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Immobilization of glucose oxidase onto a Langmuir-Blodgett ultrathin film of a cellulose derivative deposited on a self-assembled monolayer
Supramolecular Science 4 ( 1997) 279-29 I 81’1997 Else&r Science Ltd Printed in Great Britain. All rights reserved
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ELSEVIER
Immobilization of glucose oxi’dase onto a Langmuir-Blodgeti ultrathin film of a cellulose derivative deposited on a self-assembled monolayer Antbnio J. Guiomar*,
Stephen D. Evan&*
and James T. Guthrie*
*Department of Co/our Chemistry and Dyeing, The University of Leeds, Leeds LS2 9JT, UK +Department of Physics and Astronomy, The University of Leeds, Leeds LS2 9JT, UK (Received 6 September 1996; revised 17 December 19961
Glucose oxidase ‘was immobilized on a Langmuir-Blodgett film of cellulose acetate propionate deposited on a self-assembled monolayer coated substrate. These layers were characterized in terms of their ellipsometric thickness, wettability and infra-red spectra. Glucose oxidase was immobilized on this composite layer by physisorption. The presence of the enzyme on the surface was confirmed by ellipsometry, infra-red spectroscopy and by detecting its activity electrochemically. An enzyme population remained active after adsorption onto this assembly. 0 1997 Elsevier Science Ltd. All rights reserved. (Keywords:glucose oxidase; immobilization;self-assembly)
INTRODUCTION Enzyme-based
biosensors
require
close
contact
between a transducer and the enzyme. This can be achieved by immobilization of the enzyme onto the surface of the transducer. In electrochemical biosensors, the transducer surface is usually a metal. The choice of immobilization chemistry for direct attachment is thus very limited. It can be extended by depositing or creating monolayers on these metal surfaces, bringing the adv,antage of ordering, very low thickness and the possibility of controlled immobilization”2. For the biosensor to perform its task, the enzyme has to be immobilized in an active state, i.e. retaining its ability to catalyse a reaction involving its substrate (the analyte). This requires that both the immobilization step and the new micro environment into which the enzyme is placed should not cause any major conformational change in the active site of the enzyme. Glucose oxidase (GOX), a well-characterized and much used enzyme334, was used in this work. GOX is mostly used for the determination of glucose levels in body fluids. Nearly all the devices developed are of the biosensor type and the large majority are of the
amperometric subtype. GOX catalyses the oxidation of glucose to gluconic acid, coupled with the reduction of oxygen (02) to hydrogen peroxide. However, oxygen can be replaced by several synthetic electron acceptors, the so-called mediators3*5. Direct electron transfer from the enzyme Flavin Adenine Dinucleotide (FAD) centres to modified electrodes has proved elusive to attain6i3. Only a few reports have evidence that this transfer is carried out from an active population of enzyme molecules’416. The use of Langmuir-Blodgett (LB) films for the immobilization of GOX seems to have started in the late 1980s. The work reported can be divided as follows. 1. Formation
2.
i) ii)
iii) t To whom correspondence
should
be addressed
of an enzyme LB film after spreading the enzyme at the air/water interface, either in its native form or after being crosslinked17-‘9. Adsorption to a Langmuir monolayer, followed by LB transfer. The enzyme was included in the following ways: addition to the subphase before spreading the amphiphile2’; adsorption onto a Langmuir monolayer by moving it to another trough compartment containing a GOX solution, where the adsorption occurs21-23; and addition to a previously prepared Langmuir monolayer of an amphiphile (it was not disclosed how)18.
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Glucose oxidase within a self-assembled
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3. Mixing the enzyme with amphiphilic molecules, the spreading of the mixture at the air/water interface being followed by LB transfer. Reported approaches include: 9 coating the enzyme with a fatty acid, followed by spreading and LB or Langmuir-Schaefer (LS) transfer24’25; and ii) adding enzyme to a solution of polyaniline followed by spreading and LB transfer26. 4. Covalent attachment of GOX to functional groups of a preformed LB or LS lilm27. The same research group also included an amphiphilic ferrocene derivative in the system above28. 5. Specific physisorption of modified GOX to preformed LB films, containing specifically interacting molecules. The interacting pairs used were the avidin-biotin29 and the antigen-antibody3’ pairs. This work reports the non-specific physisorption of GOX to a preformed LB film of a modified polysaccharide [cellulose acetate propionate (CAP)], an approach not found in the literature. GOX is a glycoprotein, having a protein core surrounded by a carbohydrate shel13. This approach has the advantage of presenting to the enzyme an interface that is similar in nature to its own surface. This should allow preservation of the enzyme’s native conformation. Moreover, this cellulose derivative has a high degree of substitution and consequently low crystallinity. It has both hydrophilic (hydroxyl) and relatively hydrophobic groups (acetate, propionate) on its chain. It was thought that this would permit the preparation of Langmuir monolayers and LB films. The preparation and study of Langmuir monolayers of cellulose short-chain esters has long been documented in the literature31p33, although no report could be found for CAP. The transfer of long- and mediumchain cellulose esters to solid substrates has also been reported34 but no report on the transfer of their shortchain equivalents could be found. The deposition behaviour of this type of polymer is referred to as ‘very poor’35. The deposition behaviour of cellulose esters has since been improved by using derivatives with increased length of the esterilied chain. The LB method of transfer to a hydrophobic, goldcoated glass slide was used. The gold-coated glass slide was rendered hydrophobic by creating a self-assembled monolayer (SAM) of an alkylthiol (octadecylmercaptan; C 18) on its gold/air interface. Both the SAM and the LB film were characterized in terms of their ellipsometric thickness, contact angles of their surfaces with water and infra-red spectra at grazing angle of incidence. As a first approach, GOX was immobilized simply by physisorption to a preformed LB film. The presence of the enzyme on the surface was confirmed by determining the increase in thickness of the obtaining the enzyme’s multilayered system,
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vibrational spectrum and detecting its activity. The retention of enzymatic activity was detected by means of an electrochemical assay. This makes use of a coupling scheme developed for amperometric biosensors with GOX5, where the electrode reaction is coupled to a catalytic reaction (the so-called catalytic or EC’ mechanism), a mechanism readily identified and studied by cyclic voltammetry36.
METHODS Preparation of gold-coatedglass slides Gold-coated glass slides were prepared by resistive evaporation of gold to a thickness of either 100 or 200nm, by using an Edwards Auto 306 Turbo Vacuum Coater equipped with a Edwards Turbomolecular Vacuum Pump. The microscope glass slides were cleaned immediately before evaporation by rubbing with the concentrate Decon 90 and rinsing with water. All the water used in this work was of high purity, having been purified by a Millipore unit that combines a Millipore RO and a Milli Q Plus unit in sequence. The glass slides were further treated ultrasonically in a dilute Decon 90 solution (ca. 10% in water) for 15 min. Thorough rinsing with water and drying with a stream of nitrogen followed. The glass slides were subsequently cleaned by etching with an argon plasma cleaner (Plasma Preen I) for 2min, with the power dial set at 60 and an 80% cycle, rinsed with water and dried with a stream of nitrogen. After this procedure, the glass slides were completely wetted by water. The clean glass slides were placed in a holder located inside the evaporation chamber at approximately 15 cm above the holders of the metals to be evaporated. First, a very thin chromium underlayer (thickness of either 1 or 8nm, monitored with a Edwards FTMS quartz oscillator) was deposited on the glass slides by evaporation of chromium from a chromium-coated tungsten wire (Edwards). The deposition rates were 0.1-0.2 nm s-‘, at a pressure of 2.3x 10e6mbar. This was followed by the evaporation of gold from tungsten buckets, without venting the chamber. Gold (99.99%, Advent Research Materials) was evaporated at 0.24.3 nm s-l, at the same pressure and to a thickness of either 100 or 200nm. Both sides of the slides were coated. The gold slides were cleaned by a very mild plasma cleaning (15 s, power dial set at 0, 100% cycle) immediately before use. Preparation of monolayers Cl8 self-assembled monolayer (C18SAM). Freshly cleaned gold-coated slides were immersed in a 1 mM solution of octadecylmercaptan (98%, Aldrich Chemical Co.) in dichloromethane (HPLC grade, BDH) in sealed glass containers. After 1 h, the slides
SCIENCE Volume 4 Numbers 3-4 1997
Glucose
oxidase
rinsed with dichlorowere removed, thoroughly methane, dried with a stream of nitrogen and analysed. CAP Langmuir mono,layer. A 5 mgml-’ stock solution of cellulose acetate propionate (CAP 482.05, Eastman Chemical Co.; average contents: 2.5 wt% acetyl, 45 wt% propionyl and 2.6wt% hydroxyl; M, = 25 000 Daltons) in chloroform (HPLC grade, Sigma-Aldrich) was prepared. This solution was filtered through a filter with a pore diameter of l.Opm (Whatman Puradisc 25 TF). No significant change in concentration could be detected by gravimetric assessment after total solvent evaporation. These solutions were kept at 4°C in a fridge, in stoppered and sealed glass containers to prevent chloroform evaporation. Immediately prior to use, the solution was allowed to equilibrate to room temperature. Subsequently, it was accurately diluted to 0.25 mg ml-’ with chloroform. This dilute solution was used the same day it was prepared. Langmuir monolayers were prepared on a water subphase with the aid of al Fromherz trough equipped with a Wilhelmy hanging-plate surface pressure balance. The trough was Ilocated inside a ‘clean room’ and placed inside a horizontal flow cabinet. To spread a monolayer, 50~1 of the 0.25mgml-’ CAP solution were applied to a clean wa.ter surface of the fully open trough (area=318cm*). Tlhe CAP solution was added dropwise to the water surface. After spreading, a further 15 min were allowed in order for the solvent to evaporate. The monolayer was then compressed at a rate of 5.3 x 10e4 nm* (substituted anhydroglucose unit))’ s-’ and surface pressure (II) versus area isotherms were obtained. For hysteresis studies, the compression of the monolayer was immediately followed by its expansion at the same rate. CAP Langmuir-Blodgett (CAPLB) film. A Langmuir monolayer of CAP was prepared as previously described. The LB transfer was carried out at a constant surface pressure of 20 mNm_‘. The stability of this monolayer was evaluated by following the change in its area with time for 15 min. Only stable monolayers were transferred. The substrate was clamped to the holder of the motorized dipping unit, in a vertical orientation, and was immersed and withdrawn across the mfonolayer/air interface at a speed of 4 mm min- ’ . A deposition curve of area of the monolayer versus dipping-head position was recorded and used both to check the uniformity of the deposition rate and to calculate the deposition ratio.
Characterization
of the monolayers
Thickness. The average thickness of the monolayers was evaluated by ellipsometry. The ellipsometer (Beaglehole Instruments) is of the polarizer-birefringence modulator-analyse:r configuration and was
within
a self-assembled
system:
A. J. Guiomar
et al.
equipped with a 6328nm He-Ne laser. The optical constants for the clean gold layer were obtained immediately after cleaning and before forming the monolayers. The values calculated are the average of values at six different locations on the surface. After formation/deposition of the monolayer, the slides were analysed again and all thickness values calculated by using a real refractive index of 1.45 for the monolayer (in accordance with Porter et aL3’). Measurements on nine different locations on the surface were carried out. The results are presented as an average value f standard deviation. Wettability by water. Wettability was evaluated by measuring the contact angle of water droplets placed on the monolayer-covered slide. An apparatus built inhouse was used. It was composed of a sodium lamp to illuminate the sample from behind, an XYZ stage upon which the sample was placed, a mounted micrometric syringe to deliver water droplets, a video camera (Hamamatsu C3077 CCD camera) with a zoom lens with a 2x extension tube (Navitar) and a high-resolution monochrome video monitor (Hitachi VM 920K). The images were captured and analysed with appropriate software (Accuware, version 2.1) running on a 486 PC. The following contact angles were measured: advancing (0,) and receding contact angle (0,) of captive droplets, and the contact angle of a freestanding droplet (0,). To measure 8,, a small droplet was applied to the surface in such a way that the needle was kept immersed in the droplet. By using the micrometric plunger of the microsyringe, the droplet was made to advance and recede two times by adding and removing liquid, after which it was slowly advanced once again and the image frozen while the droplet boundary was moving. The Or was measured after measuring the advancing contact angle. For this, the boundary of the droplet was made to recede slowly. The image was frozen while it was moving and the contact angle was measured. For t&, the contact angle was measured after removing the needle from a droplet deposited on the surface. Contact angles were measured on both sides of the droplet and at different places on the surface (three to five locations). An average value was calculated for each sample and is presented as an average value f standard deviation. Hysteresis was defined as 13,~8,. FTZR spectra. The FTIR spectrum of a monolayer was acquired with a Bruker IFS 48 FTIR spectrometer using a grazing-angle incidence geometry. The instrument chamber was purged with dry air generated by a Nitrox Pure Air Generator unit. Light was incident on the sample surface at 80” and only the reflected ppolarized light was analysed. The number of scans accumulated was 2000 and the spectral resolution 2 cm-‘. The spectra were acquired after purging for 3 to 4 h, in order to remove water vapour and carbon
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A. J. Guiomar
dioxide from the chamber. The sample spectra were acquired against a bare gold background spectrum. The spectra were baseline-corrected by using the ‘rubber band’ algorithm. Spectra of the materials dispersed in KBr (Spectroscopic Grade, Graseby) discs were also obtained. These were acquired in transmission mode. Immobilization of GOX
The slides were immersed in a 1 mgml-’ solution of glucose oxidase (Biozyme, UK) in 0.1 M phosphate buffer at pH 7.0 and left overnight at room temperature. Before being analysed, the slides were thoroughly rinsed with water and phosphate buffer, and dried with a stream of nitrogen if required. Characterization of the immobilized GOX. The layer of physisorbed GOX was characterized by measuring its thickness and acquiring its FTIR spectrum as described above for the monolayers. Enzyme activity. The activity of GOX was evaluated by coupling the oxidation of glucose to the oxidationreduction of hydroxymethylferrocene [HMFc, prepared by MediSense (UK)]. This coupling was studied by cyclic voltammetry according to the porcedure of Cass Applied et aL5. For that, an EG&G Princeton Research Scanning Potentiostat model 362, interfaced to an Amstrad PC via an A/D converter built in-house, was used. Visualization of the voltammograms was via the Condecon 310 Cyclic Voltammetry Software (EG&G). A thermostatted (25”(Z), single-compartment, three-electrode cell (Metrohm) was connected to the potentiostat. The electrodes were constructed in-house, from metals of the highest purity available (Johnson Matthey) and were: (1) a square platinum counter electrode; (2) an Ag/AgCl (saturated KCl) reference electrode; (3) a working electrode made of a singleside-only gold-coated glass slide containing the monolayer with or without the immobilized enzyme. The chromium underlayer was 1 nm thick and the gold overlayer 200nm. Before the immobilization of GOX on the modified gold electrodes, a working area of 1.7 to 1.9cm2 was defined on the centre of it by covering the rest with Araldite Rapid (Ciba-Geigy) and allowing the curing process to occur overnight at room temperature. Electrical contact was achieved through the use of a silver-loaded epoxy resin (Circuit Works 2400 Conductive Epoxy, Planned Products) and a crocodile clip. This contact region was always kept above the solution level. The cell could be sealed and possessed a venting port that allowed purging of the test solution with water-saturated argon (Pureshield Argon, BOC) and keeping a blanket of water-saturated argon above the solution, to prevent oxygen re-entry during data acquisition. All studies were carried out in quiescent solutions that had been purged for 15 min. The electro-
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et al.
lyte was aqueous 0.09 M NaCl in 0.01 M sodium phosphate buffer, pH7.0 (referred to as PBS). HMFc was added from 0.02 M stock solutions in absolute ethanol to ease dissolution of HMFc in the electrolyte, Glucose (D-glUCOSe, Fisons) was added from a 1 M stock solution in PBS, after being left overnight to ensure complete mutarotation. GOX was added from stock solutions in 0.1 M sodium phosphate buffer, pH 7.0. GOX molarity was expressed as molarity of FAD+.
RESULTS Monolayer preparation and characterization C18SAM. The gold slides coated with C18SAM showed a hydrophobic contact angle with water with an average advancing contact angle of 114.5f2.2 (Table 1). The SAM showed uniform thickness, with an average value of 2.26f0.26 nm (Table 2). The FTIR reflection spectrum in the high-frequency region of this SAM is presented in Figure I with assignments in Table 3. The advancing contact angles obtained, the thickness and the FTIR spectrum in the high-frequency region are indicative of a well-ordered SAM. CAPLB film. The pressure/area isotherm obtained for CAP can be seen in Figure 2. The monolayer progressed from a gaseous-like state to a solid-like state upon compression, where the Wilhelmy balance plate suffered a deviation from its vertical orientation. Extrapolation of the steepest part of the curve gave an average value, for the limiting area, of 0.42f0.03 nm* per substituted anhydroglucose unit (average of values from five pressure/area isotherms). The Langmuir monolayers prepared could be compressed and expanded. Although some hysteresis occurred, especially at low surface pressures (Figure 2), it was not as large as that reported for cellulose triacetate by Hittmeier et a1.33. The LB method of monolayer transfer to a solid surface was chosen in transfer of the CAP Langmuir monolayer to a C18SAM-coated gold slide. The Langmuir-Schaefer method (horizontal lifting) was also evaluated. The transfer resulted in an irregularly deposited film, giving deposition ratios above 3, and droplets of water on its surface showed an irregular shape. A variation in the LB transfer method, beginning with the slide immersed before compression of the monolayer (crossing the surface of the subphase) and withdrawing it across the monolayer, resulted in very low deposition ratios, irregular transfer rates and a large variability in the subsequent contact angles measured at different regions of the sample surface. Therefore, transfer via immersion
‘One molecule
SCIENCE Volume 4 Numbers 3-4 1997
of GOX contains
two FAD molecules
Glucose oxidase within a self-assembled
Table 1 Contact angles of water on gold-coated with different monolayers Monolayer
CISSAM ClSSAM CAPLB
glass slides covered
Table 2 Average on the gold-coated
Backh
Front0
thickness
Monolayer
8. (“)
or (“)
0s (“)
& (“)
or (“)
0,~)
115&l
103f3
107f3
114f2
103f3
10711
73fl
53f2
68f2
70f2
50fl
65fl
“The face of the slide facing the Wilhelmy balance plate hThe face of the slide facing the moving barrier of the trough
of the monolayers
Thickness
CllSAM CAPLB film Physisorbed GOX
+
0.0015
ellipsometric glass slides
et al.
system: A. J. Guiomar
prepared
(nm)
Front”
Backh
2.10f0.16 1.41f0.35 9.95f0.58
2.42f0.21 2.88f0.59 8.71f0.78
“The face of the slide facing the Wilhelmy balance plate hThe face of the slide facing the moving barrier of the trough
1 a
0.0015
b t
8
0.8 -
0.41 3000
.
.
.
rn , 2950
.
I
I
,
, 2900
,
,
,
,
, 2850
,
,
,
,
, 2800
Wavenumber (cm-l) Figure 1 Infra-red reflection spectra of the gold-coated transmission spectrum of CAP in a KBr disc (3000-2800cm-’
slide covered region)
with
SUPRAMOLECULAR
(a) Cl8SAM
and
(b) CIISAM
+CAPLB
film;
(c) infra-red
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Glucose oxidase
within
a self-assembled
Table 3 Peak positions
and assignments in the CH stretching region”
spectra
Peak position
system:
for the CISSAM
A. J. Guiomar
infra-red
Assignment
(cm-‘)
v,(ip) C-H stretching mode of CH3 v,(FR) CH stretching mode of CH3 v, C-H stretching mode of CH2 v,(FR) CH stretching mode of CHs v, C-H stretching mode of CHs
2964.1 2931.1
2917.8 2877.3 2850.3 “Assignments
according
to ”
20 -
15 -
3 5
10 -
z 5-
’
0.
’
’
0.2
0.0
’
0.4
0.6
0.8
1.0
area per substituted anhydroglucose
Figure 2
Compressionexpansion CAP on a water subphase
surface
1
I
1.2
1.4
.
,
--I.6
unit (nm 2)
pressure/area
isotherms
of
0
et al.
slide above the monolayer, transfer occurred during both immersion and withdrawal. Accordingly, the transfer meniscus was hydrophobic during immersion and hydrophilic during withdrawal. The rate of monolayer transfer was not constant and the deposition ratios were not unity. The transfer rate was more irregular during immersion, showing first a fast transfer step when 63% of the total monolayer area deposited was transferred, followed by a slower step. Most of the film was transferred during the downward movement of the slide (82%) the total deposition ratio on immersion being 2.7. During withdrawal, the transfer rate was more regular. However, it resulted in a deposition ratio of 0.6. After deposition of the CAPLB film, the surface became more hydrophilic, showing an average advancing contact angle of 71.5f2.2” (Table I). The CAPLB film deposited on the front face of the slide (that facing the Wilhelmy balance) had an average thickness of 1.41f0.35 nm (Table 2). It was thinner than that deposited on the back face (that facing the moving barrier), which had an average thickness of 2.88f0.59nm (Table 2). The film on the back face of the slide was also more irregular than that on the front face, this being reflected in a higher standard deviation of its average thickness value. The presence of the CAP on the surface of the SAM was validated through identification of the ester C=O stretching band in the 1750 cm-’ region of the reflection FTIR spectrum and by the contribution of the C-H stretching vibrations in the high-frequency region (Figure I and Figure 4, Table 4). GOX immobilization
cd t?
cd 40 I
0
5
*
I
.
10
I
15
,
,
,
20
(
25
height of slide immersed (mm) Figure 3 Deposition curve Cl SSAM-coated gold slide
for
the
LB
transfer
of
CAP
to
a
followed by withdrawal was selected. A deposition curve is shown in Figure 3. The area of CAP Langmuir monolayer kept at a surface pressure of 20mNm-’ was constant in time. Beginning with the
284
SUPRAMOLECULAR
The presence of the enzyme on the surface after physisorption is implicit in the increase in thickness of the total assembly following the immobilization step. The adsorbed layers on both faces of the slide are of comparable thickness, with an average value of 9.33f0.97 nm (Table 2). The reflection FTIR spectrum of the physisorbed enzyme gave further support for the presence of the enzyme on the surface (Figure 5, Table 5). New vibrational bands in the amide I and II regions (170& 1600 cm-’ and 1575-1480 cm-‘, respectively) appear after immobilization of the enzyme on the C18SAM+CAPLB film assembly. This is in agreement with those seen for the enzyme in the crystalline state. The amide I band at 1656cm-’ is rather noticeable. The amide II band seems to be split and does not overlap as it does in the spectrum for the crystalline state. Activity of the immobilized GOX. The activity of the immobilized enzyme could not be determined by the conventional calorimetric activity assay for GOX. An electrochemical assay was adopted instead, using
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Glucose oxidase
within
a self-assembled
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A. J. Guiomar
et al.
0.004
a 0.003
0.8
0.8
0.21...,...,...,...,...,..., :2000 1800 1900
1700
1800
1500
1400
Wavenumber (cm-l) Figure 4 FTIR reflection spect.ra of the gold-coated slide covered with (a) Cl8SAM and (b) Cl8SAM +CAPLB film; (c) FTIR transmission spectrum of CAP in a KBr disc (:200&14OOcm-’ region)
Table 4 Peak positions spectrum0
and assignments
for the CAP infra-red
Peak position (cm-‘)
Assignment
2983 2945 2885 1749 1465 1423
C-H stretching of CHx or O-H stretching C-H stretching of CHOH, -CH*OH or CH, C-H stretching of CH or CHs C = 0 stretching of the ester group C-H deformation of CHx CHs symlmetrical bending
OAssignments according to references 38 and 39
MS 4:3/l-E
the mediator. HMFc oxidation HMFc as (E”’ = + 0.245 V) proved to be nearly reversible at an evaporated gold electrode. It showed: (1) a peak separation of 70mV, (2) the peak potential was independent of the scan rate, (3) the peak current was linearly dependent on the square root of the scan rate and (4) the ratio of anodic to cathodic current was close to 1. After the C18SAM had been formed, a strong inhibition of the reduction wave was found and the peak potential was shifted to more positive
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oxidase
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a self-assembled
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A. J. Guiomar
et al.
1.0
0.9
0.8 8 H .S
0.7
g # $
0.6
0.5
0.4
0.004
b 0.003
8 H & 0.002 $ 2 0.001
0.000 2(
I
1900
1800
I
#,‘I
I
1700
.,,.,.,,.:.,
1600
1500
1400
Wavenumber (cm-l) Figure 5 (a) FTIR transmission spectrum C18SAM +CAPLB film after immobilization
of GOX in a KBr of GOX (200(r1400cm-’
Table 5 Peak positions and assignments of the amide I and II bands of GOX immobilised on the Cl 8SAM + CAPLB and for GOX in a KBr disc Peak position
Assignment
(cm-‘)
GOX in KBr disc
Immobilized 1754.9
1655.6
1656.6
1538.9
1540.8
1517.7
1507. I
286
SUPRAMOLECULAR
GOX C = 0 stretching of the ester groups of CAP Amide I C=O stretching Amide II N-H deformation Amide II N-H deformation
disc and region)
(b)
FTIR
reflection
spectra
of the
gold
slide
coated
with
a
potentials (Figure 6b), an indication that the oxidation is being blocked. This effect was less pronounced with the ClSSAM + CAPLB (Figure 6~). However, the poor reversibility of HMFc at these modified electrodes did not prevent its role in mediating electron transfer from GOX in solution to the electrode, in the presence of glucose, even in the worst case (C18SAM) (Figure 7). The physisorbed enzyme film contained a population of active enzyme. When glucose was added to the PBS electrolyte containing HMFc, a catalytic current indicative of catalytic coupling between HMFc and GOX could be seen (Figure 8).
SCIENCE Volume 4 Numbers 3-4 1997
Glucose oxidase within a self-assembled
F
system: A. J. Guiomar et al.
b
-2.0x10-5I-
c 2.0~10-5
-2.Ox10~
-
5
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
E (V) vs. Ag/AgCl (sat. KCl) Figure 6 Cyclic voltammograms of a OSmM solution of HMFc in PBS, at a scan rate of 5mVs-‘, showing the effect of modification of the electrode on the reversibility of HMFc oxidation: (a) bare gold electrode; (b) gold electrode coated with C18SAM; (c) gold electrode coated with a Cl8SAM + CAPLB film
DISCUSSION Monolayer preparation and characterisation
C18SAM. The advancing contact angle for a smooth, packed methyl surface is blelieved to be 110-l 15” while, for a methylene surface, it is 102-103” (refs. 40 and 41). The average value of fIa= 114.5f2.2” (Table I) is indicative of a well-ordered monolayer, showing mainly methyl groups at the air interface. The average hysteresis value of 11Sf4.2” could indicate either some surface heterogeneity or a degree of surface roughness (revealed by the
standard deviation of the average contact angle value). The average thickness observed (2.26f0.26nm) is close to the value expected for a monolayer whose molecules are fully stretched but tilted 30” away from the normal to the surface (2.34f0.21 nm 40). The p-polarized light reflection FTIR spectrum also shows that the molecules are tilted away from the normal to the reflecting surface. This can be inferred from the presence of the symmetric and asymmetric vibrations of the methylene groups at 2850 and 2918 cm-’ (Figure I, Table 3). These would not be present in a arrangement normal to the reflecting
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Glucose oxidase
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A. J. Guiomar
et al.
1 a
-0.2
0.0
0.2
0.4
E (V) vs. Ag/AgCl
0.6
-0.2
(sat. KCl)
0.0
0.2
E (V) vs. AgIAgCl
0.4
0.6
(sat. KCl)
Figure 7 Cyclic voltammograms of HMFc/glucose solutions in the presence and absence of GOX (in solution), at a scan rate of 1mVs_‘: (a) gold electrode coated with CIISAM; (b) gold electrode coated with a CIISAM + CAPLB film. The composition of the solutions was: (i) 0.5 mM HMFc and 49mM glucose in PBS; (ii) as for (i) but with addition of GOX to a concentration of 7.5 nM (this amount of GOX is approximately four times that expected for a monolayer of enzyme on these electrodes)
LI.I.LI.I.t.t.t*, -0.2 -0.1
0.0
0.1
0.2
0.3
E (V) vs. Ag/AgCl
0.4
0.5
0.6
0.7
(sat. KCl)
Figure 8 Cyclic voltammograms of HMFc solutions in the presence and absence of glucose, performed with an electrode containing a Cl8SAM +CAPLB film with physisorbed GOX: (i) 0.5mM HMFc in PBS; (ii) as for (i) but with the addition of glucose to a concentration of 49 mM. Scan rates: I mV s-’
surface, since their transition dipoles would be parallel to the surface and would not be picked up by ppolarized light4’. Comparing the wavenumbers of the bands observed in the high-frequency region of the spectrum (Table 3) with those obtained for a longchain alkanethiol in the bulk crystalline phase3’, there is also further evidence that a well-ordered SAM with methylene groups existing in a crystalline-like environment, with fully extended chains in an all-trans conformation, has been formed. CAPLB film. Although a report on monolayers of CAP could not be found in a literature search, the
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average value of 0.42f0.03 nm* per substituted anhydroglucose unit for the limiting area is in accordance with published values for cellulose triacetate and cellulose tripropionate. For cellulose tripropionate on water, a limiting area of cu. 0.45nm2 has been reported3’. The published values for cellulose triacetate are rather disperse: between 0.30 and 0.54nm2 per substituted anhydroglucose unit3’-34. A detailed study of the spreading of cellulose triacetate obtained limiting areas of 0.40f0.015 nm* (ref. 33). However, the calculated area of the substituted anhydroglucose unit in cellulose triacetate is 0.69nm2 (W. T. Astbury, cited by Borgin and Johnson3*). The discrepancy encoun-
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Glucose oxidase within a self-assembled system: A. J. Guiomar et al.
tered between the calculated and the measured areas has been explained by considering that the substituted anhydroglucose rings do not lie flat on the water but are somewhat tilted3’. Localized gelation of the film, upon compression, is supposed to have occurred. This is the interpretation for the observed displacement of the Wilhelmy plate from its vertical position, a behaviour already reported for cellulose triacetate33. According to Hittmeier et a1.33, the origin of this displacement seems to be related to a high surface viscosity of the film due to gelation at low pressures. At large areas the anhydroglucose units are supposed to lie flat on the water surface, as a consequence of weak hydrogen bonds through its ester groups. During compression, chain-chain interactions occur. The hydrophobic contacts between overlapping ester methyl groups and the C-H of the anhydroglucose ring can cause tilting of the anhydroglucose rings, leading to the formation of gelated, coherent patches. The tilted conformation is stabilized by van der Waals’ interactions between adjacent pyranose rings. In a model, taking into account a limiting area of 0.40nm2 per substituted anhydroglucose unit, this unit will be in a nearly vertical orientation33. Tht: displacement of the plate during compression is supposed to be caused by the movement of gelated patches against its front face when trying to pack more closely33. The fact that CAP Langmuir monolayers could be transferred to a hydrophobic surface by crossing it vertically both during an initial downward and a subsequent upward movement (Figure 3) suggests that the CAP macromolecules are able to orient themselves at the air/water interface, as does the fact that poor transfer also occurred when a hydrophobic slide was withdrawn across it. This also shows that the monolayer possesses some flexibility. However, the existence of a fast transfer rate immediately upon contact of the slide with the monolayer (Figure 3) could indicate that the CAP monolayer has limited flex:ibility. This could lead to the transfer of collapsed patches, as indicated by a deposition ratio of 2.7. The deposition ratio of 0.6 on subsequent withdrawal indicates that an incomplete coverage of the surface occurred. The contact angle of the C18SAM surface decreased after deposition of the CAPLB film. This is expected to happen owing to the presence of the more hydrophilic hydroxyl and ester carbonyl groups in CAP. The hysteresis increased to 20f2.2”. A high hysteresis is expected for a surface containing both polar and non-polar groups, where the polar groups will interact more strongly with the water molecules than the non-polar groups. The back face of the slide showed a film with larger thickness than the front face. Considering that the back face is facing the moving barrier, it should receive a larger fraction of the monolayer since the monolayer is being compressed against it during
transfer. Correspondingly, a higher standard deviation of its average thickness was found (Table 2). The thickness values observed (1.41 f0.35 nm and 2.88f0.59nm for the front and back face, respectively) are compatible with five to seven monolayers being transferred to the back face and two or three to the front face, considering a model with the substituted anhydroglucose units vertically oriented on the surface of the subphase$. GOX immobilization A layer of GOX with an average thickness of 9.33fO.97nm was physisorbed to the Cl 8SAM + CAPLB film (Table 2). The dimensions of the GOX molecule have been published4* following the resolution of its 3D structure, albeit for a 95% deglycosylated molecule§. More realistic values should come from the Stokes radius of the whole enzyme. It has a value of 4.27nm, with an axial ratio of 2.5 (ref. 43). This is equivalent to the dimensions of 13.0 and 5.2 nm for the two axes of an ellipsoidi4. Ellipsometric workI gave the indication that the two axes of the ellipsoidal enzyme molecule would have dimensions close to 14.0 and 5.0nm. It is not possible to propose a unique model to interpret the thickness of the physisorbed GOX layers (9.95f0.58 nm and 8.71f0.78 nm, for the front and back face, respectively) by comparing its thickness with the dimensions of the enz e molecule, owing to the errors affecting these values (ny” . Additional support for the presence of the enzyme on the surface comes from the identification of its infra-red spectrum, in particular its characteristic amide bands (Figure 5, Table 5). The amide I band at 1656cm-’ was rather noticeable. The amide II band seem to be split, unlike in the crystalline state. This could indicate that a population with a different conformational state exists at the surface, although it could be an artefact resulting from the noise level. This noise is related to non-complete removal of either water vapour or adsorbed water from the sample. It is expected that improvement in purging will allow the acquisition of information about the conformational state of the physisorbed enzyme. Activity of the immobilized GOX. The failure to detect enzyme activity by the conventional calorimetric assay is thought to be due to the low amounts of ‘The dimensions of the anhydroglucose unit in cellulose are Onmx0.515nm, when the ring is lying flats’ 5 The group of researchers that solved the 3D structure of GOX have published values for the dimensions of the deglycosylated enzyme of both 6.0nmx5.2nmx7.7nm and 7.0nmx5.5nmxS.Onm (refs. 4 and Y’These thickness values were obtained by using a value of I .50 for the refractive index of the physisorbed GOX film, in accordance with published results. However, this value was calculated for a monolayer with complete coverage, from enzyme dimensions obtained from ellipsometric measurements’4. As the refractive index depends on the surface coverage, the accuracy of the thickness values will be affected4
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enzyme present. Detection of H202 production or O2 consumption was ruled out since their reduction and oxidation, respectively, are strongly inhibited even by C6SAM on bulk gold and, in the case of H202, suppressed by C 12SAM on platinum44’ 45. Hydroxymethylferrocene, a redox molecule showing reversibility at gold electrodes, was the mediator selected for mediation of the electron transfer from GOX to the electrode. Here, the oxidation of glucose is coupled to the oxidation-reduction of HMFc in solution. Electrons are thus transferred from the enzyme to the electrode via the mediator. This is a reaction scheme in which the reduced mediator (FcR) is oxidized at the electrode [equation (l)] and its oxidized form (FcR+) is catalytically put back to the reduced state by reacting with the reduced form of GOX [equation (2)] which is produced when the enzyme oxidizes glucose [equation (3)]. 2FcR H FcRf + 2e-
(1)
GOXred + 2FcR+ + GOX,, + 2FcR
(2)
glucose + GOX,, 4 gluconic acid + GOX,,d
(3)
It has been reported that HMFc shows quasireversible behaviour on C6SAM on bulk gold44. However, for C18SAM on an evaporated gold electrode, its reduction wave on reversing the potential scan was drastically reduced and the peak potential was shifted to higher potential values46. This type of suppression was also detected in this work (Figure 6b). Such suppression is a limitation to the use of this C18SAM in a sensing scheme that relies on a mediator in solution. It seems to arise from a kinetic effect since, when an electrochemical system departs from reversibility, it means that the rate of electron transfer decreases below the rate of mass transport to the electrode surface, resulting in increased peak separations4’. This is understandable if one accepts a long-range model for electron transfer across the monolayer46. This effect was less pronounced for the C18SAM coated with the CAPLB film (Figure 6~). This could be due to the introduction of some degree of disordering in the SAM, creating defects through which the mediator could have access to the gold surface. In spite of these effects, detection of the activity of GOX could be performed. The C18SAM+CAPLB system showed more sensitivity to GOX detection (Figure 7). This should be related to the fact referred to above that HMFc shows a higher degree of reversibility in this system than in the C18SAM-only system. A catalytic current was observed when an electrode with GOX immobilized on the C18SAM +CAPLB film was tested (Figure 8), showing that a population of active enzyme exists immobilized on the film surface. Comparison of the magnitude of the catalytic current obtained with the immobilized enzyme (curve
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et al.
ii of Figure 8) with that obtained with enzyme added to the solution (curve b, Figure 6) allows us to state that most of the immobilized enzyme is active”. This result is thought to be due to enzyme physisorbed to the C18SAM+CAPLB film and not to the electrode body. This is because no enzyme activity was detected in a control experiment where an electrode body, with approximately the same area but with no evaporated gold film, was treated with a GOX solution as before, inserted into the electrochemical cell and analysed with a bare gold electrode, in the same way as for the modified electrodes.
CONCLUSIONS An LB ultrathin film of cellulose acetate propionate could be transferred to a C18SAM formed on a goldcoated glass slide. Glucose oxidase was immobilized onto this assembly by simple physisorption. The activity of the enzyme could be detected electrochemically by using hydroxymethylferrocene as a redox mediator. An active enzyme population exists immobilized on this assembly.
ACKNOWLEDGEMENTS The authors thank MediSense (UK) for the gift of HMFc and GOX and Dr R. Luo (Department of Mining and Mineral Engineering, University of Leeds) for help with the electrochemical studies. A. J. Guiomar is on leave from Departamento de Bioquimica, Faculdade de CiZncias e Tecnologia, Universidade de Coimbra, Portugal. He thanks J.N.I.C.T. (Portugal) for personal financial support (Praxis XXI programme).
REFERENCES Gcpel, W. and Heiduschka, P., Biosens. Bioelectron.. 1995, 10, 853. Zull, J. E., Reed-Mundell, J., Lee, Y. W., Vezenov, D., Ziats, N. P., Anderson, J. M. and Sukenik, C. N., J. Indus/. Microbial.. 1994, 13, 137. Wilson, R. and Turner, A. P. F., Biosens. Bioelectron.. 1992, 7. 165.
‘IThe thickness value of the immobilized enzyme layer (Table 2) could be obtained by a monolayer or a bilayer of enzyme molecules, depending on the orientation of the enzyme. This allows us to compare the magnitude of the catalytic current obtained with the immobilized enzyme with that obtained when an amount equivalent to four layers of free enzyme is added to the solution and detected with a gold electrode modified with a ClSSAM+CAPLB film (Figure 6, curve b). Although direct comparison between both is not totally correct, since the current in the second case also reflects enzyme diffusion and a higher collision frequency between the enzyme and both the mediator and glucose, the magnitude of the catalytic current of the immobilized enzyme seems to indicate that most of the immobilized enzyme must be active
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4.
Hecht, H. J., Schomburg, I>., Kalisz, H. and Schmid, R. D., Biosens. Bioelectron.,
5.
6. I. 8. 9.
Cass, A. E. G., Francis, D. G., Hill, H. A. O., Aston, W. J., Hiueins. I. J.. Plotkin. E. V.. Scott. L. D. L. and Turner. A. P. F.,%ai. Chem.. 1984; 56, 667. Cenas, N. K. and Kulys, J. J., J. Electroanal. Chem., 1981, 128, 103. Sonawat, H. M., Phadke, R. S. and Govil, G., Biotechnol. Bioeng., 1984, 26, 1066. Albery, W. J., Bartlett, P. N. and Cass, A. E. G., Phil. Trans. Roy. Sot. Lond., 1987, B316, 107. Hill, B. S., Scolari, C. A. and Wilson, G. S., J. Electroanal. Chem., 1988, 252, 125.
10.
15.
Taxis du Poet, P., Miyamoto, S., Murakami, T., Kimura, J. and Karube. I.. Anal. Chim. Acta. 1990. 235. 255. Bartlett, P. ‘N: and Bradford, V. Q., J. Chem. Sot., Chem. Commun., 1990, 1135. Bartlett, P. N. and Cooper, J. M., J. Elecfroanal. Chem., 1993, 362, 1. Alvarez-Icaza, M., Kalisz, IH.M., Hecht, H. J., Aumann, K.D., Schomburg, D. and Schmid, R. D., Biosens. Bioelectron., 1995, 10, 735. Szucs, A., Hitchens, G. Di. and Bockris, J. O.‘M., Bioelectrochem. Bioenerg., 1989, 21, 133. Alvarez-Icaza, M. and Schmid. R. D., Bioelecrrochem.
16.
Jiang, L., Mcneil, C. J. and Cooper, J. M., J. Chem. Sot.,
17.
Sun, S., Ho-Si, P.-H. and Harrison, D. J., Langmuir,
18.
Zaitsev, S. Y., Coil. Surf
11. 12. 13. 14.
Bioenerg.,
30.
Ramanathan, K., Ram, M. K., Malhotra, B. D. and Murthy, S. N., Mater. Sci. Eng., 1995, C3, 159. Tsuzuki, H., Watanabe, T., Okawa, Y., Yoshida, S., Yano, S., Koumoto, K., Komiyama, M. and Nihei, Y., Chem. Le/t.. 1988, 8, 1265. Tatsuma, T., Tsuzuki, H., Okawa, Y., Yoshida, S. and Watanabe, T., Thin Solid Films, 1991, 202, 145. Lee, S. Y., Anzai, J. and Osa, T. Sens. Actuators Part BChem. 1993, 12, 153. Schuhmann, W., Heyn, S. P. and Gaub, H. E., Adv. Mater.,
31. 32. 33.
Adam, N. K., Trans. Faraday Sot., 1933, 29, 90. Borgin, K. and Johnson, P., Trans. Faraday Sot., 1953,4,956. Hittmeier, M., Sandell, L. S. and Luner, P., J. Poiym. Sci., Parr
26.
1993, 8, 197.
27. 28. 29.
1991, 3, 388.
C. 1971,36,
34. 35.
43. 44. 45.
Creager, S. E. and Olsen, K. G., Anal. Chim. Acta, 1995,307,277. Dong, X.-D., Lu, J. and Cha, C., Bioelectrochem. Bioenerg.,
46.
Creager, S. E., Hackett, L. A. and Rowe, G. K., Langmuir,
41.
Southampton Electrochemistry Group, Instrumental Methods in Elecrrochemistry. Ellis Horwood Ltd, Chichester, 1985.
38.
40. A’: Physicochem.
Eng. Aspects,
1993,
75, 211.
19.
22. 23.
Barmin, A. V., Eremenko, A. V., Sokolovskij, A. A., Chernov, S. F. and Kurochkin, I. N , Biotechnol. Appl. Biochem.. 1993, 18,369. Fiol, C., Valleton, J.-M., Delpire, N., Barbey, G., Barraud, A. and Ruaudel-Teixier. A.. Thin Solid Films. 1992. 210/211. 489. Sriyudthsak, M., Yamagishi, H. and Moriizumi, T.,’ Thin Solid Films, 1988. 160, 463. Moriizumi, T., Thin Solid Films, 1988, 160, 413. Ancelin. H.. Zhu. D. G.. Pettv. M. C. and Yarwood, J.,
24.
Okahata, Y., Tsuruta, T., Ijiro, K. and Ariga, K., Langmuir,
25.
Okahata, Y., Tsuruta, T., ljiro, K. and Ariga, K., Thin Solid Films, 1989, 180, 65.
20. 21.
Langmuir.
1990,6,‘1068.
1991, 3, 25.
Nicholson, R. S. and Shain, I., Anal. Chem., 1964, 36, 706. Porter, M. D., Bright, T. B., Allara, D. L. and Chidsey, C. E. D., J. Am. Chem. Sot., 1987, 109, 3559. Ott, E., Spurlin, H. M. and GraMin, M. W (eds), High Polvmers. Vol. V. Part IV (Cellulose and Cellulose Derivatives. ed. N. M. Bikales and L. Segal), 2nd edn. Wiley Interscience, New York, 1971. Zhbakov, R. G., Infrared Spectra of Cellulose and its Derivatives. Consultants Bureau, New York, 1966. Bain, C. D., Troughton, E. B., Tao, Y.-T., Evall, J., Whitesides, G. M. and Nuzzo, R. G., J. Am. Chem. Sot., 1989, 111, 321. Ulman, A., An Introduction to Ultrathin Organic Films-from Langmuir-Blodgett to Self-Assembly. Academic Press, Inc., New York, 1991. Hecht, H. J., Kalisz, H. M., Hendle, J., Schmid, R. D. and Schomburg, D., J. Mol. Bioi., 1993, 229, 153. Nakamura, S., Hayashi, S. and Koga, K., Biochim. Biophys.
1991, 7,
727.
Embs, R., Funhoff, D., Laschewsky, A., Licht, U., Ohst, H., Prass, W., Ringsdorf, H., Wegner, G. and Wehrmann, R., Adv. Mater.,
36. 37.
39.
1995, 1293.
H. and Fukuda, K., Thin Solid
Films, 1985, 133, 29.
1994, 33, 191.
Chem. Commun.,
267.
Kawaguchi, T., Nakahara,
41.
42.
Acta. 1976,445,
*’
294.
1995, 36, 13. 1992, 8, 854.
1988, 4, 1373.
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0968-5677/97/$17.00
ELSEVIER
Immobilization of glucose oxi’dase onto a Langmuir-Blodgeti ultrathin film of a cellulose derivative deposited on a self-assembled monolayer Antbnio J. Guiomar*,
Stephen D. Evan&*
and James T. Guthrie*
*Department of Co/our Chemistry and Dyeing, The University of Leeds, Leeds LS2 9JT, UK +Department of Physics and Astronomy, The University of Leeds, Leeds LS2 9JT, UK (Received 6 September 1996; revised 17 December 19961
Glucose oxidase ‘was immobilized on a Langmuir-Blodgett film of cellulose acetate propionate deposited on a self-assembled monolayer coated substrate. These layers were characterized in terms of their ellipsometric thickness, wettability and infra-red spectra. Glucose oxidase was immobilized on this composite layer by physisorption. The presence of the enzyme on the surface was confirmed by ellipsometry, infra-red spectroscopy and by detecting its activity electrochemically. An enzyme population remained active after adsorption onto this assembly. 0 1997 Elsevier Science Ltd. All rights reserved. (Keywords:glucose oxidase; immobilization;self-assembly)
INTRODUCTION Enzyme-based
biosensors
require
close
contact
between a transducer and the enzyme. This can be achieved by immobilization of the enzyme onto the surface of the transducer. In electrochemical biosensors, the transducer surface is usually a metal. The choice of immobilization chemistry for direct attachment is thus very limited. It can be extended by depositing or creating monolayers on these metal surfaces, bringing the adv,antage of ordering, very low thickness and the possibility of controlled immobilization”2. For the biosensor to perform its task, the enzyme has to be immobilized in an active state, i.e. retaining its ability to catalyse a reaction involving its substrate (the analyte). This requires that both the immobilization step and the new micro environment into which the enzyme is placed should not cause any major conformational change in the active site of the enzyme. Glucose oxidase (GOX), a well-characterized and much used enzyme334, was used in this work. GOX is mostly used for the determination of glucose levels in body fluids. Nearly all the devices developed are of the biosensor type and the large majority are of the
amperometric subtype. GOX catalyses the oxidation of glucose to gluconic acid, coupled with the reduction of oxygen (02) to hydrogen peroxide. However, oxygen can be replaced by several synthetic electron acceptors, the so-called mediators3*5. Direct electron transfer from the enzyme Flavin Adenine Dinucleotide (FAD) centres to modified electrodes has proved elusive to attain6i3. Only a few reports have evidence that this transfer is carried out from an active population of enzyme molecules’416. The use of Langmuir-Blodgett (LB) films for the immobilization of GOX seems to have started in the late 1980s. The work reported can be divided as follows. 1. Formation
2.
i) ii)
iii) t To whom correspondence
should
be addressed
of an enzyme LB film after spreading the enzyme at the air/water interface, either in its native form or after being crosslinked17-‘9. Adsorption to a Langmuir monolayer, followed by LB transfer. The enzyme was included in the following ways: addition to the subphase before spreading the amphiphile2’; adsorption onto a Langmuir monolayer by moving it to another trough compartment containing a GOX solution, where the adsorption occurs21-23; and addition to a previously prepared Langmuir monolayer of an amphiphile (it was not disclosed how)18.
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3. Mixing the enzyme with amphiphilic molecules, the spreading of the mixture at the air/water interface being followed by LB transfer. Reported approaches include: 9 coating the enzyme with a fatty acid, followed by spreading and LB or Langmuir-Schaefer (LS) transfer24’25; and ii) adding enzyme to a solution of polyaniline followed by spreading and LB transfer26. 4. Covalent attachment of GOX to functional groups of a preformed LB or LS lilm27. The same research group also included an amphiphilic ferrocene derivative in the system above28. 5. Specific physisorption of modified GOX to preformed LB films, containing specifically interacting molecules. The interacting pairs used were the avidin-biotin29 and the antigen-antibody3’ pairs. This work reports the non-specific physisorption of GOX to a preformed LB film of a modified polysaccharide [cellulose acetate propionate (CAP)], an approach not found in the literature. GOX is a glycoprotein, having a protein core surrounded by a carbohydrate shel13. This approach has the advantage of presenting to the enzyme an interface that is similar in nature to its own surface. This should allow preservation of the enzyme’s native conformation. Moreover, this cellulose derivative has a high degree of substitution and consequently low crystallinity. It has both hydrophilic (hydroxyl) and relatively hydrophobic groups (acetate, propionate) on its chain. It was thought that this would permit the preparation of Langmuir monolayers and LB films. The preparation and study of Langmuir monolayers of cellulose short-chain esters has long been documented in the literature31p33, although no report could be found for CAP. The transfer of long- and mediumchain cellulose esters to solid substrates has also been reported34 but no report on the transfer of their shortchain equivalents could be found. The deposition behaviour of this type of polymer is referred to as ‘very poor’35. The deposition behaviour of cellulose esters has since been improved by using derivatives with increased length of the esterilied chain. The LB method of transfer to a hydrophobic, goldcoated glass slide was used. The gold-coated glass slide was rendered hydrophobic by creating a self-assembled monolayer (SAM) of an alkylthiol (octadecylmercaptan; C 18) on its gold/air interface. Both the SAM and the LB film were characterized in terms of their ellipsometric thickness, contact angles of their surfaces with water and infra-red spectra at grazing angle of incidence. As a first approach, GOX was immobilized simply by physisorption to a preformed LB film. The presence of the enzyme on the surface was confirmed by determining the increase in thickness of the obtaining the enzyme’s multilayered system,
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vibrational spectrum and detecting its activity. The retention of enzymatic activity was detected by means of an electrochemical assay. This makes use of a coupling scheme developed for amperometric biosensors with GOX5, where the electrode reaction is coupled to a catalytic reaction (the so-called catalytic or EC’ mechanism), a mechanism readily identified and studied by cyclic voltammetry36.
METHODS Preparation of gold-coatedglass slides Gold-coated glass slides were prepared by resistive evaporation of gold to a thickness of either 100 or 200nm, by using an Edwards Auto 306 Turbo Vacuum Coater equipped with a Edwards Turbomolecular Vacuum Pump. The microscope glass slides were cleaned immediately before evaporation by rubbing with the concentrate Decon 90 and rinsing with water. All the water used in this work was of high purity, having been purified by a Millipore unit that combines a Millipore RO and a Milli Q Plus unit in sequence. The glass slides were further treated ultrasonically in a dilute Decon 90 solution (ca. 10% in water) for 15 min. Thorough rinsing with water and drying with a stream of nitrogen followed. The glass slides were subsequently cleaned by etching with an argon plasma cleaner (Plasma Preen I) for 2min, with the power dial set at 60 and an 80% cycle, rinsed with water and dried with a stream of nitrogen. After this procedure, the glass slides were completely wetted by water. The clean glass slides were placed in a holder located inside the evaporation chamber at approximately 15 cm above the holders of the metals to be evaporated. First, a very thin chromium underlayer (thickness of either 1 or 8nm, monitored with a Edwards FTMS quartz oscillator) was deposited on the glass slides by evaporation of chromium from a chromium-coated tungsten wire (Edwards). The deposition rates were 0.1-0.2 nm s-‘, at a pressure of 2.3x 10e6mbar. This was followed by the evaporation of gold from tungsten buckets, without venting the chamber. Gold (99.99%, Advent Research Materials) was evaporated at 0.24.3 nm s-l, at the same pressure and to a thickness of either 100 or 200nm. Both sides of the slides were coated. The gold slides were cleaned by a very mild plasma cleaning (15 s, power dial set at 0, 100% cycle) immediately before use. Preparation of monolayers Cl8 self-assembled monolayer (C18SAM). Freshly cleaned gold-coated slides were immersed in a 1 mM solution of octadecylmercaptan (98%, Aldrich Chemical Co.) in dichloromethane (HPLC grade, BDH) in sealed glass containers. After 1 h, the slides
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Glucose
oxidase
rinsed with dichlorowere removed, thoroughly methane, dried with a stream of nitrogen and analysed. CAP Langmuir mono,layer. A 5 mgml-’ stock solution of cellulose acetate propionate (CAP 482.05, Eastman Chemical Co.; average contents: 2.5 wt% acetyl, 45 wt% propionyl and 2.6wt% hydroxyl; M, = 25 000 Daltons) in chloroform (HPLC grade, Sigma-Aldrich) was prepared. This solution was filtered through a filter with a pore diameter of l.Opm (Whatman Puradisc 25 TF). No significant change in concentration could be detected by gravimetric assessment after total solvent evaporation. These solutions were kept at 4°C in a fridge, in stoppered and sealed glass containers to prevent chloroform evaporation. Immediately prior to use, the solution was allowed to equilibrate to room temperature. Subsequently, it was accurately diluted to 0.25 mg ml-’ with chloroform. This dilute solution was used the same day it was prepared. Langmuir monolayers were prepared on a water subphase with the aid of al Fromherz trough equipped with a Wilhelmy hanging-plate surface pressure balance. The trough was Ilocated inside a ‘clean room’ and placed inside a horizontal flow cabinet. To spread a monolayer, 50~1 of the 0.25mgml-’ CAP solution were applied to a clean wa.ter surface of the fully open trough (area=318cm*). Tlhe CAP solution was added dropwise to the water surface. After spreading, a further 15 min were allowed in order for the solvent to evaporate. The monolayer was then compressed at a rate of 5.3 x 10e4 nm* (substituted anhydroglucose unit))’ s-’ and surface pressure (II) versus area isotherms were obtained. For hysteresis studies, the compression of the monolayer was immediately followed by its expansion at the same rate. CAP Langmuir-Blodgett (CAPLB) film. A Langmuir monolayer of CAP was prepared as previously described. The LB transfer was carried out at a constant surface pressure of 20 mNm_‘. The stability of this monolayer was evaluated by following the change in its area with time for 15 min. Only stable monolayers were transferred. The substrate was clamped to the holder of the motorized dipping unit, in a vertical orientation, and was immersed and withdrawn across the mfonolayer/air interface at a speed of 4 mm min- ’ . A deposition curve of area of the monolayer versus dipping-head position was recorded and used both to check the uniformity of the deposition rate and to calculate the deposition ratio.
Characterization
of the monolayers
Thickness. The average thickness of the monolayers was evaluated by ellipsometry. The ellipsometer (Beaglehole Instruments) is of the polarizer-birefringence modulator-analyse:r configuration and was
within
a self-assembled
system:
A. J. Guiomar
et al.
equipped with a 6328nm He-Ne laser. The optical constants for the clean gold layer were obtained immediately after cleaning and before forming the monolayers. The values calculated are the average of values at six different locations on the surface. After formation/deposition of the monolayer, the slides were analysed again and all thickness values calculated by using a real refractive index of 1.45 for the monolayer (in accordance with Porter et aL3’). Measurements on nine different locations on the surface were carried out. The results are presented as an average value f standard deviation. Wettability by water. Wettability was evaluated by measuring the contact angle of water droplets placed on the monolayer-covered slide. An apparatus built inhouse was used. It was composed of a sodium lamp to illuminate the sample from behind, an XYZ stage upon which the sample was placed, a mounted micrometric syringe to deliver water droplets, a video camera (Hamamatsu C3077 CCD camera) with a zoom lens with a 2x extension tube (Navitar) and a high-resolution monochrome video monitor (Hitachi VM 920K). The images were captured and analysed with appropriate software (Accuware, version 2.1) running on a 486 PC. The following contact angles were measured: advancing (0,) and receding contact angle (0,) of captive droplets, and the contact angle of a freestanding droplet (0,). To measure 8,, a small droplet was applied to the surface in such a way that the needle was kept immersed in the droplet. By using the micrometric plunger of the microsyringe, the droplet was made to advance and recede two times by adding and removing liquid, after which it was slowly advanced once again and the image frozen while the droplet boundary was moving. The Or was measured after measuring the advancing contact angle. For this, the boundary of the droplet was made to recede slowly. The image was frozen while it was moving and the contact angle was measured. For t&, the contact angle was measured after removing the needle from a droplet deposited on the surface. Contact angles were measured on both sides of the droplet and at different places on the surface (three to five locations). An average value was calculated for each sample and is presented as an average value f standard deviation. Hysteresis was defined as 13,~8,. FTZR spectra. The FTIR spectrum of a monolayer was acquired with a Bruker IFS 48 FTIR spectrometer using a grazing-angle incidence geometry. The instrument chamber was purged with dry air generated by a Nitrox Pure Air Generator unit. Light was incident on the sample surface at 80” and only the reflected ppolarized light was analysed. The number of scans accumulated was 2000 and the spectral resolution 2 cm-‘. The spectra were acquired after purging for 3 to 4 h, in order to remove water vapour and carbon
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A. J. Guiomar
dioxide from the chamber. The sample spectra were acquired against a bare gold background spectrum. The spectra were baseline-corrected by using the ‘rubber band’ algorithm. Spectra of the materials dispersed in KBr (Spectroscopic Grade, Graseby) discs were also obtained. These were acquired in transmission mode. Immobilization of GOX
The slides were immersed in a 1 mgml-’ solution of glucose oxidase (Biozyme, UK) in 0.1 M phosphate buffer at pH 7.0 and left overnight at room temperature. Before being analysed, the slides were thoroughly rinsed with water and phosphate buffer, and dried with a stream of nitrogen if required. Characterization of the immobilized GOX. The layer of physisorbed GOX was characterized by measuring its thickness and acquiring its FTIR spectrum as described above for the monolayers. Enzyme activity. The activity of GOX was evaluated by coupling the oxidation of glucose to the oxidationreduction of hydroxymethylferrocene [HMFc, prepared by MediSense (UK)]. This coupling was studied by cyclic voltammetry according to the porcedure of Cass Applied et aL5. For that, an EG&G Princeton Research Scanning Potentiostat model 362, interfaced to an Amstrad PC via an A/D converter built in-house, was used. Visualization of the voltammograms was via the Condecon 310 Cyclic Voltammetry Software (EG&G). A thermostatted (25”(Z), single-compartment, three-electrode cell (Metrohm) was connected to the potentiostat. The electrodes were constructed in-house, from metals of the highest purity available (Johnson Matthey) and were: (1) a square platinum counter electrode; (2) an Ag/AgCl (saturated KCl) reference electrode; (3) a working electrode made of a singleside-only gold-coated glass slide containing the monolayer with or without the immobilized enzyme. The chromium underlayer was 1 nm thick and the gold overlayer 200nm. Before the immobilization of GOX on the modified gold electrodes, a working area of 1.7 to 1.9cm2 was defined on the centre of it by covering the rest with Araldite Rapid (Ciba-Geigy) and allowing the curing process to occur overnight at room temperature. Electrical contact was achieved through the use of a silver-loaded epoxy resin (Circuit Works 2400 Conductive Epoxy, Planned Products) and a crocodile clip. This contact region was always kept above the solution level. The cell could be sealed and possessed a venting port that allowed purging of the test solution with water-saturated argon (Pureshield Argon, BOC) and keeping a blanket of water-saturated argon above the solution, to prevent oxygen re-entry during data acquisition. All studies were carried out in quiescent solutions that had been purged for 15 min. The electro-
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lyte was aqueous 0.09 M NaCl in 0.01 M sodium phosphate buffer, pH7.0 (referred to as PBS). HMFc was added from 0.02 M stock solutions in absolute ethanol to ease dissolution of HMFc in the electrolyte, Glucose (D-glUCOSe, Fisons) was added from a 1 M stock solution in PBS, after being left overnight to ensure complete mutarotation. GOX was added from stock solutions in 0.1 M sodium phosphate buffer, pH 7.0. GOX molarity was expressed as molarity of FAD+.
RESULTS Monolayer preparation and characterization C18SAM. The gold slides coated with C18SAM showed a hydrophobic contact angle with water with an average advancing contact angle of 114.5f2.2 (Table 1). The SAM showed uniform thickness, with an average value of 2.26f0.26 nm (Table 2). The FTIR reflection spectrum in the high-frequency region of this SAM is presented in Figure I with assignments in Table 3. The advancing contact angles obtained, the thickness and the FTIR spectrum in the high-frequency region are indicative of a well-ordered SAM. CAPLB film. The pressure/area isotherm obtained for CAP can be seen in Figure 2. The monolayer progressed from a gaseous-like state to a solid-like state upon compression, where the Wilhelmy balance plate suffered a deviation from its vertical orientation. Extrapolation of the steepest part of the curve gave an average value, for the limiting area, of 0.42f0.03 nm* per substituted anhydroglucose unit (average of values from five pressure/area isotherms). The Langmuir monolayers prepared could be compressed and expanded. Although some hysteresis occurred, especially at low surface pressures (Figure 2), it was not as large as that reported for cellulose triacetate by Hittmeier et a1.33. The LB method of monolayer transfer to a solid surface was chosen in transfer of the CAP Langmuir monolayer to a C18SAM-coated gold slide. The Langmuir-Schaefer method (horizontal lifting) was also evaluated. The transfer resulted in an irregularly deposited film, giving deposition ratios above 3, and droplets of water on its surface showed an irregular shape. A variation in the LB transfer method, beginning with the slide immersed before compression of the monolayer (crossing the surface of the subphase) and withdrawing it across the monolayer, resulted in very low deposition ratios, irregular transfer rates and a large variability in the subsequent contact angles measured at different regions of the sample surface. Therefore, transfer via immersion
‘One molecule
SCIENCE Volume 4 Numbers 3-4 1997
of GOX contains
two FAD molecules
Glucose oxidase within a self-assembled
Table 1 Contact angles of water on gold-coated with different monolayers Monolayer
CISSAM ClSSAM CAPLB
glass slides covered
Table 2 Average on the gold-coated
Backh
Front0
thickness
Monolayer
8. (“)
or (“)
0s (“)
& (“)
or (“)
0,~)
115&l
103f3
107f3
114f2
103f3
10711
73fl
53f2
68f2
70f2
50fl
65fl
“The face of the slide facing the Wilhelmy balance plate hThe face of the slide facing the moving barrier of the trough
of the monolayers
Thickness
CllSAM CAPLB film Physisorbed GOX
+
0.0015
ellipsometric glass slides
et al.
system: A. J. Guiomar
prepared
(nm)
Front”
Backh
2.10f0.16 1.41f0.35 9.95f0.58
2.42f0.21 2.88f0.59 8.71f0.78
“The face of the slide facing the Wilhelmy balance plate hThe face of the slide facing the moving barrier of the trough
1 a
0.0015
b t
8
0.8 -
0.41 3000
.
.
.
rn , 2950
.
I
I
,
, 2900
,
,
,
,
, 2850
,
,
,
,
, 2800
Wavenumber (cm-l) Figure 1 Infra-red reflection spectra of the gold-coated transmission spectrum of CAP in a KBr disc (3000-2800cm-’
slide covered region)
with
SUPRAMOLECULAR
(a) Cl8SAM
and
(b) CIISAM
+CAPLB
film;
(c) infra-red
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283
Glucose oxidase
within
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Table 3 Peak positions
and assignments in the CH stretching region”
spectra
Peak position
system:
for the CISSAM
A. J. Guiomar
infra-red
Assignment
(cm-‘)
v,(ip) C-H stretching mode of CH3 v,(FR) CH stretching mode of CH3 v, C-H stretching mode of CH2 v,(FR) CH stretching mode of CHs v, C-H stretching mode of CHs
2964.1 2931.1
2917.8 2877.3 2850.3 “Assignments
according
to ”
20 -
15 -
3 5
10 -
z 5-
’
0.
’
’
0.2
0.0
’
0.4
0.6
0.8
1.0
area per substituted anhydroglucose
Figure 2
Compressionexpansion CAP on a water subphase
surface
1
I
1.2
1.4
.
,
--I.6
unit (nm 2)
pressure/area
isotherms
of
0
et al.
slide above the monolayer, transfer occurred during both immersion and withdrawal. Accordingly, the transfer meniscus was hydrophobic during immersion and hydrophilic during withdrawal. The rate of monolayer transfer was not constant and the deposition ratios were not unity. The transfer rate was more irregular during immersion, showing first a fast transfer step when 63% of the total monolayer area deposited was transferred, followed by a slower step. Most of the film was transferred during the downward movement of the slide (82%) the total deposition ratio on immersion being 2.7. During withdrawal, the transfer rate was more regular. However, it resulted in a deposition ratio of 0.6. After deposition of the CAPLB film, the surface became more hydrophilic, showing an average advancing contact angle of 71.5f2.2” (Table I). The CAPLB film deposited on the front face of the slide (that facing the Wilhelmy balance) had an average thickness of 1.41f0.35 nm (Table 2). It was thinner than that deposited on the back face (that facing the moving barrier), which had an average thickness of 2.88f0.59nm (Table 2). The film on the back face of the slide was also more irregular than that on the front face, this being reflected in a higher standard deviation of its average thickness value. The presence of the CAP on the surface of the SAM was validated through identification of the ester C=O stretching band in the 1750 cm-’ region of the reflection FTIR spectrum and by the contribution of the C-H stretching vibrations in the high-frequency region (Figure I and Figure 4, Table 4). GOX immobilization
cd t?
cd 40 I
0
5
*
I
.
10
I
15
,
,
,
20
(
25
height of slide immersed (mm) Figure 3 Deposition curve Cl SSAM-coated gold slide
for
the
LB
transfer
of
CAP
to
a
followed by withdrawal was selected. A deposition curve is shown in Figure 3. The area of CAP Langmuir monolayer kept at a surface pressure of 20mNm-’ was constant in time. Beginning with the
284
SUPRAMOLECULAR
The presence of the enzyme on the surface after physisorption is implicit in the increase in thickness of the total assembly following the immobilization step. The adsorbed layers on both faces of the slide are of comparable thickness, with an average value of 9.33f0.97 nm (Table 2). The reflection FTIR spectrum of the physisorbed enzyme gave further support for the presence of the enzyme on the surface (Figure 5, Table 5). New vibrational bands in the amide I and II regions (170& 1600 cm-’ and 1575-1480 cm-‘, respectively) appear after immobilization of the enzyme on the C18SAM+CAPLB film assembly. This is in agreement with those seen for the enzyme in the crystalline state. The amide I band at 1656cm-’ is rather noticeable. The amide II band seems to be split and does not overlap as it does in the spectrum for the crystalline state. Activity of the immobilized GOX. The activity of the immobilized enzyme could not be determined by the conventional calorimetric activity assay for GOX. An electrochemical assay was adopted instead, using
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Glucose oxidase
within
a self-assembled
system:
A. J. Guiomar
et al.
0.004
a 0.003
0.8
0.8
0.21...,...,...,...,...,..., :2000 1800 1900
1700
1800
1500
1400
Wavenumber (cm-l) Figure 4 FTIR reflection spect.ra of the gold-coated slide covered with (a) Cl8SAM and (b) Cl8SAM +CAPLB film; (c) FTIR transmission spectrum of CAP in a KBr disc (:200&14OOcm-’ region)
Table 4 Peak positions spectrum0
and assignments
for the CAP infra-red
Peak position (cm-‘)
Assignment
2983 2945 2885 1749 1465 1423
C-H stretching of CHx or O-H stretching C-H stretching of CHOH, -CH*OH or CH, C-H stretching of CH or CHs C = 0 stretching of the ester group C-H deformation of CHx CHs symlmetrical bending
OAssignments according to references 38 and 39
MS 4:3/l-E
the mediator. HMFc oxidation HMFc as (E”’ = + 0.245 V) proved to be nearly reversible at an evaporated gold electrode. It showed: (1) a peak separation of 70mV, (2) the peak potential was independent of the scan rate, (3) the peak current was linearly dependent on the square root of the scan rate and (4) the ratio of anodic to cathodic current was close to 1. After the C18SAM had been formed, a strong inhibition of the reduction wave was found and the peak potential was shifted to more positive
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Glucose
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et al.
1.0
0.9
0.8 8 H .S
0.7
g # $
0.6
0.5
0.4
0.004
b 0.003
8 H & 0.002 $ 2 0.001
0.000 2(
I
1900
1800
I
#,‘I
I
1700
.,,.,.,,.:.,
1600
1500
1400
Wavenumber (cm-l) Figure 5 (a) FTIR transmission spectrum C18SAM +CAPLB film after immobilization
of GOX in a KBr of GOX (200(r1400cm-’
Table 5 Peak positions and assignments of the amide I and II bands of GOX immobilised on the Cl 8SAM + CAPLB and for GOX in a KBr disc Peak position
Assignment
(cm-‘)
GOX in KBr disc
Immobilized 1754.9
1655.6
1656.6
1538.9
1540.8
1517.7
1507. I
286
SUPRAMOLECULAR
GOX C = 0 stretching of the ester groups of CAP Amide I C=O stretching Amide II N-H deformation Amide II N-H deformation
disc and region)
(b)
FTIR
reflection
spectra
of the
gold
slide
coated
with
a
potentials (Figure 6b), an indication that the oxidation is being blocked. This effect was less pronounced with the ClSSAM + CAPLB (Figure 6~). However, the poor reversibility of HMFc at these modified electrodes did not prevent its role in mediating electron transfer from GOX in solution to the electrode, in the presence of glucose, even in the worst case (C18SAM) (Figure 7). The physisorbed enzyme film contained a population of active enzyme. When glucose was added to the PBS electrolyte containing HMFc, a catalytic current indicative of catalytic coupling between HMFc and GOX could be seen (Figure 8).
SCIENCE Volume 4 Numbers 3-4 1997
Glucose oxidase within a self-assembled
F
system: A. J. Guiomar et al.
b
-2.0x10-5I-
c 2.0~10-5
-2.Ox10~
-
5
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
E (V) vs. Ag/AgCl (sat. KCl) Figure 6 Cyclic voltammograms of a OSmM solution of HMFc in PBS, at a scan rate of 5mVs-‘, showing the effect of modification of the electrode on the reversibility of HMFc oxidation: (a) bare gold electrode; (b) gold electrode coated with C18SAM; (c) gold electrode coated with a Cl8SAM + CAPLB film
DISCUSSION Monolayer preparation and characterisation
C18SAM. The advancing contact angle for a smooth, packed methyl surface is blelieved to be 110-l 15” while, for a methylene surface, it is 102-103” (refs. 40 and 41). The average value of fIa= 114.5f2.2” (Table I) is indicative of a well-ordered monolayer, showing mainly methyl groups at the air interface. The average hysteresis value of 11Sf4.2” could indicate either some surface heterogeneity or a degree of surface roughness (revealed by the
standard deviation of the average contact angle value). The average thickness observed (2.26f0.26nm) is close to the value expected for a monolayer whose molecules are fully stretched but tilted 30” away from the normal to the surface (2.34f0.21 nm 40). The p-polarized light reflection FTIR spectrum also shows that the molecules are tilted away from the normal to the reflecting surface. This can be inferred from the presence of the symmetric and asymmetric vibrations of the methylene groups at 2850 and 2918 cm-’ (Figure I, Table 3). These would not be present in a arrangement normal to the reflecting
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et al.
1 a
-0.2
0.0
0.2
0.4
E (V) vs. Ag/AgCl
0.6
-0.2
(sat. KCl)
0.0
0.2
E (V) vs. AgIAgCl
0.4
0.6
(sat. KCl)
Figure 7 Cyclic voltammograms of HMFc/glucose solutions in the presence and absence of GOX (in solution), at a scan rate of 1mVs_‘: (a) gold electrode coated with CIISAM; (b) gold electrode coated with a CIISAM + CAPLB film. The composition of the solutions was: (i) 0.5 mM HMFc and 49mM glucose in PBS; (ii) as for (i) but with addition of GOX to a concentration of 7.5 nM (this amount of GOX is approximately four times that expected for a monolayer of enzyme on these electrodes)
LI.I.LI.I.t.t.t*, -0.2 -0.1
0.0
0.1
0.2
0.3
E (V) vs. Ag/AgCl
0.4
0.5
0.6
0.7
(sat. KCl)
Figure 8 Cyclic voltammograms of HMFc solutions in the presence and absence of glucose, performed with an electrode containing a Cl8SAM +CAPLB film with physisorbed GOX: (i) 0.5mM HMFc in PBS; (ii) as for (i) but with the addition of glucose to a concentration of 49 mM. Scan rates: I mV s-’
surface, since their transition dipoles would be parallel to the surface and would not be picked up by ppolarized light4’. Comparing the wavenumbers of the bands observed in the high-frequency region of the spectrum (Table 3) with those obtained for a longchain alkanethiol in the bulk crystalline phase3’, there is also further evidence that a well-ordered SAM with methylene groups existing in a crystalline-like environment, with fully extended chains in an all-trans conformation, has been formed. CAPLB film. Although a report on monolayers of CAP could not be found in a literature search, the
288
SUPRAMOLECULAR
average value of 0.42f0.03 nm* per substituted anhydroglucose unit for the limiting area is in accordance with published values for cellulose triacetate and cellulose tripropionate. For cellulose tripropionate on water, a limiting area of cu. 0.45nm2 has been reported3’. The published values for cellulose triacetate are rather disperse: between 0.30 and 0.54nm2 per substituted anhydroglucose unit3’-34. A detailed study of the spreading of cellulose triacetate obtained limiting areas of 0.40f0.015 nm* (ref. 33). However, the calculated area of the substituted anhydroglucose unit in cellulose triacetate is 0.69nm2 (W. T. Astbury, cited by Borgin and Johnson3*). The discrepancy encoun-
SCIENCE Volume 4 Numbers 3-4 1997
Glucose oxidase within a self-assembled system: A. J. Guiomar et al.
tered between the calculated and the measured areas has been explained by considering that the substituted anhydroglucose rings do not lie flat on the water but are somewhat tilted3’. Localized gelation of the film, upon compression, is supposed to have occurred. This is the interpretation for the observed displacement of the Wilhelmy plate from its vertical position, a behaviour already reported for cellulose triacetate33. According to Hittmeier et a1.33, the origin of this displacement seems to be related to a high surface viscosity of the film due to gelation at low pressures. At large areas the anhydroglucose units are supposed to lie flat on the water surface, as a consequence of weak hydrogen bonds through its ester groups. During compression, chain-chain interactions occur. The hydrophobic contacts between overlapping ester methyl groups and the C-H of the anhydroglucose ring can cause tilting of the anhydroglucose rings, leading to the formation of gelated, coherent patches. The tilted conformation is stabilized by van der Waals’ interactions between adjacent pyranose rings. In a model, taking into account a limiting area of 0.40nm2 per substituted anhydroglucose unit, this unit will be in a nearly vertical orientation33. Tht: displacement of the plate during compression is supposed to be caused by the movement of gelated patches against its front face when trying to pack more closely33. The fact that CAP Langmuir monolayers could be transferred to a hydrophobic surface by crossing it vertically both during an initial downward and a subsequent upward movement (Figure 3) suggests that the CAP macromolecules are able to orient themselves at the air/water interface, as does the fact that poor transfer also occurred when a hydrophobic slide was withdrawn across it. This also shows that the monolayer possesses some flexibility. However, the existence of a fast transfer rate immediately upon contact of the slide with the monolayer (Figure 3) could indicate that the CAP monolayer has limited flex:ibility. This could lead to the transfer of collapsed patches, as indicated by a deposition ratio of 2.7. The deposition ratio of 0.6 on subsequent withdrawal indicates that an incomplete coverage of the surface occurred. The contact angle of the C18SAM surface decreased after deposition of the CAPLB film. This is expected to happen owing to the presence of the more hydrophilic hydroxyl and ester carbonyl groups in CAP. The hysteresis increased to 20f2.2”. A high hysteresis is expected for a surface containing both polar and non-polar groups, where the polar groups will interact more strongly with the water molecules than the non-polar groups. The back face of the slide showed a film with larger thickness than the front face. Considering that the back face is facing the moving barrier, it should receive a larger fraction of the monolayer since the monolayer is being compressed against it during
transfer. Correspondingly, a higher standard deviation of its average thickness was found (Table 2). The thickness values observed (1.41 f0.35 nm and 2.88f0.59nm for the front and back face, respectively) are compatible with five to seven monolayers being transferred to the back face and two or three to the front face, considering a model with the substituted anhydroglucose units vertically oriented on the surface of the subphase$. GOX immobilization A layer of GOX with an average thickness of 9.33fO.97nm was physisorbed to the Cl 8SAM + CAPLB film (Table 2). The dimensions of the GOX molecule have been published4* following the resolution of its 3D structure, albeit for a 95% deglycosylated molecule§. More realistic values should come from the Stokes radius of the whole enzyme. It has a value of 4.27nm, with an axial ratio of 2.5 (ref. 43). This is equivalent to the dimensions of 13.0 and 5.2 nm for the two axes of an ellipsoidi4. Ellipsometric workI gave the indication that the two axes of the ellipsoidal enzyme molecule would have dimensions close to 14.0 and 5.0nm. It is not possible to propose a unique model to interpret the thickness of the physisorbed GOX layers (9.95f0.58 nm and 8.71f0.78 nm, for the front and back face, respectively) by comparing its thickness with the dimensions of the enz e molecule, owing to the errors affecting these values (ny” . Additional support for the presence of the enzyme on the surface comes from the identification of its infra-red spectrum, in particular its characteristic amide bands (Figure 5, Table 5). The amide I band at 1656cm-’ was rather noticeable. The amide II band seem to be split, unlike in the crystalline state. This could indicate that a population with a different conformational state exists at the surface, although it could be an artefact resulting from the noise level. This noise is related to non-complete removal of either water vapour or adsorbed water from the sample. It is expected that improvement in purging will allow the acquisition of information about the conformational state of the physisorbed enzyme. Activity of the immobilized GOX. The failure to detect enzyme activity by the conventional calorimetric assay is thought to be due to the low amounts of ‘The dimensions of the anhydroglucose unit in cellulose are Onmx0.515nm, when the ring is lying flats’ 5 The group of researchers that solved the 3D structure of GOX have published values for the dimensions of the deglycosylated enzyme of both 6.0nmx5.2nmx7.7nm and 7.0nmx5.5nmxS.Onm (refs. 4 and Y’These thickness values were obtained by using a value of I .50 for the refractive index of the physisorbed GOX film, in accordance with published results. However, this value was calculated for a monolayer with complete coverage, from enzyme dimensions obtained from ellipsometric measurements’4. As the refractive index depends on the surface coverage, the accuracy of the thickness values will be affected4
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enzyme present. Detection of H202 production or O2 consumption was ruled out since their reduction and oxidation, respectively, are strongly inhibited even by C6SAM on bulk gold and, in the case of H202, suppressed by C 12SAM on platinum44’ 45. Hydroxymethylferrocene, a redox molecule showing reversibility at gold electrodes, was the mediator selected for mediation of the electron transfer from GOX to the electrode. Here, the oxidation of glucose is coupled to the oxidation-reduction of HMFc in solution. Electrons are thus transferred from the enzyme to the electrode via the mediator. This is a reaction scheme in which the reduced mediator (FcR) is oxidized at the electrode [equation (l)] and its oxidized form (FcR+) is catalytically put back to the reduced state by reacting with the reduced form of GOX [equation (2)] which is produced when the enzyme oxidizes glucose [equation (3)]. 2FcR H FcRf + 2e-
(1)
GOXred + 2FcR+ + GOX,, + 2FcR
(2)
glucose + GOX,, 4 gluconic acid + GOX,,d
(3)
It has been reported that HMFc shows quasireversible behaviour on C6SAM on bulk gold44. However, for C18SAM on an evaporated gold electrode, its reduction wave on reversing the potential scan was drastically reduced and the peak potential was shifted to higher potential values46. This type of suppression was also detected in this work (Figure 6b). Such suppression is a limitation to the use of this C18SAM in a sensing scheme that relies on a mediator in solution. It seems to arise from a kinetic effect since, when an electrochemical system departs from reversibility, it means that the rate of electron transfer decreases below the rate of mass transport to the electrode surface, resulting in increased peak separations4’. This is understandable if one accepts a long-range model for electron transfer across the monolayer46. This effect was less pronounced for the C18SAM coated with the CAPLB film (Figure 6~). This could be due to the introduction of some degree of disordering in the SAM, creating defects through which the mediator could have access to the gold surface. In spite of these effects, detection of the activity of GOX could be performed. The C18SAM+CAPLB system showed more sensitivity to GOX detection (Figure 7). This should be related to the fact referred to above that HMFc shows a higher degree of reversibility in this system than in the C18SAM-only system. A catalytic current was observed when an electrode with GOX immobilized on the C18SAM +CAPLB film was tested (Figure 8), showing that a population of active enzyme exists immobilized on the film surface. Comparison of the magnitude of the catalytic current obtained with the immobilized enzyme (curve
290
SUPRAMOLECULAR
et al.
ii of Figure 8) with that obtained with enzyme added to the solution (curve b, Figure 6) allows us to state that most of the immobilized enzyme is active”. This result is thought to be due to enzyme physisorbed to the C18SAM+CAPLB film and not to the electrode body. This is because no enzyme activity was detected in a control experiment where an electrode body, with approximately the same area but with no evaporated gold film, was treated with a GOX solution as before, inserted into the electrochemical cell and analysed with a bare gold electrode, in the same way as for the modified electrodes.
CONCLUSIONS An LB ultrathin film of cellulose acetate propionate could be transferred to a C18SAM formed on a goldcoated glass slide. Glucose oxidase was immobilized onto this assembly by simple physisorption. The activity of the enzyme could be detected electrochemically by using hydroxymethylferrocene as a redox mediator. An active enzyme population exists immobilized on this assembly.
ACKNOWLEDGEMENTS The authors thank MediSense (UK) for the gift of HMFc and GOX and Dr R. Luo (Department of Mining and Mineral Engineering, University of Leeds) for help with the electrochemical studies. A. J. Guiomar is on leave from Departamento de Bioquimica, Faculdade de CiZncias e Tecnologia, Universidade de Coimbra, Portugal. He thanks J.N.I.C.T. (Portugal) for personal financial support (Praxis XXI programme).
REFERENCES Gcpel, W. and Heiduschka, P., Biosens. Bioelectron.. 1995, 10, 853. Zull, J. E., Reed-Mundell, J., Lee, Y. W., Vezenov, D., Ziats, N. P., Anderson, J. M. and Sukenik, C. N., J. Indus/. Microbial.. 1994, 13, 137. Wilson, R. and Turner, A. P. F., Biosens. Bioelectron.. 1992, 7. 165.
‘IThe thickness value of the immobilized enzyme layer (Table 2) could be obtained by a monolayer or a bilayer of enzyme molecules, depending on the orientation of the enzyme. This allows us to compare the magnitude of the catalytic current obtained with the immobilized enzyme with that obtained when an amount equivalent to four layers of free enzyme is added to the solution and detected with a gold electrode modified with a ClSSAM+CAPLB film (Figure 6, curve b). Although direct comparison between both is not totally correct, since the current in the second case also reflects enzyme diffusion and a higher collision frequency between the enzyme and both the mediator and glucose, the magnitude of the catalytic current of the immobilized enzyme seems to indicate that most of the immobilized enzyme must be active
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Glucose oxidase within a self-assembled system: A. J. Guiomar et al.
4.
Hecht, H. J., Schomburg, I>., Kalisz, H. and Schmid, R. D., Biosens. Bioelectron.,
5.
6. I. 8. 9.
Cass, A. E. G., Francis, D. G., Hill, H. A. O., Aston, W. J., Hiueins. I. J.. Plotkin. E. V.. Scott. L. D. L. and Turner. A. P. F.,%ai. Chem.. 1984; 56, 667. Cenas, N. K. and Kulys, J. J., J. Electroanal. Chem., 1981, 128, 103. Sonawat, H. M., Phadke, R. S. and Govil, G., Biotechnol. Bioeng., 1984, 26, 1066. Albery, W. J., Bartlett, P. N. and Cass, A. E. G., Phil. Trans. Roy. Sot. Lond., 1987, B316, 107. Hill, B. S., Scolari, C. A. and Wilson, G. S., J. Electroanal. Chem., 1988, 252, 125.
10.
15.
Taxis du Poet, P., Miyamoto, S., Murakami, T., Kimura, J. and Karube. I.. Anal. Chim. Acta. 1990. 235. 255. Bartlett, P. ‘N: and Bradford, V. Q., J. Chem. Sot., Chem. Commun., 1990, 1135. Bartlett, P. N. and Cooper, J. M., J. Elecfroanal. Chem., 1993, 362, 1. Alvarez-Icaza, M., Kalisz, IH.M., Hecht, H. J., Aumann, K.D., Schomburg, D. and Schmid, R. D., Biosens. Bioelectron., 1995, 10, 735. Szucs, A., Hitchens, G. Di. and Bockris, J. O.‘M., Bioelectrochem. Bioenerg., 1989, 21, 133. Alvarez-Icaza, M. and Schmid. R. D., Bioelecrrochem.
16.
Jiang, L., Mcneil, C. J. and Cooper, J. M., J. Chem. Sot.,
17.
Sun, S., Ho-Si, P.-H. and Harrison, D. J., Langmuir,
18.
Zaitsev, S. Y., Coil. Surf
11. 12. 13. 14.
Bioenerg.,
30.
Ramanathan, K., Ram, M. K., Malhotra, B. D. and Murthy, S. N., Mater. Sci. Eng., 1995, C3, 159. Tsuzuki, H., Watanabe, T., Okawa, Y., Yoshida, S., Yano, S., Koumoto, K., Komiyama, M. and Nihei, Y., Chem. Le/t.. 1988, 8, 1265. Tatsuma, T., Tsuzuki, H., Okawa, Y., Yoshida, S. and Watanabe, T., Thin Solid Films, 1991, 202, 145. Lee, S. Y., Anzai, J. and Osa, T. Sens. Actuators Part BChem. 1993, 12, 153. Schuhmann, W., Heyn, S. P. and Gaub, H. E., Adv. Mater.,
31. 32. 33.
Adam, N. K., Trans. Faraday Sot., 1933, 29, 90. Borgin, K. and Johnson, P., Trans. Faraday Sot., 1953,4,956. Hittmeier, M., Sandell, L. S. and Luner, P., J. Poiym. Sci., Parr
26.
1993, 8, 197.
27. 28. 29.
1991, 3, 388.
C. 1971,36,
34. 35.
43. 44. 45.
Creager, S. E. and Olsen, K. G., Anal. Chim. Acta, 1995,307,277. Dong, X.-D., Lu, J. and Cha, C., Bioelectrochem. Bioenerg.,
46.
Creager, S. E., Hackett, L. A. and Rowe, G. K., Langmuir,
41.
Southampton Electrochemistry Group, Instrumental Methods in Elecrrochemistry. Ellis Horwood Ltd, Chichester, 1985.
38.
40. A’: Physicochem.
Eng. Aspects,
1993,
75, 211.
19.
22. 23.
Barmin, A. V., Eremenko, A. V., Sokolovskij, A. A., Chernov, S. F. and Kurochkin, I. N , Biotechnol. Appl. Biochem.. 1993, 18,369. Fiol, C., Valleton, J.-M., Delpire, N., Barbey, G., Barraud, A. and Ruaudel-Teixier. A.. Thin Solid Films. 1992. 210/211. 489. Sriyudthsak, M., Yamagishi, H. and Moriizumi, T.,’ Thin Solid Films, 1988. 160, 463. Moriizumi, T., Thin Solid Films, 1988, 160, 413. Ancelin. H.. Zhu. D. G.. Pettv. M. C. and Yarwood, J.,
24.
Okahata, Y., Tsuruta, T., Ijiro, K. and Ariga, K., Langmuir,
25.
Okahata, Y., Tsuruta, T., ljiro, K. and Ariga, K., Thin Solid Films, 1989, 180, 65.
20. 21.
Langmuir.
1990,6,‘1068.
1991, 3, 25.
Nicholson, R. S. and Shain, I., Anal. Chem., 1964, 36, 706. Porter, M. D., Bright, T. B., Allara, D. L. and Chidsey, C. E. D., J. Am. Chem. Sot., 1987, 109, 3559. Ott, E., Spurlin, H. M. and GraMin, M. W (eds), High Polvmers. Vol. V. Part IV (Cellulose and Cellulose Derivatives. ed. N. M. Bikales and L. Segal), 2nd edn. Wiley Interscience, New York, 1971. Zhbakov, R. G., Infrared Spectra of Cellulose and its Derivatives. Consultants Bureau, New York, 1966. Bain, C. D., Troughton, E. B., Tao, Y.-T., Evall, J., Whitesides, G. M. and Nuzzo, R. G., J. Am. Chem. Sot., 1989, 111, 321. Ulman, A., An Introduction to Ultrathin Organic Films-from Langmuir-Blodgett to Self-Assembly. Academic Press, Inc., New York, 1991. Hecht, H. J., Kalisz, H. M., Hendle, J., Schmid, R. D. and Schomburg, D., J. Mol. Bioi., 1993, 229, 153. Nakamura, S., Hayashi, S. and Koga, K., Biochim. Biophys.
1991, 7,
727.
Embs, R., Funhoff, D., Laschewsky, A., Licht, U., Ohst, H., Prass, W., Ringsdorf, H., Wegner, G. and Wehrmann, R., Adv. Mater.,
36. 37.
39.
1995, 1293.
H. and Fukuda, K., Thin Solid
Films, 1985, 133, 29.
1994, 33, 191.
Chem. Commun.,
267.
Kawaguchi, T., Nakahara,
41.
42.
Acta. 1976,445,
*’
294.
1995, 36, 13. 1992, 8, 854.
1988, 4, 1373.
SUPRAMOLECULAR
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