Anal. Chem. 1997, 69, 5059-5066
Formation and Imaging of Microscopic Enzymatically Active Spots on an Alkanethiolate-Covered Gold Electrode by Scanning Electrochemical Microscopy Gunther Wittstock*,† and Wolfgang Schuhmann‡
Institute of Physical and Theoretical Chemistry, University of Leipzig, Linne´ strasse 2, D-04103 Leipzig, Germany, and Chair of General Chemistry and Biochemistry, Technical University Munich, Vo¨ ttingerstrasse 40, D-85350 Freising, Germany
Microscopic, enzymatically active spots on self-assembled monolayers (SAMs) of alkanethiolates on gold were obtained by a combination of localized desorption induced using the scanning electrochemical microscope (SECM) followed by chemical derivatization. Starting from a SAM of dodecanethiolate on gold, localized desorption of alkanethiolates creates microscopic areas of an uncovered gold surface surrounded by a dense Au alkanethiolate layer. The renewed gold surface chemisorbs an aminoderivatized disulfide (cystaminium dihydrochloride) in a second step. Periodate-oxidized glucose oxidase was attached covalently to the terminal amino functions to create a stable, catalytically active pattern of the enzyme on the alkanethiolate SAM. The enzymatic activity was mapped using the imaging capabilities of SECM. The generator-collector mode (amperometric H2O2 detection) was advantageously used, as the feedback mode leads to interferences due to concurrence between mediator regeneration by the enzymatic reaction and by the heterogeneous electron transfer at the gold regions from which the blocking dodecanethiolate layer had been desorbed. Rising backgrounds due to H2O2 accumulation in the bulk solution can be prevented by adding minute amounts of the enzyme catalase to the working solution. By catalyzing the H2O2 decomposition, the lifetime of H2O2 is adjusted to prevent its accumulation in the bulk phase yet to allow its diffusion across the gap between the enzyme-modified region and the collecting electrode. Perspectives for creating miniaturized multienzyme structures, which will become accessible by repeating the desorption and covalent enzyme immobilization steps using different enzymes in each cycle, are highlighted. Strong efforts toward the development of miniaturized biosensors target new application fields in which the total sensor size is of major concern or in which different biorecognition elements have to be united into one multianalyte sensor. Besides overcoming size constraints, experiences with unmodified microelectrodes additionally raise the hope for advantageous response character* To whom correspondence should be addressed. Fax: (+49-341) 97 36399. E-mail:
[email protected]. † University of Leipzig. ‡ Technical University Munich. Present address: Chair of Analytical Chemistry, Group of Biosensor and Electroanalytical Chemistry, Ruhr-University Bochum, Universita¨tsstrasse 150, D-44780 Bochum, Germany. S0003-2700(97)00504-0 CCC: $14.00
© 1997 American Chemical Society
istics such as short response times or low convection dependence,1 which may be achieved in the case of microbiosensors as compared to millimeter-sized sensors. Recently, Ratcliff et al.2 emphasized the potential advantages which may be achieved by microscopic compartmentalization, i.e., the immobilization of different functional units at distinct yet closely spaced locations to allow communication between them by diffusing reaction products. While there are several established methods for the production of microstructured inorganic transducers, such as thinfilm technology and screen printing, the localized modification of these transducer surfaces with biological recognition elements, redox mediators, etc. still represents a significant problem. It is becoming increasingly urgent to find a solution to permit the development of miniaturized sensors or sensor arrays. Fabrication techniques that allow the modification of a large number of sensor elements on one support with different biological recognition elements and nonmanual techniques for patterning of surfaces, locally resolving immobilization of biomolecules, and microscopically characterizating the immobilized layers have to be invented. To accomplish this goal, the use of chemisorbed monolayers of thiol-containing compounds (RSH) on gold surfaces seems to be very promising.3 If a clean gold surface is exposed to n-alkanethiols, a well-ordered self-assembled monolayer (SAM) is formed. By varying the terminal group of R, surface properties like wetting behavior,4-6 permeability,7,8 and chemical reactivity9-12 can be tuned to the need of a particular application. The ω-functionality can also serve as an anchor site for the immobilization of biomolecules.13,14 Such protocols allow the binding of the biological recognition elements at a small yet defined distance (1) Pons, S., Rollison, D. R., Schmidt, P. P., Eds. Ultramicroelectrodes; Datatech Systems Inc. Science Publishers: Morganton, NC, 1987. (2) Ratcliff, B. B.; Klancke, J. W.; Koppang, M. D.; Engstrom, R. C. Anal. Chem. 1996, 68, 2010-2014. (3) Allara, D. L. Biosens. Bioelectron. 1995, 10, 771-783. (4) Bain, C. D.; Whitesides, G. M. Langmuir 1989, 5, 1370-1378. (5) Sondag-Huethorts, J. A. M.; Fokkink, L. G. J. Langmuir 1994, 10, 43804387. (6) Abbott, N. L.; Gorman, C. B.; Whitesides, G. M. Langmuir 1995, 11, 1618. (7) Miller, C.; Cuendet, P.; Gra¨tzel, M. J. Phys. Chem. 1991, 95, 877-886. (8) Hockett, L. A.; Creager, E. S. Langmuir 1995, 11, 2318-2321. (9) Chidsey, C. E. D. Science 1991, 251, 919-922. (10) Duevel, R. V.; Corn, R. M. Anal. Chem. 1992, 64, 337-342. (11) Katz, E.; Itzhak, N.; Willner, I. Langmuir 1993, 9, 1392-1396. (12) Schlereth, D. D.; Katz, E.; Schmidt, H.-L. Electroanalysis 1995, 7, 46-54. (13) Collison, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247-1250. (14) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-10966.
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from the electrode surface, and direct electron transfer with high rate constants has been demonstrated for selected systems.15,16 While the vertical structuring of the modification layer occurs spontaneously due to the formation of gold alkanethiolates (AuSR), lateral structuring of alkanethiolate layers has been the subject of numerous investigations. Manipulation of individual alkanethiolate units is possible by nanolithography using scanning tunneling microscopy17 or atomic force microscopy.18 Formation of pattern sizes in the submicrometer to micrometer range, which seem to be more suitable for sensor applications, has been achieved by writing an autophobic alkanethiol ink with a drawing pen,19 microcontact printing,20 micromachining,21 conducting photochemical reactions,22 or using particle beams.23 In particular, microcontact printing has led to small pattern size and high shape definition concomitantly with the simultaneous formation of a large number of microstructures.24 It is based on wetting a defined fraction of the surface with an alkanethiol and rinsing the surface with another, ω-functionalized thiol, thus creating a bifunctional pattern. Subsequently, biological recognition elements may be bound to the areas modified with the ω-functionalized thiol. However, all binding functionalities would be derivatized in one step with this approach, which would prevent the formation of distinct regions with different immobilized biochemical recognition elements. Recently, we demonstrated the structuring of gold alkanethiolate layers using the scanning electrochemical microscope (SECM).25 SECM can be used either for electrochemically induced microfabrication or for imaging the distribution of (electrochemical) reactivity on surfaces.26 Imaging of enzymatic activity is performed by detecting reaction products of an enzymatic reaction using an amperometric27 or potentiometric28 probe electrode (generator-collector (GC) mode). Higher spatial resolution has been achieved with the enzyme-generated feedback mode in the case of oxidases.29 In oxygen-free solution, the electrons are transferred to the oxidized form (Ox) of the SECM mediator if Ox can react as an artificial electron acceptor for the investigated enzyme. Recently, the extension of these concepts to imaging the distribution of nonmetabolizing biomolecules (e.g., antibodies) has been demonstrated by forming a complex with (15) Jiang, L.; McNeil, C. J.; Cooper, J. M. J. Chem. Soc., Chem. Commun. 1995, 1293-1295. (16) Lo¨tzbeyer, T.; Schuhmann, W.; Schmidt, H.-L. Sens. Actuators B 1996, 33, 50-54. (17) Schoer, J. K.; Zamborini, F. P.; Crooks, R. M. J. Phys. Chem. 1996, 100, 11086-11091. (18) LaGraff, J. R.; Gewirth, A. A. J. Phys. Chem. 1995, 99, 10009-10018. (19) Kumar, A.; Biebuyck, H. A.; Abbott, N. L.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 9188-9189. (20) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 14981511. (21) Abbott, N. L.; Folkers, J. P.; Whitesides, G. M. Science 1992, 257, 13801382. (22) Rozsnyai, L. F.; Wrighton, M. S. Langmuir 1995, 11, 3913-3920. (23) Berggren, K. K.; Bard, A.; Wilbur, J. L.; Gillaspy, J. D.; Helg, A. G.; McClelland, J. J.; Rolston, S. L.; Phillips, W. D.; Prentiss, M.; Whitesides, G. M. Science 1995, 269, 1255-1257. (24) Kumar, A.; Abbott, N. L.; Kim, E.; Biebuyck, H. A.; Whitesides, G. M. Acc. Chem. Res. 1995, 28, 219-226. (25) Wittstock, G.; Hesse, R.; Schuhmann, W. Electroanalysis 1997, 9, 746750. (26) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. in Electroanalytical Chemistry, Vol. 18, Bard, A. J., Ed.; Marcel Dekker: New York, 1994; Chapter 3. (27) Lee, C.; Kwak, J.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 89, 17401743. (28) Horrocks, B. R.; Mirkin, M. V.; Pierce, D. T.; Bard, A. J.; Nagy, G.; Toth, K. Anal. Chem. 1993, 65, 1213-1224. (29) Pierce, D. T.; Bard, A. J. Anal. Chem. 1993, 65, 3598-3604.
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an enzyme-labeled antigen and mapping the activity of the immobilized enzyme label with one of the above-mentioned working modes.30,31 So far, microstructuring of biochemically active layers with SECM has been demonstrated by localized generation of a strong oxidizer, which deactivates microscopic regions of an initially uniform enzyme-modified electrode,32 or by utilizing the potential drop between a macroscopic working electrode and the microscopic auxiliary electrode for the formation of defined structures of conducting polymers in two33 and three dimensions34 to which enzymes may be attached covalently.35 This article focuses on the successive application of the SECM as a microfabrication tool and as a microscope for selective mapping of enzymatic activity. For this purpose, microscopic areas of uncovered gold surrounded by a dense n-alkanethiolate SAM are formed by localized electrochemical desorption of AuSR. The region of the renewed gold surface is used to chemisorb an ω-functionalized alkanethiolate to which the enzyme is bound covalently in a subsequent step. The result of the modification step as well as intermediate stages can be optimized using the imaging capabilities of the SECM. By incorporating a homogeneous enzymatic reaction as a drain for H2O2 and as a competing process for the amperometric detection, an improved version of the GC mode is proposed, which confirms the specificity of the measured currents and limits rising background currents and diffusional blurring. The main advantage of the general strategy is seen in the opportunity to form miniaturized multienzyme structures by repeating the modification protocol and, thereby, to immobilize a different enzyme in each cycle. EXPERIMENTAL SECTION Chemicals and Preparation of Test Plates. Glass slides (25 × 25 mm2) pretreated with octadecyltrichlorosilane (Fluka, Buchs, Switzerland) were coated with a 500 Å thick layer of gold (Goodfellow), evaporated by resistive heating in a high-vacuum chamber (BA360, Balzer-Union, Liechtenstein) at pressures below 5 × 10-6 mmHg. A mask was placed in front of the glass slides, which resulted in a gold-coated surface of 22 mm diameter. The slides were then dipped into a solution made from 100 mL of propanol-2 (Merck) and 10 µL of dodecanethiol (Aldrich). The slides were dipped on one half only to retain an uncovered gold surface, necessary to establish the open-circuit potential (OCP) governed by the redox species in solution, for reference measurements and for electrical contacting during the subsequent experiments. All chemicals were of analytical grade and were used as received. Aqueous solutions were prepared from deionized water (Millipore). Instrumentation. The SECM instrument has been described before.36 Microelectrodes with radius r ) 25 µm were used throughout the study. They were produced by sealing a Pt wire into a pulled capillary. After the assembly was embedded into a polymer cast which was ground and dissolved, disk-shaped (30) Wittstock, G.; Yu, K.; Halsall, H. B.; Ridgway, T. H.; Heineman, W. R. Anal. Chem. 1995, 67, 3578-3582. (31) Shiku, H.; Matsue, T.; Uchida, I. Anal. Chem. 1996, 68, 1276-1278. (32) Shiku, H.; Takeda, T.; Yamada, H.; Matsue, T.; Uchida, I. Anal. Chem. 1995, 67, 312-317. (33) Kranz, C.; Ludwig, M.; Gaub, H. E.; Schuhmann, W. Adv. Mater. 1995, 7, 568-571. (34) Kranz, C.; Gaub, H. E.; Schuhmann, W. Adv. Mater. 1996, 8, 634-637. (35) Kranz, C.; Wittstock, G.; Wohlschla¨ger, H.; Schuhmann, W. Electrochim. Acta 1997, 42, 3105-3111. (36) Wittstock, G.; Kranz, C.; Strike, D.; Schuhmann, W.; Schmidt, H.-L. Microsc. Anal. 1996 (Nov), 5-7.
x
x
Figure 2. Reaction sequence for creating a microscopic spot of immobilized glucose oxidase (GOx) on a patterned gold alkanethiolate layer.
Figure 1. (a) Principle of the localized electrochemical desorption of alkanethiolate layers from a macroscopic Au surface; UME, ultramicroelectrode. (b) Line scans in the feedback mode across renewed gold surfaces obtained using (1) 5 mM KOH or (2) 50 mM KOH during the desorption step. Imaging was made in 20 mM [Ru(NH3)]Cl3 + 1 M phosphate buffer.
electrodes with a total diameter of 100-150 µm (shielding + active electrode area) were obtained.37 The comparatively large microelectrode had to be used because the sensitivity of the potentiostat (10 nA V-1) is too low to detect the enzymatic activity with smaller electrodes. For all the samples presented here, the microelectrode was not changed or reactivated between operation as a microauxiliary electrode during the desorption and subsequent imaging steps, where it functioned as a working electrode. The three-electrode cell was completed with a saturated calomel reference electrode (SCE; +241 mV vs NHE) and a platinum wire auxiliary electrode. All potentials are quoted with respect to the SCE. Vertical Positioning of the Microelectrode and Imaging of the Renewed Gold Surface within the SAM. Vertical positioning (z approach) of the microelectrode as well as imaging of the renewed gold surfaces within the alkanethiolate SAM was performed in the feedback mode (probe potential ET ) -400 mV, sample at OCP, vertical scan speed 1 µm s-1, lateral scan speed vt ) 10 µm s-1) with 20 mM [Ru(NH3)6]Cl3 (Aldrich) as mediator in 1 M phosphate buffer (pH 7.4). Usually, the z approach was interrupted when the steady-state microelectrode current (iT) over the gold-dodecanethiolate layer had decreased to 50% of its initial value (iT∞) in the bulk phase of the solution (current at quasiinfinite probe-sample distance). Using the calculated data38 for the response over an insulator and assuming a ratio of 10 between the radius of the insulating shielding (s) and the radius (r) of the active part of the microelectrode, a ratio iT/iT∞ ) 0.5 corresponds to a distance d ) 0.89 r (23 µm for r ) 25 µm). In the case of negative feedback, the transformation of iT/iT∞ ratios to distances is not as accurate as for the positive feedback because the negative feedback depends more strongly on the difficult-to-control s/r ratio (37) Ludwig, M.; Kranz, C.; Schuhmann, W.; Gaub, H. E. Rev. Sci. Instrum. 1995, 66, 2857-2860. (38) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227.
than the positive feedback38 and because of the longer time τ to establish a steady-state current in case of the negative feedback.39 Therefore, and because our s/r ratio was smaller than the tabulated data, experimental approach curves to insulators tend to give higher currents than expected from theory. Comparing experimental approach curves toward glass vs those to AuSR surfaces with one and the same probe electrode, slightly higher currents were observed in the case of AuSR surfaces, which also showed slight variations depending on the preparation batch. We attribute this to the influence of defects within AuSR layers. For these reasons, the d values calculated above represent an upper limit. From approach curves at AuSR and uncovered Au surfaces performed until the probe mechanically touched the sample surface, d is estimated to be 12-18 µm. The OCP is established at the uncovered half of the Au surface, which is located several millimeters away from the scanning microelectrode. This enables feedback imaging of gold patterns within the alkanethiolate-covered part of the Au support at OCP, even if probe and pattern sizes are comparable. SAM Modification. For the electrochemical modification of the thiolate monolayer, the macroscopic gold surface was used as the working electrode, and the microelectrode served as the auxiliary electrode. Desorption of the chemisorbed alkanethiolate monolayer was performed in 5 or 50 mM KOH. Two cyclic voltammograms (CVs) between -1200 and +1200 mV were performed, starting and finishing at 0 mV. The microelectrode was then moved laterally away from the modified region by 300 µm in the x and y directions. This position was used later to initiate the imaging after enzyme immobilization. From this position, the microelectrode was retracted out of the electrochemical cell by moving a defined distance in the z direction. The cell was rinsed extensively with water. Afterward, cystaminium dihydrochloride (Merck) was chemisorbed onto the renewed gold surface from a 100 mM solution in water during 30 min. Following rinsing, a suspension of periodate-oxidized glucose oxidase (GOx), prepared according to ref 40, was incubated over 4 h at room temperature. Unbound enzyme was rinsed off with 0.1 M phosphate buffer (pH 7.4) containing 0.25 % (v/v) poly(oxyethylene) sorbitan monolaureate (Tween 20, Serva, Heidelberg, Germany). During all chemical steps, the sample remained mounted in the open SECM (39) Bard, A. J.; Denuault, G.; Friesner, R. A.; Dornblaser, B.; Tuckerman, L. S. Anal. Chem. 1991, 63, 1282-1288. (40) Srere, P.; Uyeda, K. in Methods in Enzymology; Mosbach, K., Colowick, S. P., Kaplan, N. D., Eds.; Academic Press: San Diego, CA, 1976; pp. 11-19.
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Figure 3. Detection of GOx activity: (a) feedback mode on an insulating support, (b) feedback mode on a conductive support, and (c) generatorcollector mode; GOx, glucose oxidase; UME, ultramicroelectrode or probe electrode.
cell, which was equipped with a peristaltic pump to facilitate frequent solution exchange and rinsing steps. Finally, the microelectrode was brought into the start position for imaging by moving it in the vertical direction back into the electrochemical cell to the position before the chemical modification steps (vide supra). Imaging of the Enzyme-Modified Surface. Imaging of the enzyme-modified surface was performed using the GC mode in an air-saturated solution of 50 mM glucose + 0.1 M phosphate buffer (pH 7.4) by detecting the production of H2O2 at the scanning amperometric probe electrode (ET ) +400 mV, νt ) 15 µm s-1). If desired, minute quantities of a catalase suspension (from beef liver, 20 mg mL-1 protein, Boehringer, Mannheim, Germany) were dissolved in the working solution. RESULTS AND DISCUSSION Localized Desorption of n-Alkanethiolates from Gold Surfaces and Secondary Modifications. The localized electrochemical desorption of n-alkanethiolates from a gold electrode has been introduced in a previous communication.25 Regeneration of the bare gold surface occurs by reductive desorption (eq 1) in the cathodic branch of the CV and by oxidative decomposition (eq 2) as well as formation/reduction of gold oxides in the anodic half cycle.41 To restrict these reactions to a small region
AuSR + e- f Au(0) + RS(0)
AuSR + 2H2O f Au
(1)
+ RSO2 + 3e + 4H , -
-
+
pH > 7 (2)
perpendicularly beneath the microelectrode, the macroscopic gold electrode was operated as a working electrode and the microelectrode as an auxiliary electrode in close proximity to the working electrode (Figure 1a). Line scans across the renewed gold surface in the feedback mode result in high currents above the gold surface and low currents above the AuSR layer, which blocks the regeneration of [Ru(NH3)]3+ at the gold surface (Figure 1b). Scans as large as 1000 × 1000 µm2 did not show any damage outside the area situated directly beneath the microelectrode during the desorption step if the desorption was carried out in (41) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-359.
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Figure 4. (a, top) Optical microphotograph of the investigated test sample. The dark region is the enzyme-modified gold surface. The wire of 50 µm diameter serves as scale. (b, bottom) SECM GC image of the enzymatic activity; working solution, air-saturated 50 mM glucose + 0.1 M phosphate buffer.
KOH solution.25 In addition to these results, it was found that the use of lower concentration KOH solutions gives smaller features and reduces the variation between different batches of alkanethiolate-covered gold surfaces (Figure 1b). Therefore, desorption was performed in 5 mM KOH for the following experiments. No damage to the microelectrodes was observed during the desorption step, where they may have experienced extreme anodic or cathodic potentials. Desorbed sulfur-containing organics are diluted by diffusion into the desorption solution. A partial readsorption cannot be
excluded. It is, however, limited by the time until the desorption solution is removed from the cell, which occurred within 2-3 min after the end of the electrochemical desorption procedure. The extreme potentials occurring at the auxiliary microelectrode may also help to prevent passivation of the microelectrode, which could be used for subsequent imaging without intermediate cleaning procedures. Cystaminium dihydrochloride was allowed to chemisorb on the renewed gold surface as secondary adsorbate, whereby a patterned SAM was obtained. The terminal amino groups were used to form a Schiff base with carbaldehyde groups of periodateoxidized GOx. The reaction sequence is summarized in Figure 2. Imaging of GOx Activity Covalently Bound to an ω-Functionalized AuSR Monolayer. Immobilized GOx catalyzes reaction 3.
sample:
GOx
glucose + O2 98 gluconic acid + H2O2
(3)
The activity of GOx immobilized on an insulating surface has been followed with the SECM using the feedback mode (Figure 3a).29 A mediator is added to the bulk phase of the solution, and the probe electrode is operated at a potential where the diffusioncontrolled oxidation of the mediator causes a steady-state current at the probe electrode in the bulk phase of the solution. Imaging is achieved by monitoring the modulation of this steady-state current as the probe electrode is moved laterally in close proximity to the sample surface. Ferrocene derivatives (Fc) represent suitable mediators for the feedback imaging of GOx because their oxidized forms (Fc+) generated at the probe (eq 4) act as artificial electron acceptors for GOx in the absence of O2, and reaction 5 occurs at the immobilized enzyme if the probe electrode is located directly above the enzyme-modified region. The feedback mode
probe: sample:
Fc f Fc+ + e-
(4) + GOx
glucose + 2Fc 98 gluconic acid + 2Fc
(5)
is often preferred because of its better lateral resolution compared to that of the GC mode. If GOx is, however, immobilized on a metallic support, the heterogeneous electrochemical reaction (eq 6) at the metal-solution interface causes the reduction of Fc+ to Fc. This conventional positive feedback results in a high iT onto
sample:
Fc+ + e- f Fc
(6)
which the (small) contribution of the enzyme-mediated feedback (eq 5) is superimposed (Figure 3b). As proposed in ref 35, the contribution due to the enzymatic activity can be estimated as the difference between two feedback images taken in the presence and absence of glucose. In the latter case, only reaction 6 occurs, while in the presence of glucose, both reactions 5 and 6 proceed. As can be seen from Figure 1b, the contribution due to the heterogeneous electron transfer (eq 6) is not uniform over the investigated area due to the localized desorption of the blocking gold-dodecanethiolate SAM. The immobilized GOx layer is less effective as a diffusion barrier than the AuSR SAM, and highfeedback currents are measured above the modified area, even
Figure 5. SECM GC images of GOx covalently bound to a patterned SAM. Working solution: (a) air-saturated 50 mM glucose + 0.1 M phosphate buffer; (b) air-saturated 0.1 M phosphate buffer without glucose; (c) same as (a).
without any enzymatic activity. Thus, a map of the enzymatic activity is difficult to obtain with the feedback mode if GOx is immobilized on a conducting support, which is usually the case in biosensor applications. This problem can be circumvented by using the GC mode. An air-saturated phosphate buffer containing glucose is used. The probe electrode is held at a potential at which neither O2 nor glucose undergoes an electrochemical reaction. Therefore, no probe current is measured in the bulk phase of the solution. The enzymatic reaction (eq 3) at the GOx-modified surface proceeds continuously, since both the enzyme substrate (glucose) and an electron acceptor (O2) are present in the solution bulk. For Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Figure 6. Imaging of GOx activity with the GC mode coupled to a homogeneous decomposition of H2O2 by the enzyme catalase; working solution, air-saturated 50 mM glucose + 0.1 M phosphate buffer (pH 7.4). Additions of catalase in microliter of catalase suspension (20 mg mL-1 protein) per milliliter of working solution (a, top left) none, (b, middle left) 0.005, (c, bottom left) 0.03, (d, top right) 0.15, (e, bottom right) none.
imaging purposes, H2O2 formed during the enzymatic reaction is detected amperometrically, whereby imaging becomes independent of the conductivity of the support onto which GOx is immobilized. For evaluation, cystaminium dihydrochloride was chemisorbed on a clean, macroscopic gold surface, and oxidized GOx was bound to the amino groups (Figure 2). All but a 450 × 500 µm2 region of the modified gold surface was removed from the glass slide with a scalpel. Figure 4a shows an optical microphotograph of the obtained test structure. The GOx-modified gold region and the surrounding glass were subsequently imaged in the GC mode (Figure 4b). Reaction 3 occurs only at the modified gold surface, and H2O2 is detected amperometrically at +400 mV at the platinum microelectrode. No other component of the working solution is redox-active in this potential region. The background current increases from the front (start of imaging) to the rear part of Figure 4b. This is due to the slowly increasing bulk concentration of H2O2. However, the GOx-modified region can be easily identified. Imaging of GOx Activity at Patterned SAMs. After the localized desorption of an Au dodecanethiolate monolayer from a Au surface and the possibility of imaging the activity of GOx covalently bound to chemisorbed, amino-functionalized thiolates on gold had been demonstrated, specimens were prepared by 5064 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
combining both approaches. After localized desorption, the regenerated gold area was further modified as described in the Experimental Section to yield a microscopic area modified by covalently bound GOx (Figure 2). The distribution of the enzymatic activity bound to the gold surface was mapped in the GC mode as described above. The presence of immobilized GOx can be concluded from the high currents in the middle of Figure 5a. The signal width at half-height is about 300 µm for the enzyme-modified region in the GC images. This is slightly larger than the signal width obtained from the feedback images of the renewed Au regions after localized desorption (Figure 1b). Because of the lower lateral resolution of the GC mode as compared to that of the feedback mode, it cannot be concluded whether the GOx-modified regions extend slightly beyond the limits of the uncovered Au surface as identified by feedback imaging after desorption. As in the case of the test sample described above, a rising background due to H2O2 accumulation in the bulk solution is observed (image starts at (0,0), and line scans are parallel to the x axis). By recording an image of the same region with identical scan parameters in glucose-free buffer, the specific nature of the detected signal is highlighted (Figure 5b). If the solution does not contain the enzyme substrate glucose, the enzymatic glucose oxidation does not commence, and hence no H2O2 oxidation
Figure 7. Profiles across GC images of Figure 6. (a-c) Location of cuts for Figures 6a-c, and (d) current profiles (1) for (a), (2) for (b), and (3) for (c). The fwhm is given for each curve.
current is measured at the probe. It is important to exclude the possibility that GOx has lost its activity or has been washed off during scanning and frequent solution exchanges. Therefore, glucose was added to the working solution again. The recorded image shows the restored signal due to enzymatic H2O2 production (Figure 5c). Figure 5 parts a and c were recorded under exactly the same conditions. However, Figure 5c was recorded 3 h after Figure 5a. The similarity between both images proves the stability of the immobilized GOx, which is expected from a covalent attachment. Improved Imaging of GOx Activity in the GC Mode. The rising background currents in GC images shown above are due to the H2O2 accumulation in the bulk solution. To avoid the background signal drift and to enhance the quality of GC imaging, a homogeneous reaction which converts H2O2 to a redox-inactive substance should be useful. The H2O2 disproportionation catalyzed by the enzyme catalase was expected to accomplish this goal. A microscopic spot of immobilized GOx was formed as described above. Imaging was initiated in air-saturated, glucosecontaining buffer (Figure 6a). Afterward, minute quantities of the enzyme catalase were added to the solution (Figure 6b-d). The enzyme catalyzes the decomposition of H2O2 in the solution phase. This is equivalent to limiting the lifetime (τ(H2O2)) of H2O2. Consequently, the rising background is not observed when imaging is performed in the presence of trace amounts of dissolved catalase, while the signal height measured as peak-tobackground is reduced only slightly (Figure 7d). The amount of catalase in the electrolyte solution has to be tuned exactly to make full use of the effect. The time needed for diffusional transport across the gap between enzyme-modified regions to the probe electrode positioned perpendicularly above the surface is of the order of τ ≈ d2/D, where D is the diffusion coefficient of H2O2.
τ(H2O2) should be large enough that H2O2 generated at the sample may reach the probe electrode but short enough to prevent H2O2 transport over considerably larger distances. Unfortunately, the kinetics of enzymatic H2O2 disproportionation do not follow a firstorder law in the micromolar concentration range. This makes a quantitative estimation of the required enzyme activity difficult. Instead, an empirical adjustment is carried out by successive addition of trace amounts of catalase. A further increase of the catalase concentration (Figure 6c,d) eventually leads to a nearly complete quenching of the GOxspecific signal (Figure 6d). At that catalase concentration, H2O2 is decomposed before it reaches the probe electrode. The signal quenching by catalase confirms that H2O2 is the only compound oxidized at the probe electrode and proves the specificity of the recorded signals for immobilized GOx activity. Considering the full width at half-maximum (fwhm) of the cross sections in Figure 7, it seems attractive to explore the possibility of resolution enhancement in GC imaging by introducing a homogeneous reaction that consumes the detected product. For samples investigated in this study, the limited sensitivity of the potentiostat currently available to us (10 nA V-1) does not allow us to sacrifice intensity for better spatial resolution. Therefore, resolution enhancement cannot be demonstrated. However, this opportunity may be attractive in any applications where signal height is not a matter of concern. Here, the advantages of adding catalase are the suppression of rising background currents and the opportunity to prove that probe currents are exclusively due to H2O2 oxidation and, hence, are a measure of GOx activity. CONCLUSION SECM provides new possibilities to form and characterize miniaturized biochemically active layers for sensor applications or biomolecular devices. A SAM of long-chain n-alkanethiolates on gold is used as the starting material. By localized electrochemical desorption of the alkanethiolate, a microscopic area of uncovered gold is formed, onto which an ω-functionalized thiol is chemisorbed in a second step. The enzyme is bound covalently to the terminal group of the secondary adsorbate. The possibility to repeat the procedure at different spots is seen as the main advantage over existing protocols. By using different enzymes in each modification cycle, a route to multienzyme structures is envisaged. The mapping of enzymatic activity for such samples is achieved by the GC mode of the SECM, while the feedback mode is not suitable because of the interference between positive feedback due to the enzymatic reaction and that due to heterogeneous electron transfer at the gold surface. The independence of GC imaging of the nature of the support onto which the biomolecules are immobilized makes it a very flexible tool for the analysis of biosensing surfaces. It is also easier to achieve a high sensitivity. The mapping of the GOx activity was achieved for a periodateoxidized GOx coupled to a monolayer of amino-terminated adsorbates using a potentiostat with a sensitivity of 10 nA V-1 and a 14 bit AD board. This compares favorably with reports about feedback imaging where a much higher enzyme activity per apparent surface area was immobilized within enzyme gels.29 Rising backgrounds due to H2O2 accumulation in the bulk phase can be avoided by adding small amounts of catalase to the solution. Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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ACKNOWLEDGMENT This paper is dedicated to Prof. Dr. Gerhard Werner (Leipzig) on the occasion of his 65th birthday. G.W. gratefully acknowledges a grant from the Alexander von Humboldt Foundation, which allowed a research stay under the supportive mentorship of Prof. Dr. H.-L. Schmidt at the Lehrstuhl fu¨r Allgemeine Chemie und Biochemie of the Technische Universita¨t Mu¨nchen, as well as cooperation with Dr. C. Kranz (SECM setup, protocols for
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microelectrode fabrication) and technical assistance of H. Wohlschla¨ger (preparation of oxidized GOx). Received for review May 15, 1997. Accepted September 25, 1997.X AC970504O X
Abstract published in Advance ACS Abstracts, November 1, 1997.
