Microscopic and Voltammetric Characterization of Bioanalytical

May 10, 2006 - A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is ...
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Langmuir 2006, 22, 5443-5450

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Microscopic and Voltammetric Characterization of Bioanalytical Platforms Based on Lactate Oxidase A. Parra,† E. Casero,† L. Va´zquez,‡ J. Jin,§ F. Pariente,† and E. Lorenzo*,† Departamento de Quı´mica Analı´tica y Ana´ lisis Instrumental, UniVersidad Auto´ noma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain, Instituto de Ciencia de Materiales de Madrid (CSIC), C/Sor Juana Ine´ s de la Cruz, no. 3, 28049 Madrid, Spain, and Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell UniVersity, Ithaca, New York 14853-1301 ReceiVed January 19, 2006. In Final Form: March 3, 2006 A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is presented. In this context, emphasis is placed on amperometric biosensors based on LOx that have been immobilized by direct absorption on carbon surfaces, in particular, glassy carbon (GC) and highly ordered pyrolytic graphite (HOPG). The immobilized LOx layers have been characterized using atomic force microscopy (AFM) under liquid conditions and cyclic voltammetry. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). In the presence of lactate with hydroxymethylferrocene (HMF) as a redox mediator in solution, biosensors obtained by direct adsorption of LOx onto GC electrodes exhibited a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. The proposed biosensor also exhibits good operating performance in terms of linearity, detection limit, and lifetime.

1. Introduction Development of bioanalytical platforms based on immobilized enzymes, retaining their full activity and stability, is one of the most interesting prospects in the field of biotechnology. The morphology of the immobilized enzyme could have a great influence on its catalytic properties, as it is well-known that some enzymes lose part of their activity when they are immobilized on metallic surfaces. In this sense, an understanding of the basic physicochemical behavior of the resulting enzyme monolayer after the immobilization process is a crucial factor in the biosensor development process. Several scanning probe microscopy techniques have been applied to the characterization of the biological nanomaterials, providing a unique insight into their structure and properties. Among them, we can highlight atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM). During the past several years, AFM has been widely used to visualize biological molecules, allowing morphological and mechanical information at the nanometer level to be obtained. Particularly, since the development of tappingmode operation in aqueous environment,1 AFM has achieved particular relevance as a characterization technique able to provide morphological data on protein deposits without damaging the sample surface.2 In addition, AFM provides very important information on protein-protein and protein-surface interactions, through force spectroscopy measurements.3 SECM is an in situ scanning probe microscopy that has achieved great importance for studying biological systems during the past 10 years. It allows spatially resolved information to be obtained on a wide range of electrochemical processes occurring at interfaces.4 Signal * To whom correspondence should be addressed. [email protected]. † Universidad Auto ´ noma de Madrid. ‡ Instituto de Ciencia de Materiales de Madrid (CSIC). § Cornell University.

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(1) Putman, C. A.; van der Werf, K. O.; de Grooth, B. G.; van Hulst, N. F.; Greve, J. Appl. Phys. Lett. 1994, 64, 2454. (2) Forbes, J. G.; Jin, A. J.; Wang, K. Langmuir 2001, 17, 3067. (3) Feldman, K.; Ha¨hner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134.

generation in SECM is based on surface-induced changes of a Faraday current that result from the hemispherical diffusion of a redox-active species to a microelectrode that is scanned across the sample surface. Recently, SECM has been applied successfully to obtain enzyme activity images. In the present article, we describe the immobilization by direct adsorption of lactate oxidase (LOx) on carbon electrodes, in particular, glassy carbon (GC) and highly orderered pyrolytic graphite (HOPG), to prepare (construct) bioanalytical devices, such as biosensors. The aim of this work is to develop a simple and general immobilization approach, using oxidoreductase enzymes. In this context, emphasis has been placed on monitoring and controlling the deposition conditions and procedures, as the immobilization process can induce a substantial decrease of the enzyme activity. For this purpose, AFM, SECM, and cyclic voltammetry (CV) have been used to correlate deposition procedures to changes in morphology and biological activity. LOx was chosen as a model enzyme because, to the best of our knowledge, very few studies on its physicochemical behavior and activity when immobilized on metallic surfaces have been conducted. However, its substrate (lactate) is of great importance in several fields including clinical studies and food analysis. Because of its intrinsic interest, several methods to determine lactate have been proposed in the literature. Among them, we can highlight amperometric LOx-based biosensors. These devices combine the inherent sensitivity of the electrochemical techniques with the high selectivity provided by the specific binding of the analyte to the active-site regions of the enzyme.5-21 When O2 is used as a mediator, biosensors are based (4) Bard, A. J., Mirkin, M. V., Eds. Scanning Electrochemical Microscopy; Marcel Dekker: New York, 2001. (5) Yang, Q.; Atanasov, P.; Wilkins, E. Electroanalysis 1998, 10, 752. (6) Palmisano, F.; Rizzi, R.; Centonze, D.; Zambonin, P. G. Biosens. Bioelectron. 2000, 15, 531. (7) Palmisano, F.; Quinto, M.; Rizzi, R.; Zambonin, P. G. Analyst 2001, 126, 866. (8) Garjonyte, R.; Yigzaw, Y.; Meskys, R.; Malinauskas, A.; Gorton, L. Sens. Actuators B 2001, 79, 33. (9) Aydin, G.; C¸ elebi, S. S.; O ¨ zyo¨ru¨k, H.; Yıldız, A. Sens. Actuators B 2002, 87, 8.

10.1021/la060184g CCC: $33.50 © 2006 American Chemical Society Published on Web 05/10/2006

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on the direct determination of H2O2 production. However, the determination of lactate in real samples by direct amperometric measurement of hydrogen peroxide can be affected by interfering species that could be oxidized at the high potential required for H2O2 detection. To minimize the contribution of interfering substances to the biosensor response, several approaches have been reported in the literature, including electrode modification by a catalyst,8 coimmobilization of horseradish peroxidase with lactate oxidase,19 and replacement of the natural electron acceptor (O2) by an artificial mediator.12,14,18 In this sense, we have reported that hydroxymethylferrocene (HMF) is a suitable electron mediator for lactate determination.22 The use of HMF allows us to operate at low electrode potential (E ) +0.30 V), circumventing the possible interference caused by oxidizable compounds that could be present in the solution. In this context, after an exhaustive physicochemical characterization of the resulting enzyme monolayer, we have focused our attention on LOx-based biosensor applications using HMF as a redox mediator. Finally, the applicability of the designed biosensor to lactate determination is demonstrated. 2. Experimental Section 2.1. Materials. Lactate oxidase (EC 232-841-6 from Pediococcus species) lyophilized powder containing 41 units/mg of solid was obtained from Sigma Chemical Co. (St. Louis, MO). Stock solution was prepared dissolving 1.3 mg of the LOx lyophilized powder in 250 µL of 0.1 M phosphate buffer (pH 7.0), was aliquoted (10 µL), and was stored at -30 °C. Under these conditions, the enzymatic activities remain stable for several weeks. L-(+)-Lactic acid lithium salt (97%), hydroxymethylferrocene, ferricyanide, hexaaminruthenium, and potassium chloride were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used as received. Sodium phosphate (Merck) was employed for the preparation of buffer solutions (0.1 M, pH 7.0). Water was purified with a Millipore Milli-Q system. All solutions were prepared just prior to use. 2.2. Experimental Techniques and Procedures. Atomic Force Microscopy (AFM). The atomic force microscope was a Nanoscope IIIa apparatus from Digital Instruments (DI, Santa Barbara, CA) used with a scanner with a maximum scan range of ∼14 µm. Measurements were carried out using Si3N4 cantilevers (nominal spring constant of 0.38 N/m) from Digital Instruments. HOPG and GC substrates were used as supports for protein adsorption. The samples were imaged with different cantilevers to ensure that the imaged structures were not due to tip artifacts. Image acquisition times were between 2 and 6 min for images with pixel resolutions of 512 × 512. All measurements were carried out in aqueous buffered solutions because protein structure can be distorted by the drying process and the biological relevance of dry samples might be questioned. Moreover, imaging under ambient conditions implies the application of higher forces on the protein, which could damage or distort the imaged structures. (10) Liu, H.; Zhang, Z.; Zhang, X.; Qi, D.; Liu, Y.; Yu, T.; Deng, J. Electrochim. Acta 1997, 42, 349. (11) Iwuoha, E. I.; Rock, A.; Smyth, M. R. Electroanalysis 1999, 11, 367. (12) Park, T. M.; Iwuoha, E. I.; Smyth, M. R.; Freaney, R.; Mcshane, A. J. Talanta 1997, 44, 973. (13) Palmisano, F.; de Benedetto, G. E.; Zambonin, C. G. Analyst 1997, 122, 365. (14) Haccoun, J.; Piro, B.; Tran, L. D.; Dang, L. A.; Pham, M. C. Biosens. Bioelectron. 2004, 19, 1325. (15) Serra, B.; Reviejo, A. J.; Parrado, C.; Pingarro´n, J. M. Biosens. Bioelectron. 1999, 14, 505. (16) Herrero, A. M.; Requena, T.; Reviejo, A. J.; Pingarro´n, J. M. Eur. Food Res. Technol. 2004, 219, 556. (17) Kulys, J.; Wang, L.; Maksimoviene, A. Anal. Chim. Acta 1993, 274, 53. (18) Boujtita, M.; Chapleaua, M.; El Murr, N. Electroanalysis 1996, 8, 485. (19) Ledru, S.; Boujtita, M. Bioelectrochemistry 2004, 64, 71. (20) Rubianes, M. D.; Rivas, G. A. Electroanalysis 2005, 17, 73. (21) Suman, S.; Singhal, R.; Sharma, A. L.; Malthotra, B. D.; Pundir, C. S. Sens. Actuators B 2005, 107, 768. (22) Parra, A.; Casero, E.; Va´zquez, L.; Pariente, F.; Lorenzo, E. Anal. Chim. Acta 2006, 555, 308.

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Figure 1. Tapping-mode AFM image of an incomplete LOx layer adsorbed on a HOPG substrate under buffer conditions on a 500 nm × 500 nm area. Tapping-mode AFM imaging was used to image the different protein deposits and substrates. In this mode, the cantilever oscillates in the aqueous buffers at a frequency close to its resonant frequency, f0, in the ∼9-11 kHz range with amplitude A0 (close to 15 nm) above the surface. Contact-mode AFM imaging under liquid environment was also employed, but it distorted the protein structures for incomplete protein monolayer coverage. For this reason, we used tapping-mode AFM imaging in this study. The substrate was first imaged in buffer solution to ensure that the surface was flat and clean before the protein immobilization was performed. Both sample and cantilever were located within a Plexiglas fluid cell to which extremely small volumes (∼50 µL) of buffer were added. The enzyme adsorption was carried out by immersing the support into a solution of LOx (0.2 µM in 0.1 M phosphate buffer at pH 7.0). Before being imaged by AFM, the samples were rinsed thoroughly with deionized water to remove any loosely bound residue of LOx. Scanning Electrochemical Microscopy (SECM). SECM measurements were carried out using CH Instruments model 900B equipment. The setup was formed by an electrochemical cell located on an XYZ positioning stage. The distance between the tip and the sample was controlled using a stepper motor (coarse approach) combined with a piezoelectric micropositioning system (fine approach). The threeor four-electrode cell was formed by a Ag/AgCl reference electrode; a platinum wire auxiliary electrode; a substrate (GC); and a Pt ultramicroelectrode (UME) with radius of 10 µm, usually termed the tip or probe electrode. The substrate was modified with the enzyme by placing 10 µL of LOx stock solution onto the surface. Measurements were performed in the feedback mode (probe potential E ) +0.30 V, sample at open circuit) in 0.1 M phosphate buffer (pH 7.0) + 0.1 M KCl containing 1 mM of HMF and 2 mM of lactic acid. Previously, the assay solution was thoroughly deoxygenated in the SECM cell. Electrochemical Measurements. Cyclic voltammetric and amperometric studies were carried out with a BAS CV-27 potentiostat connected to a BAS X-Y recorder. The electrochemical experiments were carried out in a three-compartment electrochemical cell with standard taper joints so that all compartments could be hermetically sealed with Teflon adapters. A glassy carbon disk electrode (geometric area of 0.2 cm2) was used as the working electrode. A large-area coiled platinum wire was employed as a counter electrode. All potentials are reported against a sodium-saturated calomel electrode (SSCE) without taking into account the liquid junction. All solutions were deaerated with nitrogen gas before use, and the gas flow was maintained over the solutions during experiments. Prior to each experiment, GC electrodes were polished with 1-µm diamond paste (Buehler) and rinsed with water. The enzyme adsorption was carried out by placing 10 µL of LOx stock solution onto the surface. After being dried in the air, the enzyme electrode was washed with water to remove any weakly bound enzyme. The immobilized enzyme (LOx) oxidizes the L-lactate to pyruvate in the presence of HMF, which acts as a soluble mediator in solution.

3. Results and Discussion 3.1. AFM Imaging of Lactate Oxidase Immobilized on Carbon Surfaces. AFM measurements were carried out to study

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Figure 2. Morphological analysis of Figure 1: (A) Height histogram of the LOx structures, (B) autocorrelation, (C) cross-sectional profile measured along the dashed line of part B.

Figure 4. 500 nm × 500 nm tapping-mode AFM image recorded under buffer conditions of a complete LOx layer adsorbed on a HOPG substrate.

Figure 3. Sequential 250 nm × 250 nm tapping-mode AFM images recorded under buffer conditions of an incomplete LOx layer adsorbed on a HOPG substrate. The same area was imaged at different A/A0 ratios: (A) 0.92, (B) 0.78, (C) 0.92. Topographical surface profiles of the same LOx structures from images A-C are shown in part D.

the LOx monolayers adsorbed on GC and HOPG substrates in order to obtain morphological and mechanical information. Contact-mode AFM imaging of an incomplete LOx monolayer deposited on HOPG substrate was unsuccessful. This kind of imaging leads to extremely flat images that correspond rather to the substrate surface (not shown) because of the poor proteinHOPG interaction, which leads to removal of the LOx protein by the tip. These results led us to employ tapping-mode imaging under liquid environment (Figure 1). In this figure, nanometer features due to the adsorption of LOx proteins are observed. They appear as both isolated and aggregated features. In tapping mode, the low tip-sample

interactions allow the reproducible and repetitive imaging of the same surface area without any apparent distortion. In contrast, the higher tip-sample lateral interactions in contact-mode AFM hamper the reliable imaging of the LOx structures. It should be noted that the lateral dimensions of the isolated LOx features could be enhanced by tip convolution effects. In Figure 2A is plotted the histogram of the heights of the protein structures. The height values span from 2.4 to 6.2 nm, which suggests different adsorption geometries as well as, likely, different protein-substrate interactions. The most probable height range (almost 75% of the structures have heights in this range) was found to be 3.5-4.5 nm. Interestingly, the LOx structures have heights in the range of the monolayer thickness (see below). This fact indicates that LOx proteins tend to aggregate laterally rather than vertically, which, for longer adsorption times, leads to quite homogeneous LOx monolayers. In Figure 2B is shown the autocorrelation of the AFM image of Figure 1. A central bright spot, around 40 nm wide, can be seen, indicating the extent of the size of the adsorbed structures. This central bright spot is the only noticeable feature of the

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Figure 5. 1.2 µm × 1.2 µm tapping-mode AFM images recorded under buffer conditions of a LOx layer adsorbed on (A) a HOPG substrate and (B) a GC surface after removal of LOx structures from a 300 nm × 300 nm central area of the sample by sweeping in contact mode at relatively high loads. The corresponding profiles measured along the scratched area are shown in parts C and D, respectively.

image autocorrelation. This behavior is better seen in the cross section (Figure 2C) taken along the horizontal line depicted in Figure 2B. In this section, we observe a high central peak with no other appreciable feature along the profile. This behavior indicates the disordered character of the LOx adsorption process, in contrast to that of xanthine oxidase on gold surfaces, in which a clear gap distance between next-neighbor proteins was found at the initial stages of deposition.23 The incomplete monolayer coverage of the LOx deposit on HOPG allows us to study the mechanical response of the LOx structures to the tip load by tapping-mode AFM. Thus, we scanned the same area initially at the lowest load compatible with stable imaging of the sample surface, which usually corresponds to a ratio in the 0.91-0.93 range between the set point, A, and the free-amplitude oscillation, A0. Then, we increased this load to A/A0 ≈ 0.78 and finally imaged the same area at the initial low load. This study is shown in Figure 3, in which images, corresponding to the same 250 nm × 250 nm area, obtained under the above conditions are shown. The arrows indicate two clear LOx aggregated features that help to identify the LOx features in the different images, as a lateral drift of 0.25 nm/s was present in the experiment. It is worth noting that, although this drift can distort the lateral dimensions of the LOx features, it should not affect their heights. This fact implies that any differences in (23) Casero, E.; Martı´nez G. de Quesada, A.; Jin, J.; Quintana, M. C.; Pariente, F.; Abrun˜a, H. D.; Va´zquez, L.; Lorenzo, E. Anal. Chem. 2006, 78, 530.

height should be associated, in principle, with the different tip loads. It can be seen from Figure 3A-C that some protein features are slightly distorted by the tip action both vertically and laterally. However, an abrupt or dramatic distortion of the LOx features can be discarded. These effects can be also seen in Figure 3D, which shows the surface profile corresponding to the same LOx features under the initial low load (bottom profile), the high load (middle), and again at the low load (top). Despite the existing drift, the profiles are rather similar. These results allow us to conclude that the LOx protein features, under the present experimental conditions, are not deformed to a great extent because of the tip load, although some lateral distortions can be present. Having imaged the incomplete LOx monolayer, we studied the full LOx monolayer deposits on HOPG. The results are shown in Figure 4, in which a continuous, homogeneous, and relatively smooth (surface roughness of 0.5 nm) morphology is observed. The protein lateral dimension can be extracted and was found to be in the 5-8 nm range. The AFM technique allows for the estimation of the thickness of the LOx deposit on HOPG. This is important because the standard quartz crystal microbalance (QCM) technique is more suitable for measurements on metallic substrates. To carry out this measurement, we imaged a 300 nm × 300 nm area of the sample in contact mode, at relatively high loads, to remove the LOx structures. Afterward, we imaged a larger area around this spot in tapping mode. The tip used in the whole experiment was

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Figure 6. (A) 410 nm × 410 nm tapping-mode AFM image recorded under buffer conditions of a LOx layer adsorbed on a GC substrate. Both the protein structures and the substrate are visible. (B) Contrast phase image measured simultaneously. (C) A/A0 ratio versus tip-sample relative distance approach curves obtained with the same cantilever on a LOx region (see P1 in part A) and a GC region (see P2 in part A). (D) Phase shift versus relative tip-sample distance approach curves measured on GC domains (P1) and LOx-packed domains (P2).

the same, which implies that the tapping-mode image quality was not the same as that obtained with unused clean tips, but the image quality was good enough to obtain an estimation of the LOx thickness. This value is an overestimation of the thickness because the tip scanning at high loads could also remove some carbon atoms from the HOPG surface. This final tapping-mode image for the case of HOPG is shown in Figure 5A, and the corresponding surface profile across the scratched window is shown in Figure 5C. The resulting thickness estimation is in the 4-6 nm range, which is close to the range of the lateral dimensions of the LOx proteins imaged by tapping-mode AFM (Figure 4). The same procedure was followed to estimate the thickness of the LOx monolayer deposited on GC (Figure 5B and D). In this case, the assessment of this value becomes more difficult because of the inherent roughness of the glassy surface, which is considerably greater than that of the atomically flat HOPG surfaces. In any case, the thickness estimation seems to be consistent with that obtained on HOPG substrates, i.e., 6-8 nm. Finally, we also studied incomplete LOx monolayer deposits on GC substrates by tapping-mode AFM. The corresponding topographic image (Figure 6A) showed uncovered glassy substrates regions (P1 in Figure 6A) surrounded by areas in which well-packed LOx structures were easily observed (P2 in Figure 6A). The size of the protein structures is in the 5-8 nm range, which agrees with that obtained for LOx deposits on HOPG. The difference between these two regions becomes more evident in the corresponding phase-contrast image (Figure 6B). In this image, the bare GC (P1) regions appear brighter that those covered by LOx proteins (P2). In fact, the phase shift is 6° higher on GC than on the LOx domains. This result is consistent with that found in air for the tapping-mode imaging of purple membrane

islands on HOPG surfaces, in which a larger phase shift was also found on HOPG.24 The coexistence of LOx-free and LOx-containing regions allows us to perform force volume imaging of this sample in tapping mode. This kind of imaging allows one to obtain tip amplitude versus tip-sample relative distance curves on both regions with the same tip and under the same experimental conditions. These curves are displayed in Figure 6C. It should be noted that the glassy substrate is relatively hard, in any case, quite harder than HOPG, which allows us to analyze the indentation of the LOx proteins by the same tip for different A/A0 ratios. This estimation can be done by measuring the lateral shift of the P1 curve with respect to the P2 curve at a given tip load (i.e., A/A0 ratio) in Figure 6C. Thus, for A/A0 ) 0.93, which is the minimum load compatible with stable imaging conditions, the indentation of the LOx structure is already 0.6-0.7 nm, whereas for A/A0 ) 0.78, it increases slightly to 0.7-0.8 nm. This small difference is consistent with the slight differences found in the surface profiles obtained through the same LOx proteins at these A/A0 ratios in Figure 3D. In Figure 6D are shown the phase shift versus relative tip-sample distance plots measured on GC domains (P1) and LOx-packed domains (P2). It should be noted that these curves and those of Figure 6C were not obtained simultaneously. In agreement with the contrast phase image of Figure 6B, the phase shift is higher on GC than on LOx regions. It is clear that the phase shift decreases with tip-sample distance (i.e., with tapping amplitude; see Figure 6C) in both LOx and GC; however, this decrease is larger on LOx. Tapping-mode experiments in fluids differ significantly from those in air.1 Thus, to date, several attempts with different degrees (24) Tamayo, J.; Garcı´a, R. Appl. Phys. Lett. 1998, 73, 2926.

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performed over a 700 µm × 700 µm area under the conditions described above. After addition of lactate, the enzymatic reaction occurs according to the following pathway

+ (LOx)ox f pyruvate + (LOx)red

(2)

(LOx)red + (HMF)ox f (LOx)ox + (HMF)red

(3)

(HMF)red f (HMF)ox + e-

(4)

L-lactate

Figure 7. SECM surface-plot image (700 µm × 700 µm) of LOx/ GC system. Positive feedback with HMF mediator (1 mM) at ET ) +0.3 V vs Ag/AgCl in pH 7.0 (0.1 M) phosphate buffer solution in the (A) absence and (B) presence of 2 mM lactate.

of complexity to understand such experiments have been reported.25-27 Because of the complexity of tapping-mode experiments in fluids, as a first rough approximation, we can, in principle, use the expression proposed in ref 24 for the phase shift, φ, which, for given resonance and excitation frequencies and cantilever characteristics (i.e., force constant and quality factor), reads as

sin φ ∝ A + Edis/A

(1)

where A is the working amplitude or set point, which changes with the tip-sample distance (Figure 6C), and Edis represents the dissipated energy. Thus, the larger phase shift for the bare glassy domains would be consistent, in a first approximation, with larger energy dissipation on these domains compared to that on the LOx regions. 3.2. SECM Measurements. The activity of LOx directly adsorbed on GC substrates was investigated using feedback mode. In this mode, the probe electrode is immersed in a buffer solution (pH 7.0) containing a mediator, in our case, HMF (1 mM). At a probe potential of +0.3 V, a steady-state current (Iss) due to the diffusion-controlled oxidation of HMF is obtained. The imaging of the surface consists of following the modulation of this current value when the probe electrode is moved laterally over the surface in close proximity. Figure 7A shows the scan (25) Chen, G. Y.; Warmack, R. J.; Oden, P. I.; Thundat, T. J. Vac. Sci. Technol. B 1996, 14, 1313. (26) van Noort, S. T. J.; Willemsen, O. H.; van der Werf, K. O.; de Grooth, B. G.; Greve, J. Langmuir 1999, 15, 7101. (27) Legleiter, J.; Kowalewski, T. Appl. Phys. Lett. 2005, 87, 163120.

Reaction 4 takes place on the electrode surface. Thus, when the probe is located on a LOx-modified region of the GC substrate, an enhancement of the current detected by the tip is observed, as can be seen in Figure 7B. The enzymatic activity can be assessed from the difference between the feedback images in the presence and absence of lactate. The variations in feedback current, which would correspond to variations in enzymatic activity, can be understood in terms of the spatial distribution of the enzyme and its activity. As mentioned in the Experimental Section, we employed direct adsorption for enzyme immobilization. This method often gives rise to a nonuniform spatial distribution of enzyme molecules on the surface. In addition, it is often found that immobilized enzyme layers have a nonuniform activity, reflecting variations in the specifics of adsorption (and conformation) for particular locations. Thus, such spatially inhomogeneous enzymatic activity would, in fact, be anticipated, as we indeed observed. 3.3. Blocking Characteristics of a LOx Monolayer on Carbon. From the AFM and SECM studies, one can conclude that a LOx monolayer adsorbed on a GC surface is not very tightly packed, as some pinholes can be observed. To further characterize the physicochemical properties of LOx monolayers on GC, in terms of packing degree and permeability, we carried out cyclic voltammetric studies of various redox probes including ferricyanide (1.0 mM), HMF (1.0 mM), and [Ru(NH3)6]3+ (1.0 mM) on bare and LOx-modified electrodes. These molecules were chosen so as to be able to assess the effects of the charge (negative, neutral, and positive) of the redox probe on the charge of the adsorbed layer of LOx because of its isoelectric point. Because the isoelectric point of LOx is around 4.6, one would anticipate that the permeability of the LOx layer to the various probes would be pH-dependent. At pH 2.0, below the isoelectric point, the enzyme is positively charged. Thus, the electrochemical responses of the negative (ferricyanide) and neutral (HMF) probes were virtually identical to those observed for bare carbon electrode (Figure 8A,B). In contrast, the cyclic voltammetric response for the positively charged probe ([Ru(NH3)6]3+) was extremely attenuated, with a very large ∆Ep value (Figure 8C). Such behavior can be explained by a strong electrostatic repulsion between the positively charged enzyme layer and the positively charged metal complex. At pH 7.0, which was used because it is the optimum pH for the enzymatic reaction, the LOx layer is negatively charged. In this case, the cyclic voltammetric responses of HMF and [Ru(NH3)6]3+ remained virtually unchanged, suggesting little, if any, change in the permeability, as would be anticipated (Figure 8E,F). In contrast, the response of the ferricyanide probe was significantly attenuated, with a very large ∆Ep value (Figure 8D). We attribute such behavior to a strong electrostatic repulsion arising from the negative charge of the protein layer. These observations would suggest that the adsorption of LOx on carbon surfaces gives rise to a layer that is quite permeable to different compounds, which is very interesting for biosensor applications. In such devices, enzyme substrates, products, and artificial redox mediators must reach the electrode surface to undergo oxidation or reduction.

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Figure 8. Cyclic voltammograms (V ) 100 mV/s) of a bare GC electrode (solid line) and a LOx/GC electrode (dashed line) in phosphate buffer containing 1.0 mM [Fe(CN)6]3- at pH (A) 2.0 and (D) 7.0, 1.0 mM HMF at pH (B) 2.0 and (E) 7.0, and 1.0 mM [Ru(NH3)6]3+ at pH (C) 2.0 and (F) 7.0.

In addition, the electrostatic properties of the enzymatic layer can reduce access of negatively charged electroactive species to the electrode surface and avoid potential interferences, in particular, ascorbate. 3.4. Enzyme Electrode Response. The response of a LOx electrode to lactate is based on the oxidation of L-lactate to pyruvate, catalyzed by LOx, in the presence of HMF, which acts as a soluble mediator in solution. Figure 9A depicts the cyclic voltammetric response from -0.1 to +0.5 V at 10 mV/s for LOx adsorbed on a GC electrode in contact with a 0.1 M phosphate buffer solution containing 1.0 mM HMF in the absence (curve a) and in the presence (curve b) of the substrate. In the absence of substrate, as would be anticipated, a well-behaved redox response ascribed to the ferrocene/ferrocinium process is observed. Upon addition of lactate (to a final concentration of 3 mM), the cyclic voltammogram exhibits an enhancement of the anodic peak current concomitant with a decrease of the cathodic peak current. This behavior is consistent with a strong electrocatalytic effect. To confirm the role of the enzyme in the catalytic response of the biosensor, the cyclic voltammetric responses of a bare GC electrode in contact with a 0.1 M phosphate buffer solution containing 1.0 mM HMF in the absence and in the presence of lactate were recorded (data not shown). As one would expect, the results obtained showed that the presence of lactate did not change the cyclic voltammetric response associated with the ferrocene/ferrocinium process. In the biosensor assembly developed, the immobilized LOx oxidizes lactate to pyruvate in the presence of HMF, which acts as an acceptor of electrons generated in the enzymatic reaction and is transformed to its reduced form. This, in turn, diffuses to the electrode, where is reoxidized back into its oxidized form. The electrode serves as a secondary acceptor of electrons able to regenerate the redox mediator used in the enzymatic reaction, so the magnitude of this catalytic current can be employed as the analytical signal in the determination of the substrate (lactate)

Figure 9. (A) Cyclic voltammetric response for LOx adsorbed on a GC electrode in contact with a 0.1 M phosphate buffer solution containing 1.0 mM HMF in the absence (curve a) and in the presence (curve b) of 3 mM of lactate. The potential was cycled between -0.1 and +0.5 V, and the scan rate was 10 mV/s. (B) Calibration curve obtained from chronoamperometric measurements (E ) +0.30 V). (C) Linear part of plot in B.

concentration. The steady-state current response obtained for a LOx/GC electrode poised at E ) +0.30V, where oxidation of

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4. Conclusions

Table 1. Analytical Properties of Lactate Biosensor LOx/GC biosensor equation (Iss vs [lactate]) linear response (mM) sensitivity (µA mM-1) detection limit (µM) determination limit (µM) reproducibility (RSD) repeatability (RSD) stability

y ) 6.07x - 0.02 (R ) 0.998) up to 0.2 6.07 ( 0.21 0.9 69 3% 2% g1 month

HMF is assured, was plotted as a function of the bulk concentration of lactate in solution (Figure 9B). The analytical propertiess linear response, sensitivity, and detection limitsare obtained from the linear part of the plot of the electrocatalytic current versus lactate concentration (Figure 9C), and the results are summarized in Table 1. The sensitivity, obtained from the slope of the plot, was found to be 6.07 ( 0.21 µA mM-1. The detection and determination limits were calculated as the concentrations that gave a signal equal to 3 and 10 times the standard deviation of background current, respectively. A determination limit of 69 µM was found. The reproducibility was evaluated by comparing the analytical signals obtained using four different biosensors prepared from the same enzyme batch for a lactate concentration of 1.0 × 10-4 mol L-1. A relative standard deviation of 3% was calculated. In addition, the repeatability was evaluated from three different measurements of a lactate concentration (1.0 × 10-4 mol L-1) with the same electrode. Under these conditions, a relative standard deviation (RSD) of 2% was obtained. One of the main properties of the enzyme-modified electrodes to be considered for biosensors application is stability. Concerning the stability of LOx-based electrodes, it was found that the response decreases by about 50% of its initial value after one assay and, afterward, it remains constant for more than 1 month. In light of the results summarized in Table 1, we conclude that the biosensor developed is quite robust and has enough sensitivity to be used to determine lactate in several samples.

A comprehensive microscopic and voltammetric study of bioanalytical platforms based on lactate oxidase for biosensor applications has been described. In particular, emphasis has been placed on the study of the influence of the immobilization on the morphological and catalytic properties of the enzyme. LOx was immobilized on both bare GC and HOPG by direct adsorption. AFM measurements afforded morphological characterizations of the resulting enzymatic layers, providing data concerning the protein distribution on the substrate and the tip-induced deformation during AFM imaging. AFM measurements showed that, on HOPG surfaces, LOx molecules tend to aggregate laterally rather than vertically and that disorder is characteristic of the LOx adsorption process. Electrochemical and SECM assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization and exhibits a spatially inhomogeneous enzymatic activity, as one would expect given that we employed direct adsorption for enzyme immobilization. Finally, we studied the analytical features of the amperometric lactate biosensor. The operating potential was reduced to +300 mV (versus SSCE) from the typical voltage (+0.70 V) required for H2O2 electrooxidation by the use of a mediator (HMF) in solution. The electrocatalytic response, stable and reproducible, shows a linear dependence on the concentration of L-lactate in solution. Lactate can be determined with a detection limit of 69 µM and good reproducibility (RSD 3%). Moreover, the useful lifetime and the simplicity of the preparation represent two notable analytical properties. Acknowledgment. This work was partially supported by the Ministerio de Educacio´n y Ciencia of Spain through Projects CTQ2005-02816/BQU and BFM-2003-07749-C05-2, by Comunidad Auto´noma de Madrid-Universidad Auto´noma de Madrid through Project 12/TES/001. LA060184G