1037. 11 P.N. Bartlett and R. G. Whitaker, J. Eleetroanal. Chem., 224 (1987) 37.
72
12 13 14 15
Y. OKAHATA et al.
T. Murakami, S. Nakamoto, J. Kimura, T. Kuriyama and I. Karube, Anal. Lett, 16 (1986) 1973. S. Suzuki and I. Karube, J. Mol. Catal., 6 (1979) 251. P.H.S. Tse and D. A. Gough, Anal. Chem., 59(1987) 2339. G.J. Moody, G. S. Sanghera and J. D. R. Thomas, Analyst, 111 (1986) 605.
65
P R E P A R A T I O N S O F L A N G M U I R - B L O D G E T T FILMS OF E N Z Y M E - L I P I D COMPLEXES: A G L U C O S E SENSOR M E M B R A N E * Y. OKAHATA, T. TSURUTA, K. IJIRO AND K. ARIGA
Department of Polymer Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152 (Japan) (Received April 25, 1989; accepted May 16, 1989)
A stable monolayer of water-soluble enzyme (glucose oxidase (GOD)) could be prepared by spreading an organic solution of lipid-coated G O D on a water subphase. Langrnuir-Blodgett (LB) films of the G O D - l i p i d monolayers could be deposited on a platinum electrode and these acted as a glucose-sensing ultrathin membrane with a high sensitivity and a short response time. The complexing of enzyme with lipid molecules in advance is a useful technique for producing a waterinsoluble monolayer and LB films of water-soluble enzymes. This technique will become a new tool not only for preparing sensor membrane but also for assembling water-soluble proteins on biological electrical components, so-called "biochips".
1. INTRODUCTION Recently it has been of interest to use monolayer and Langmuir-Blodgett (LB) films of enzymes and antibodies as biosensors or biomolecular switches because of their high selectivity for substrates and antigens respectively 1'2. However, it seems to be difficult to prepare stable monolayers of water-soluble proteins on a water subphase because of their solubility in the subphase and their easy denaturation at the air-water interface a. Recently we developed a new lipid-coated enzyme which could be prepared as a precipitate by mixing aqueous solutions of enzyme and synthetic dialkyl amphiphiles, and the obtained enzyme-lipid complex was soluble only in organic solvents without being denatured (Fig. 1). For example, the lipaselipid complex has been found to function vigorously as a catalyst for esterification in a highly enantioselective manner in homogeneous and non-aqueous isooctane 4' 5. In this paper, we report that the enzyme-lipid complex forms a stable monolayer on a water subphase and LB films can be transferred onto a substrate (platinum electrode). When glucose oxidase (GOD) is employed as an enzyme, the GOD-immobilized LB films (2-10 layers) on platinum electrodes act as a sensitive and ultrathin glucose sensor membrane. * Enzyme-lipidcomplex4. For part 3 of this series(a preliminaryreport), see Y. Okahata, T. Tsuruta, K.. ljiro and K. Ariga, Langmuir, 4 (1988) 1373.
0040-6090/89/$3.50
© ElsevierSequoia/Printedin The N e t h e r l a n d s
Y. OKAHATAet al.
66
Synthetic
~
dialky._.~l amphiphile...._..ffi~s
CHa(CH2)ls'OC~-NHCO'CHz'N? -CH3
J/[/~\
CH3(CH2)ls-O~ ---/ O 2C1sgluN*
Br"
CH3
CH3(CHzhr'O-~ CH3(CH2)17-O--~ CH 2ClsOH CH3(CH2)17"O-~ ~-O.CH=.COO" Na ÷ CH3(CH2)17-O-~ 2CleCOO-
Fig. 1. A schematicillustration of the GOD-lipid complex. 2. EXPERIMENTAL PROCEDURES
2.1. Formation of the glucose oxidase-lipid complex Preparations of the monolayer-forming amphiphiles dihexadecyl N-(trimethylammoniomethylcarbonyl)glutamate bromide (2C16gluN+) 6, 1,3-dioctadecyl-2glycerol (2CtsOH) 7 and 1,3-dioctadecylglycero-2-oxymethylcarboxylic acid (2C 18COOH) 7 have been reported elsewhere, A buffer solution (25 ml, 0.01 M acetate, p H 5.6) of 25-50 mg of G O D (157 000 unit g - 1; Amano Pharmaceutical Co., Japan) and an aqueous dispersion of 100 mg of cationic 2C16gluN ÷, anionic 2 C 1 8 C O O - or non-ionic 2CI s O H amphiphiles was mixed, and the precipitates obtained after incubation at 4°C for 1 day were iyophilized. The characterizations of the pale yellow powder obtained are summarized in Table I. The protein content in the complex was determined from elemental analysis (carbon, hydrogen and nitrogen) and UV absorption of aromatic residues of proteins at 270 nm in chloroform. TABLE I PREPARATIONAND CHARACTERIZATIONOF GLUCOSEOXIDASE--LIPIDCOMPLEXES Lipid
Cationic 2C16gluN+ Non-ionic 2C~sOH Anionic 2C1sCOO-
GOD content (wt.%) b
Preparation a Amount of lipid (mg)
GOD (mg)
Yield(rag(%))
100 100 100
25 50 50
105 (84) 45 (30) 9 (6)
24 3.0 > 0.1
a 25 ml of aqueous solutions of GOD and lipid moleculesweremixed and the precipitate was lyophilized. bObtained from the elemental analysisand the UV absorption at 270 nm,
2.2. Langmuir-Blodgett films of the glucose oxidase-lipid complex A benzene or chloroform solution (1.0 ml rag-1) of the G O D - l i p i d complex was spread on Milli-Q water (Millipore Co.) at 20 °C in a Teflon-coated trough (475mm × 1 5 0 m m ) with a microprocessor-controlled Teflon barrier (San-Esu
67
GLUCOSE-SENSING ENZYME-LIPID LB FILMS
Keisoku Co., Fukuoka, Japan) 7' 8. The monolayer was continuously compressed at a speed of 180mm2s -1 and surface x-area A isotherms at 20°C were stored automatically in a microcomputer (NEC, PC 9801 model). The substrate (platinum electrode or quartz crystal microbalance (QCM)) was lowered and raised at a speed of 5-100mmmin -1 through the monolayer on the subphase at 4 0 m N m -1 Transfer ratios were confirmed from the frequency decrease of the QCM (8 mm x 8 mm, AT cut, 9 MHz) which constitutes a very sensitive mass measuring device because the resonance frequency change AF (Hz) on deposition of a given mass Am (g) on the QCM electrode is as followsS-~°: Am = (1.27 _+0.01) × 10 -9 AF
(1)
The LB films of GOD-lipid complex were transferred onto a platinum electrode as well as onto a QCM plate.
2.3. Detection of glucose The platinum electrode deposited with LB films of GOD-lipid complexes was placed in stirred 0.01 M acetate buffer solution (50 ml, pH 5.6) at 25 °C together with an Ag[AgCI counterelectrode. The potential of +0.6 V vs. AglAgCI was applied to the GOD/Pt electrode in order to detect H202 produced by the enzymic oxidation of glucose: OOD glucose + 02 , gluconic acid (2) gluconic acid + H 2 0 2
on electrode) 2H+
+ 02 "[-2e-
The electric current was recorded in response to the addition of glucose (10- 6-10- 3 M) into the buffer solution. 3.
RESULTS AND DISCUSSION
3.1. Characterization of the glucose oxidase-lipid complex Table I shows the yield and the GOD content of the complex prepared from GOD and cationic 2Cx6gluN ÷, anionic 2ClaCOO- , or non-ionic 2C18OH amphiphiles. The cationic 2Ca6gluN + amphiphiles gave the complex containing 24 wt.% proteins in a high yield of 84%. The pale yellow powder obtained was soluble in organic solvents such as benzene, chloroform and isooctane but insoluble in aqueous solutions. The complex was calculated to involve 150-250 lipid molecules per GOD molecule, which is roughly consistent with the number (ca. 200) of lipid molecules required to cover the surface of GOD as a lipid monolayer. Considering that the GOD-lipid complex is soluble only in organic solvents, we assume that cationic head groups of 2C16gluN ÷ bind to the negatively charged surface of GOD proteins with electrostatic interactions and that lipophilic dialkyl tails of lipids solubilize the complex in organic solvents as schematically illustrated in Fig. 1. When the non-ionic dialkyl 2C18OH amphiphiles were employed instead of the cationic 2C 16gluN+, the complex was obtained in the relatively low yield of 30% and the content of proteins was only 3 wt.%, probably because of the lack of an
68
Y. OKAHATA et
al.
electrostatic interaction between the hydrophilic head groups of the lipids and the enzyme surface. In the case of anionic 2C~8COO- amphiphiles, the formation of a complex with G O D was hardly observed because of the electrostatic repulsion.
3.2. Langmuir-Blodgett films of the glucose oxidase-lipid complex rc-A isotherms of the G O D - l i p i d complexes and the pure lipid molecules are shown in Fig. 2. The monolayer of the 2C 16gluN + 24~oGOD complex exhibited two steep rises in the curve corresponding to the expanded liquid phase and the condensed solid phase, which was stable up to 50 mN m - 1 and very similar to that of the pure 2C16gluN + amphiphiles (Fig. 2(a)). In the case of the monolayer of the 2C~ sOH-3~oGOD complex, the limiting area per molecules at zero surface pressure was 0.52 nm z which was larger than that of the pure lipid molecules. The monolayers of the GOD-lipid complex were stable without undergoing denaturation for at least 6 h on the air-water interface.
60 luN+/ GOD-24% ~-: 20 2C~sgluN ~ , ~ . ~ . _
(a)
~
.
6O
z,O
20
(b)
.
0. . . .
°o~z
,80H
" , ~ 2 C , 8 O H / GOD-3%
.I
0.4 a6 o,s 1.o tz t4 ts A / nmZrnolec:~
Fig. 2. Surface pressure ~-area A isotherms of GOD--lipid complexes and pure lipid molecules at 20 °C.
Only two layers of the monolayer of the 2C16gluN+-24?/oGOD complex could be transferred (Y type; transfer ratio, 1.02+0.01) when a platinum electrode (1 cm x 1 cm x 0.1 cm) was lowered and raised through the monolayer at a surface pressure of 40 mN m - 1 and at a dipping speed of 100 mm m i n - 1. On the contrary, more than 30 layers (Y type) of the monolayer of the 2 C 1 8 O H - 3 ~ G O D complex could be transferred with a good transfer ratio (1.01 _+0.02) at a surface pressure of 40 mN m - 1 and a dipping speed of 5 mm m i n - 1. The true transferred amount was determined from the frequency decrease (mass increase) of the Q C M plate as the substrate according to eqn. (1)s: the masses of two layers of 2C16gluN+-24~GOD and ten layers of 2 C 1 8 O H - 3 ~ G O D were calculated to be 201 ng (48 ng of G O D in the complex) and 1320 ng (41 ng of G O D in the complex) respectively. The obtained masses of LB films on the Q C M plate agreed well with the theoretical value calculated from the area per molecule on the subphase and the area (23.8 mm 2) of the QCM electrode.
3.3. Electric current response to glucose Typical amperometric responses of the platinum electrode (1 c m × 1 cm)
69
GLUCOSE-SENSING ENZYME-LIPID LB FILMS
deposited with two layers of the 2C16gluN+-24~oGOD LB film (GOD, 203 ng) to the addition of glucose are shown in Fig. 3. A steady state current increase of 0.05 ___0.01 IxA was observed within 5 s of each addition of 70 IxM glucose. Figure 4 shows good linear correlations between the current and the glucose concentration in the range 0-1.5 mM glucose. Although the slope for the 2C16gluN+-24~oGOD LB films (two layers; GOD, 203 ng) was larger than that of2C~sOH-3~GOD LB films (two layers; GOD, 27 ng) the current per microgram of GOD and for a millimolar glucose concentration was calculated to be 3.2-3.6 I~A for both LB films. Thus the GOD seems not to be denatured during complexation with lipids and the reactivity in the LB film is independent of head group charges or structures of lipid molecules.
i
I 0
10
20 30 time/sec
Fig. 3. Electric current response of the 2Ct6gluN+-24%GOD LB films (two layers) on a platinum electrode to the addition of 70 IxM glucose (marked by the arrows) in aqueous solution (pH 5.6, 0.01 M acetate) at 25 °C.
1.2'
,o ~=08
2CIBgtu~
0.6
d 0.4 0.2 0.5
1.0 [Glucose]/mM
I. 5
Area per molecule/nrn 2 0.95 Q8 0.58 0.46
O.E
OA
~O.E
0.3 0
E
~-o.~
0.2 -~
;o.~
0.t
°O
~b
2'0
3'0
4b
0
Surface pressure / mN.m-~
Fig. 4. Linear correlation between the electric current of two layers of 2C16gluN+-24~GOD or 2G8OH-3%GOD LB films on a platinum electrode and the concentration of glucose in solution at 25 °C. Fig. 5. Effect of surface pressure during the transfer of the 2C16gluN +-24%GOD LB films (two layers) on the electric current response to 1 mM glucose.
Figure 5 shows the effects of the surface pressure during the transfer of the 2C16gluN+-24~oGOD LB films (two layers) on the current responding to 1 mM glucose. The amount of LB films transferred (GOD content) was confirmed to increase with increasing surface pressure from the frequency measurements of the QCM plate as a substrate 8. On the contrary, the electric current exhibited a large increase at low surface pressures although the current increased gradually with increasing surface pressure (GOD amount) in the region of high surface pressures. At low surface pressures, the molecular packing of LB films may be loose and glucose
70
v. OKAHATAet al.
molecules may easily penetrate into the LB films. As a result, the current response to glucose was relatively large at the low surface pressure although the G O D content on the electrode was low. Figure 6 shows the effect of the number of layers of the 2C18OH-3~oGOD LB films on the electric current response to glucose and H202. The current response to the addition of 1 mM glucose increased with increasing number of layers (GOD content) below 10 layers. However, the current decreased gradually for more than 20 layers, which can be explained by the barrier effect of thick LB films for the penetration of glucose molecules into LB films. The current response to the addition of H202 reflects the penetration behaviour of H202 molecules into LB films, since H202 molecules are reduced directly on the electrode without GOD. The current for H202 decreased substantially for more than ten layers of LB films. This indicates that the penetration of glucose or H202 molecules is disturbed by the GOD-lipid LB films more than ten layers thick. Amount of GOD/IJg
0.1
E
0.2
0.3
0.4
0.5
% q~
0.4
80o
0.3
600
0.2
400
< "~
0.1
200
"6
M
0
10
20
30
U
Number of layers
Fig. 6. Effect of the number of layers of 2C1 s O H - 3 % G O D LB films on the electric current response to the addition of glucose and H202 (1 mM) into aqueous solution at 25 °C.
The barrier effect of LB films is clearly shown in the current response to glucose for the double-decker-type LB films in Fig. 7. When some layers of the pure 2C16gluN ÷ LB films were deposited over two layers of the 2C16gluN÷-24~GOD LB films on the electrode, the current for glucose decreased linearly with increasing number of layers of the covered pure lipid LB films (see Fig. 7(a)). This indicates that the upper pure lipid LB films act as a barrier for the penetration of glucose to G O D molecules buried in the lower LB films. When two layers of 2C16gluN+-24~GOD were deposited over some layers of the pure 2C16gluN + LB films on the electrode, the current for glucose decreased also with increasing number of layers of pure lipid LB films (see Fig. 7(b)). In this case, the permeability to the electrode of H202 produced by the reaction of G O D with glucose is reduced with increasing number of layers of pure lipid LB films underneath the complex films. Figure 7 indicates that glucose and H202 molecules cannot penetrate through pure lipid LB films more than six layers thick. The GOD-immobilized LB films on the platinum electrode were stable, in that enzymes were not solubilized into aqueous solutions, and they exhibited a good reproducibility (more than 30 times) of the amperometric response even after 3 months. When the complex-coated electrode was prepared without the use of the LB
GLUCOSE-SENSING ENZYME-LIPID LB FILMS
71
1.0 0.8
~,,.,-
~o 0A, •2 (a) ~
G~u¢ose pa
,
1.0
L 0.4 ~
B,a~
layers
0.2
(b)
CO
2
4
6
8
10
Number of layers
Fig. 7. Barrier effect of double-decker-type LB films formed from 2Ct6gluN+-24%GOD LB films (two layers) and pure lipid molecules on the current response to 1 mM glucose at 25 °C.
method, the amperometric response was not reproducible and diminished with time, probably because of leaching out of the complex (or enzyme) from the electrode. 4. CONCLUSION The GOD-immobilized LB film has the following features as a sensor membrane compared with currently available GOD-immobilized thick polymer films11-15: (i) a short response time, (ii) a small amount of enzyme required for preparations, and (iii) a simple ultrathin membrane. We can also prepare easily double- or triple-decker-type LB films in each layer of which different enzymes are incorporated. The complexing of enzyme with lipids in advance is a useful technique not only for producing a good sensor membrane but also for assembling other water-soluble proteins on biological electrical components, so-called "biochips". REFERENCES 1 G.G. Guilbault, Analytical Uses oflmmobilized Enzymes, Dekker, New York, 1984. 2 T.M.S. Chang, Biomedical Application oflmmobilized Enzymes and Proteins, Plenum, New York, 1977. 3 T. Ishii and M. Muramatsu, Bull. Chem. Soc. Jpn., 44 (1971) 679. 4 Y. Okahata and K. Ijiro, J. Chem. Soc., Chem. Commun., (1988) 1392. 5 Y. Okahata, Y. Fujimoto and K. Ijiro, Tetrahedron Lett., 29 (1988) 5133. 6 Y. Okahata, S. Hachiya and T. Seki, J. Polym. Sci., Polym. Lett. Edn., 22 (1984) 595. 7 Y. Okahata, K. Ariga, H. Nakahara and K. Fukuda, J. Chem. Soc.. Chem. Commun., (1986) 1069. K. Ariga and Y. Okahata, J. Am. Chem. Soc., 111 (1989) 5618. 8 Y. Okahata and K. Ariga, Jr. Chem. Soc., Chem. Commun., (1987) 1535. 9 G. Sauerbrey, Z. Phys., 155 (1959) 206. 10 Y. Okahata, H. Ebato and K. Taguchi, J. Chem. Soc., Chem. Commun., (1987) 1363. Y. Okahata and O. Shimizu, Langmuir, 3 (1987) 1171. Y. Okahata, H. Ebato and X. Ye, J. Chem. Soc., Chem. Commun., (1988) 1037. 11 P.N. Bartlett and R. G. Whitaker, J. Eleetroanal. Chem., 224 (1987) 37.
72
12 13 14 15
Y. OKAHATA et al.
T. Murakami, S. Nakamoto, J. Kimura, T. Kuriyama and I. Karube, Anal. Lett, 16 (1986) 1973. S. Suzuki and I. Karube, J. Mol. Catal., 6 (1979) 251. P.H.S. Tse and D. A. Gough, Anal. Chem., 59(1987) 2339. G.J. Moody, G. S. Sanghera and J. D. R. Thomas, Analyst, 111 (1986) 605.