Anal. Chem. 2004, 76, 4665-4671
Articles
A Superoxide Sensor Based on a Multilayer Cytochrome c Electrode Moritz K. Beissenhirtz,†,‡ Frieder W. Scheller,†,‡ and Fred Lisdat*,†,§
Analytical Biochemistry, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, H. 25, 14476 Golm, Germany, International Max Planck Research School on Biomimetic Systems, Golm, Germany, and Biosystems Technology, Wildau University of Applied Science, Bahnhofstrasse 1, 15745 Wildau, Germany
A novel multilayer cytochrome c electrode for the quantification of superoxide radical concentrations is introduced. The electrode consists of alternating layers of cytochrome c and poly(aniline(sulfonic acid)) on a gold wire electrode. The formation of multilayer structures was proven by SPR experiments. Assemblies with 2-15 protein layers showed electrochemical communication with the gold electrode. For every additional layer, a substantial increase in electrochemically active cytochrome c (cyt. c) was found. For electrodes of more than 10 layers, the increase was more than 1 order of magnitude as compared to monolayer electrode systems. Thermodynamic and kinetic parameters of the electrodes were characterized. The mechanism of electron transfer within the multilayer assembly was studied, with results suggesting a protein-protein electron-transfer model. Electrodes of 2-15 layers were applied to the in vitro quantification of enzymatically generated superoxide, showing superior sensitivity as compared to a monolayerbased sensor. An electrode with 6 cyt. c/PASA layers showed the highest sensitivity of the systems studied, giving an increase in sensitivity of half an order of magnitude versus the that of the monolayer electrode. The stability of the system was optimized using thermal treatment, resulting in no loss in sensor signal or protein loading after 10 successive measurements or 2 days of storage. The quantitative detection of superoxide radicals and the characterization of the influence of antioxidants on their concentration are of great interest. The radical’s high reactivity to proteins, DNA, and lipid cell compartments and the fact that it is produced in the human body as a byproduct of several enzyme reactions give the background for its implication in diseases,1,2 * To whom correspondence should be addressed. E-mail: flisdat@ rz.uni-potsdam.de. Fax: (+49) 331-977 5053. † University of Potsdam. ‡ International Max Planck Research School on Biomimetic Systems. § Wildau University of Applied Science. (1) Fulbert, J. C.; Cals, M. J. Pathol. Biol. 1992, 40, 66-77. (2) Cadenas, E. Annu. Rev. Biochem. 1989, 28, 8. 10.1021/ac049738f CCC: $27.50 Published on Web 07/17/2004
© 2004 American Chemical Society
such as cancer,3,4 heart failure,5,6 neural diseases,7 and general aging processes.8,9 Sensorial detection of superoxide can further the understanding of the radical’s role in degenerative processes and optimize the use of antioxidants in food production, disease prevention, and therapeutics.10,11 The most critical factors for a quantification of superoxide radical concentrations are its short half-life (in the millisecond to second range) and the low concentrations in which it occurs in the body. Several methods of instrumental analysis have been applied to the study of superoxide radicals and their interaction with antioxidants, such as chromatography,12 EPR spin trapping,13 chemiluminescence dyes,14 and the quantification and repair of oxidative damage to biomolecules.15 One promising and relatively simple approach to the quantification of the superoxide radical both in vitro and in vivo is based on a promotor-modified gold electrode on which cytochrome c is immobilized.16,17 Superoxide radicals have been shown to reduce the protein, which can be reoxidized by the electrode at a suitable potential, leading to an oxidation current proportional to the superoxide concentration in solution. This current can serve as a sensor signal to determine the radical concentration in in vivo (3) Bostwick, D. G.; Alexander, E. E.; Singh, R.; Shan, A.; Qian, J. Q.; Santella, R. M.; Oberley, L. W.; Yan, T.; Zhong, W. X.; Jiang, X. H.; Oberley, T. D. Cancer 2000, 89, 123-134. (4) Poka, R.; Szucs, S.; Adany, R.; Szikszay, E. Eur. J. Obstet. Gynecol. Reprod. Biol, 2000, 89, 55-57. (5) Wattanapitayakul, S. K.; Bauer, J. A. Pharmacol. Ther. 2001, 89, 187-206. (6) Mak, S.; Newton, G. E. Chest 2001, 120, 2035-2046. (7) Leonard, B. E. Int. J. Dev. Neurosci. 2001, 19, 305-312. (8) Hensley, K.; Floyd, R. A. Arch. Biochem. Biophys. 2002, 397, 377-383. (9) Youdim, K. A.; Joseph, J. A. Free Radical Biol. Med. 2001, 30, 583-594. (10) Vendemiale, G.; Grattagliano, I.; Altomare, E. Int. J. Clin. Lab. Res. 1999, 29, 49-55. (11) Thomas, M. J. Crit. Rev. Food Sci. Nutr. 1995, 35, 21-39. (12) Zhang, W.; Danielson, N. D. Anal. Chim. Acta 2003, 493, 167-177. (13) Vasquez-Vivar, J.; Joseph, J.; Karoui, H.; Zhang, H.; Miller, J.; Martasek, P. Analusis 2000, 28, 487-492. (14) Sohn, H. Y.; Gloe, T.; Keller, M.; Schoenafinger, K.; Pohl, U. J. Vasc. Res. 1999, 36, 456-464. (15) Salles, B.; Sattler, U.; Bozzato, C.; Calsou, P. Food Chem. Toxicol. 1999, 37, 1009-1014. (16) Lisdat, F.; Ge, B.; Ehrentreich-Forster, E.; Reszka, R.; Scheller, F. W. Anal. Chem. 1999, 71, 1359-1365. (17) Cooper, J. M.; Greenough, K. R.; McNeil, C. J. J. Electroanal. Chem. 1993, 347, 267-275.
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experiments,18,19 as well as for a quantitative study of the reaction of a chosen antioxidant with superoxide in vitro.20 Advantages of this method include the possibility of an on-line, spatially resolved measurement at low cost, with little equipment and minimum disturbance of the sample. However, current sensor systems are limited by the amount of protein immobilized on the electrode surface. The study of such monolayer electrodes has shown that the sensitivity of the sensor is directly proportional to the surface density of protein on the electrode.21 Therefore, an increase in protein loading on the sensor surface could promise a significant improvement in sensitivity. To meet this goal, we have developed a new type of multilayer electrode consisting of an assembly of cytochrome c and a polyelectrolyte. The layer-by-layer technique introduced by Decher and co-workers22,23 is a useful tool to increase protein content on the electrode surface. Successive incubation steps of a solid substrate in solutions of polyions of alternating charge lead to the formation of multilayer structures stabilized by electrostatic interactions. This strategy has in the past been employed also for the immobilization of proteins. Campa`s and O’Sullivan have recently reviewed the application of such layer-by-layer approaches to biosensoring.24 For the application of redox proteins, however, an effective electron transport through the protein-polyelectrolyte assembly to the electrode is critical. For proteins such as hemoglobin,25,26 myoglobin,27,28 and Cyt P450cam,29 only 2-5 layers have been reported to be electroactive in such an arrangement. Thus, the questions of whether a further increase in electrode-addressable protein loading on the electrode is possible and how this increase determines the sensor’s response to analyte molecules interacting with the redox protein are obvious. In this study, multilayer assemblies of up to 15 layers of cytochrome c (cyt. c) and polyelectrolyte were constructed on a modified gold surface and characterized electrochemically and via surface plasmon resonance studies. The drastic increase on electroactive cyt. c reached by this technique will be discussed. The multilayer electrode was applied as a sensor for superoxide radicals and has shown a profound increase in sensitivity. The influence of the number of cyt. c/PASA layers on the sensitivity parameter will also be discussed. EXPERIMENTAL SECTION Materials. Horse-heart cytochrome c (cyt. c), bovine erythrocyte superoxide dismutase (SOD), and 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) were purchased from Sigma (18) Scheller, W.; Jin, W.; Ehrentreich-Forster, E.; Ge, B.; Lisdat, F.; Buttemeier, R.; Wollenberger, U.; Scheller, F. W. Electroanalysis 1999, 11, 703-706. (19) Buttemeyer, R.; Philipp, A. W.; Mall, J. W.; Ge, B. X.; Scheller, F. W.; Lisdat, F. Microsurgery 2002, 22, 108-113. (20) Lisdat, F.; Ge, B.; Reszka, R.; Kozniewska, E. Fresenius J. Anal. Chem. 1999, 365, 494-498. (21) Ge, B.; Lisdat, F. Anal. Chim. Acta 2002, 454, 53-64. (22) Decher, G.; Lehr, B.; Lowack, K.; Lvov, Y.; Schmitt, J. Biosens. Bioelectron. 1994, 9, 677-684. (23) Decher, G. Science 1997, 277, 1232-1237. (24) Campas, M.; O’Sullivan, C. Anal. Lett. 2003, 36, 2551-2569. (25) Shang, L. B.; Liu, X. J.; Zhong, J.; Fan, C. H.; Suzuki, I.; Li, G. X. Chem. Lett. 2003, 32, 296-297. (26) Wang, L. W.; Hu, N. F. Bioelectrochemistry 2001, 53, 205-212. (27) Ma, H. Y.; Hu, N. F.; Rusling, J. F. Langmuir 2000, 16, 4969-4975. (28) Li, Z.; Hu, N. F. J. Colloid Interface Sci. 2002, 254, 257-265. (29) Lvov, Y. M.; Lu, Z. Q.; Schenkman, J. B.; Zu, X. L.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080.
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(Steinheim, Germany). 11-Mercapto-1-undecanoic acid (MUA), 11mercapto-1-undecanol (MU), and poly(anilinesulfonic acid) (PASA) and poly(styrenesulfonic acid) (PSS) were provided by Aldrich (Taufkirchen, Germany). Xanthine oxidase (XOD) from cow milk was purchased from Roche (Mannheim, Germany). Uric acid and ascorbic acid were provided by VWR (Darmstadt, Germany). Gold wire electrodes with a diameter of 0.5 mm were purchased from Goodfellow (Bad Nauheim, Germany). Apparatus. All electrochemical experiments were performed in a custom-made measuring cell with 1-mL volume using an Ag/ AgCl/1 M KCl reference electrode (Biometra, Germany) and a Pt-wire counter electrode. All potentials reported here refer to this reference electrode. As the working electrode, a modified gold wire electrode was used. All cyclic voltammetric experiments were performed with the Autolab System (Methrohm, Germany). Amperometric measurements were conducted using a Model 720A electrochemical analyzer purchased from CH Instruments (UK). A Biacore 2000 (Biacore AB, Sweden) was used for SPR experiments. Buffers. Phosphate buffers were prepared from di-potassium phosphate and potassium di-phosphate, with the pH adjusted with potassium hydroxide or phosphoric acid, respectively. Buffer 1: 0.5 mM potassium phosphate, pH 5.0, was used for the preparation, washing, and storage of the multilayer systems. Buffer 2: 5 mM potassium phosphate, pH 7.0, served for the preparation of the cyt. c monolayer electrodes and for recording of cyclic voltammograms of the multilayer electrodes. For experiments at different pH values, the pH was adjusted with potassium hydroxide or phosphoric acid, where needed. Buffer 3: 5 mM potassium phosphate, pH 7.5, was used during the amperometric experiments and for the stock solutions of ascorbic acid and uric acid. Monolayer Electrode Preparation. Gold wire electrodes with a diameter of 0.5 mm were cleaned by boiling in 2 M KOH and successive incubation in concentrated H2SO4 (overnight) and concentrated HNO3 (10 min). A careful rinsing with Millipore water followed each successive step. The cleaned electrodes were incubated in a mixture of 5 mM mercaptoundecanioc acid and 5 mM mercaptoundecanol with a volume ratio of 1:3 for 48 h. After having been rinsed carefully with water, they were incubated in 500 µL of 20 µM cyt. c in 5 mM potassium phosphate (pH 7) for 2 h and finally rinsed carefully with water and buffer 2. Where specifically mentioned, the cyt. c molecules were covalently linked to the promotor layer by 30-min exposure to 2.5 mM EDC after preparation of the monolayer. Multilayer Electrode Preparation. Freshly prepared monolayer electrodes were successively incubated in PASA solution (0.2 mg/mL, 0.5 mM potassium phosphate, pH 5.0) and cyt. c solution (20 µM, 0.5 mM potassium phosphate, pH 5.0) for 10 min each, with five washing steps in buffer 1 in between. Incubation time was controlled by robotics. Apo-cyt. c was used in the same buffer where mentioned. Preparation of Apo-cyt. c. Apo-cyt. c was prepared by chemical removal of the heme group from the protein as described by Stellwagen and Rysavy.30 The lack of the heme group in the apo-cyt. c was verified by spectroscopy (380-440 nm). Cyclic Voltammetry. Cyclic voltammograms of the electrodes were recorded in buffer 2 at a scan rate of 100 mV/s if not noted (30) Stellwagen, E.; Rysavy, R.; Babul, G. J. Biol. Chem. 1972, 247, 8074-8077.
otherwise. Five scans between -350 and +350 mV were recorded in buffer 2 at pH 7.0, unless specified otherwise. The formal potential of the immobilized protein was calculated as the midpoint potential of oxidation and reduction peaks determined by the peak search function of the Autolab software (GPES Version 4.8). The heterogeneous electron-transfer rate constant ks was determined by studying the increase in peak separation with increased scanning speed (from 50 mV/s to 20 V/s) in cyclic voltammograms, following the method of Laviron.31 Amperometric Sensor Measurements. Nine hundred ninety microliters of 5 mM buffer 3 were pipetted into the measuring cell (volume, 1 mL). Under constant stirring, a potential of +150 mV (vs Ag/AgCl/1 M KCl) was applied and current recording was started on the Electrochemical Analyzer Software (CH Instruments, UK). Ten microliters of a hypoxanthine stock solution was added to result in a final concentration of 100 µM in the cell. Within 5-8 s after the beginning of current recording, a stable background current was established. Thus, the establishment of a stable baseline was marginally slower than the one found for a monolayer electrode. Only the first measurement after the preparation of the multilayer electrode showed slower kinetics before reaching a steady baseline (50-70 s). After establishment of such a stable background signal, XOD was added to a final activity of 20 mU/mL in the cell. The resulting oxidation current was measured as a function of time. At the end of the measurement, data was transferred to the software program Microcal Origin for evaluation. The sensor signal was calculated by subtraction of the background current from the steady-state signal obtained after XOD addition. The antioxidative properties of ascorbic acid were studied by triple additions of 1-5 µL of an ascorbic acid stock solution to the cell after establishment of the radical-induced oxidation current at the sensor electrode. The ascorbic acid concentration necessary for a 50% decrease of the radical’s current signal was calculated and termed IC50 analogous to enzyme inhibition studies. Possible interference by uric acid was tested by adding aliquots of a suitable stock solution to the sensor cell operating in the amperometric mode without XOD and HX present. SPR Measurements. A clean Biacore PIONEER Sensor Chip J1 was incubated in a mixture of 5 mM mercaptoundecanoic acid and 5 mM mercaptoundecanol with a volume ratio of 1:3 for 48 h. Then the chip was rinsed with Millipore water and mounted into the SPR flow system. Cyt. c and PASA solutions and buffer were used as described for the multilayer electrode preparation. The solutions containing cyt. c and PASA were successively pumped through the cell for 2.5 min each, with 10 min of buffer 1 in between, at a flow rate of 1 µL/min. The change of the resonance signal versus time was recorded. RESULTS AND DISCUSSION Characterization of the Formation of Structure. Surface plasmon resonance experiments were conducted to prove the formation of multilayers consisting of positively charged cytochrome c (cyt. c), and sulfonated polyaniline (PASA), which bears a negative net charge. An MUA/MU modified gold chip was flushed sequentially with a cyt. c and a PASA solution, intermitted by running buffer (0.5 mM potassium phosphate, pH 5.0)). One (31) Laviron, E. J. Electroanal. Chem. 1978, 101, 19-28.
Figure 1. SPR experiment showing successive deposition rounds of cyt. c and PASA on an MUA/MU (1:3)-modified gold chip. Conditions: 2.5 min of 20 µM cyt. c, 2.5 min of 1 mg/mL PASA, in 500 µM phosphate buffer, pH 5.0. Cyt. c addition at 150 s and PASA at 870 s. Numbers denote the subsequent deposition rounds.
example is given in Figure 1, showing that each alternating deposition round increases the surface loading. This rise in mass on the chip is due to the formation of a multilayer structure in which alternating layers of polyelectrolyte and protein bind to each other and thus are immobilized on the modified gold surface. The concentrations of the polyions, pH of the solution, and deposition times were varied to find optimum conditions for the design of a sensor electrode. Best results were obtained using 0.5 mM phopshate buffer at pH 5.0. At neutral pH or in buffers of higher ionic strength, no stable multilayer structures were observed. Concentrations higher than 0.2 mg/mL PASA and 20 µM cyt. c yielded no further gain in deposited material, while lower concentrations showed less increase in the SPR signal. Electrochemical Parameters of the Multilayer Electrode. The formation of a multilayer assembly is a precondition for the construction of a superoxide sensor. However, it has to be tested whether the protein molecules within the polyelectrolyte network can communicate electrochemically with the electrode. This is essential for transferring the information from the analyterecognition element interaction to the sensing electrode. To investigate this, the same assembly was built up on gold wire electrodes as was built on the SPR chips starting from a cyt. c monolayer electrode. A clean gold electrode was modified with the alkanethiol promotor and a monolayer of cyt. c, as described in previous studies.21 This modified electrode was then alternatingly incubated in solutions of PASA and cyt. c until the desired number of layers was deposited. The voltammetric investigation of the multilayer electrodes showed a quasi-reversible electron transfer between protein molecules immobilized within the assembly and the gold electrode. Cyclic voltammograms revealed a significant increase in oxidation and reduction peak currents with a growing number of layers, which corresponds to an increase in the amount of electroactive cyt. c molecules immobilized on the surface with each deposition round. Figure 2 displays cyclic voltammograms of several such multilayer electrodes. From an integration of the peak currents, the amount of cyt. c communicating with the electrode was calculated (inset in Figure 2), showing a large increase with Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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Figure 2. Cyclic voltammograms of multilayer electrodes of PASA and cyt. c. In increasing order: monolayer, 4, 6, 8, 12, and 15 layers of cyt. c. Measuring conditions: 5 mM phosphate buffer, pH 7.0, scan rate 100 mV/s, reference electrode Ag/AgCl, 1 M KCl. Inset: Amount of electroactive cyt. c immobilized in 2-15 layers of polyelectrolyte and cyt. c. Error bars were derived from averaging five electrodes per data point.
each additional protein layer. These results show that the cyt. c molecules in the protein/PASA network can exchange electrons with the electrode, even when they are located in greater distance from the electrode surface. Control experiments in which either of the charged species was substituted by buffer showed a cyt. c loading on the surface no different than that for a monolayer electrode, proving the necessity of both compounds for the construction of the multilayer structure. The formal potential of the protein for up to 15 layers, E0′, was found to be independent of the number of layers. The value for all multilayer electrodes investigated was -15 ( 7 mV vs Ag/ AgCl/1 M KCl, which is within the error of the value previously obtained for Au-MUA/MU-cyt. c electrodes (-19 ( 5 mV). The cyclic voltammograms also showed an increase in the peak width at half peak height from 128 ( 7 mV for a monolayer electrode to 188 ( 11 mV for the multilayer electrode, disregarding the number of layers deposited. This can be explained by the differing microenvironments of the protein molecules bound within the multilayer assembly, resulting in a distribution of redox states. A variation of the scan rate was performed to investigate the kinetics of electron transfer. With increased scan rate, the peak separation grew, as to be expected for an immobilized protein. However, the amount of electrode-addressable protein changed drastically, as shown in Figure 3. While between 50 and 100 mV/s the surface density of electroactive cyt. c remained constant, an increase of scanning speed to 125 mV/s already caused a loss of electron transport to 40% of the proteins electrochemically active at lower scan rates. This tendency continued with an exponential dependency, until at 10 V/s only 10% of the cyt. c molecules showed communication with the electrode. This behavior strongly suggests that a scan rate of more than 125 mV/s surpasses the minimum rate of electron transfer within the multilayer assembly. While for a monolayer electrode a ks value of 75 ( 3 s-1 could be calculated by the method of Laviron,31 the electron-transfer rate 4668 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
Figure 3. Surface density of electrode-addressable cyt. c within a multilayer electrode at different scan rates. Density is given in percent of the value at 0.1 V/s, which is set at 100%. Filled squares, 4-layer electrode; empty squares, 8-layer electrode.
Figure 4. Peak separation of mono- and multilayer electrodes at varying pH (100 mV/s, 5 mM phosphate buffer). Filled squares, 4-layer electrode; empty squares, 8-layer electrode. Circles: 2 Au-MUA/MUcyt. c monolayer electrodes.
within the multilayer assembly is significantly lower and changes with distance to the electrode surface. On the basis of these measurements, we estimated a minimum ks for the protein molecules within the bulk of the network to be 1.5 ( 0.3 s-1. The potential mechanism behind this will be discussed below. A quantification of the electrochemical behavior at different pH values showcased further differences between cyt. c molecules immobilized in mono- and multilayer electrodes. While the formal potential for both systems decreased in a similar way with rising pH values, the peak separation for a cyt. c monolayer remained constant between pH 5 and pH 9. Peak separation is connected with the rate of electron transfer and was found to be pHdependent for the multilayer electrode. It showed a minimum at pH 7, with values increasing toward both higher and lower pH. This is depicted in Figure 4. The influence of proton concentration on the redox properties of such a multilayer system can be understood to be due to the fact that both polyions are bound to one another by electrostatic interactions caused by their opposite net charges. A pH difference in the solution could be expected to influence the inner structure of the system, resulting in a changed flexibility or differing
microenvironment for the redox protein. Since an increase in peak separation (as found at a pH higher or lower than 7) corresponds to slower redox kinetics, thus the electron-transfer rate constant of the multilayer electrode is pH-dependent and is at its highest when working at neutral pH. Electron-Transfer Mechanism within the Multilayer Assembly. On the basis of these findings, the question arose for the background of the efficient electron transfer within the cyt. c/PASA network. Since no mediator was present and the voltammograms showed no formation of redox-active species or catalytic current, basically two mechanisms have to be taken into account: Either the polyelectrolyte may act as a “wire” along which the electrons can be transported, since under certain conditions sulfonated polyanilin can behave as conducting polymers,32,33 or a protein-protein electron transfer takes place in which the polyelectrolyte acts as a stabilizer of the structure without being involved in redox reactions. To test these hypotheses, apo-cyt. c was used to replace some cyt. c in the PASA/protein assembly. Apo-cyt. c contains no heme and can therefore not participate in cyt. c redox reactions. Thus, an electrode was constructed based on a cyt. c monolayer electrode which was covered by 8-12 layers of apo-cyt. c/PASA followed by an additional 4 layers of cyt. c/PASA. The resulting voltammograms showed no oxidation and reduction peaks and thus the loss of electrochemical communication between protein and electrode. These findings show that a sufficient barrier of heme-free protein can inhibit the electron transfer between outerlayer cyt. c and the electrode surface. Since SPR studies showed that apo-cyt. c and PASA interact in the same manner as cyt. c and PASA, this prevention of electrochemical communication suggests that the heme centers of the assembly play a vital role in electron transfer. Experiments with a cyt. c monolayer electrode and cyt. c in solution verified a reaction of immobilized cyt. c with the freely diffusible protein. However, precondition for such a mechanism within the multilayer assembly is a rotational mobility of cyt. c since a face-to-face orientation is considered as the most efficient pathway for electron transfer with cyt. c reaction partners. An approach to limit the flexibility of the assembly is the interconnection of the building blocks by a cross-linking agent. In this study EDC was used to activate carboxylic groups and interconnect them with available amino groups within the assembly. As a result, a clear decrease in the electrode-addressable amount of cyt. c was found. One example is given in Figure 5. Additionally, the peak separation increased drastically, indicating a much slower electron transfer within the EDC-treated assembly. This drop in electron-transfer rate would again point to an active involvement of cyt. c in the electron transport. Rotational flexibility of cyt. c in the immobilized state has been reported in the literature.34 Recently, the electron transfer to the protein in a monolayer arrangement could be enhanced by preventing protein rotation using a surface complexing agent.35 (32) Wei, X. L.; Wang, Y. Z.; Long, S. M.; Bobeczko, C.; Epstein, A. J. J. Am. Chem. Soc. 1996, 118, 2545-2555. (33) Yue, J.; Wang, Z. H.; Cromack, K. R.; Epstein, A. J.; Macdiarmid, A. G. J. Am. Chem. Soc. 1991, 113, 2665-2671. (34) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766. (35) Wei, J. J.; Liu, H. Y.; Dick, A. R.; Yamamoto, H.; He, Y. F.; Waldeck, D. H. J. Am. Chem. Soc. 2002, 124, 9591-9599.
Figure 5. Cyclic voltammograms of 8-layer electrodes (Au-MUA/ MU-cyt. c-[PASA/cyt. c]8) in 5 mM potassium phosphate buffer, pH 7.0 (scan rate 100 mV/s). Continuous line, cross-linked by a 30-min exposure to EDC; dotted line, not cross-linked.
These all may give some arguments for a protein-protein interaction. However, further investigations will be necessary. Application as a Superoxide Radical Sensor. The electrochemical properties detailed above warrant an application of the multilayer electrode for detection of superoxide radicals. The formal potential of cyt. c is not much shifted and the electrontransfer rate is still high enough for an efficient reoxidation of the protein molecules. Thus, amperometric experiments were conducted to investigate the multilayer assemblies’ response to the radical. The electrodes were polarized at +150 mV (vs Ag/ AgCl/1 M KCl), and the xanthine oxidase-catalyzed oxidation of hypoxanthin was used to generate superoxide. Before the start of the enzymatic production of radicals, a constant background current of a few nanoamperes was recorded, which was rather similar compared to that of the monolayer electrode. After the start of superoxide generation, the current rose sharply and reached a maximum plateau, which remained unchanged for several minutes. Such a constant radical concentration has been found for XOD-catalyzed superoxide production and is explained as a steady-state between generation and dismutation of the radical proceeding at an equal rate. With beginning depletion of the substrate (HX), the current signal decreased. These measurements showed that the protein in the multilayer electrode was reduced by the radical and subsequently reoxidized by the electrode, proving the functioning of the signal chain: radical-protein-electrode. Since the signal could be completely depressed by SODsthe most effective scavenger of superoxides it is clear that the multilayer electrode responded to the superoxide radical only and is not influenced by other reaction products present. To clarify the concentration range of interference-free measurements with uric acid present, additions of this reducing substance to the solution have been investigated in the absence of superoxide. No influence on the sensor current was found for concentrations of up to 2 mM. Ascorbic acid is an effective superoxide scavenger. Thus, it was tested whether the scavenging reaction can be followed by the multilayer electrode. For these measurements, ascorbic acid was added to the solution with the superoxide generating system, and the decreased superoxide sensor current was evaluated. The IC50 (50% inhibition of the superoxide signal) was determined to Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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Figure 6. Sensor response of mono- and multilayer electrodes to superoxide. 6-layer electrode (a), 10-layer elecrode (b), and monolayer electrode (c). Amperometric measurement at +150 mV vs Ag/ AgCl/1 M KCl. Conditions: 20 mU/mL XOD, 100 µM HX, 5 mM phosphate buffer, pH 7.5.
Figure 7. Dependency of the superoxide response of multilayer electrodes on the number of cyt. c/PASA layers. 0 layers denotes the Au-MUA/MU-cyt. c monolayer electrode. Conditions: 20 mU/mL XOD, 100 µM HX, 5 mM phosphate buffer, pH 7.5.
be 2 ( 0.3 µM, which was comparable with the analysis using the monolayer electrode under the same experimental conditions (2.2 ( 0.3 µM). These experiments show that the multi- and monolayer electrodes behave rather similarly toward the analyte, interfering substances, and antioxidants. This can be explained by the use of the same recognition element (cyt. c) and the same blocking layer (mercaptoundecanoic acid/mercaptoundecanol) on top of the gold electrode for both types of sensors. Typical amperometric sensor curves are depicted in Figure 6. Compared to the monolayer electrode, the new type of sensor showed a strong increase in sensitivity for all multilayer systems investigated (see Figure 7). The change of the sensor’s response to a constant superoxide concentration with an increasing amount of cyt. c deposited followed a defined dependency. Between 2 and 6 layers, each additional deposition round yields a substantial and linear increase in the sensor signal, which is at its highest for electrodes of 6 layers. Electrodes containing more than 6 cyt. 4670 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
Figure 8. Stability of 6-layer sensor electrodes determined as the peak charge of electroactive cyt. c in voltammetric experiments between sequential amperometric measurements. Charge is given in percent of value before the first amperometric measurements, which is set at 100%. Gray columns: electrodes incubated at 45 °C for 30 min before application. White columns: no thermal treatment after layer deposition.
c/PASA layers showed no further signal enhancement, but a tendency for a gradual decline in sensitivity. These experimental findings support on one hand the idea of increasing sensitivity with an increasing amount of the electroactive recognition element. However, since not all the protein molecules can participate in the reaction with the radical at higher protein loadings, the experiments show on the other hand that accessibility of the recognition element by the analyte is also important. For electrodes with more than 6 layers, an increasing amount of protein is obviously not taking part in the reaction with superoxide radicals. This may be due to the short half-life time of the radical and the reduced permeability for diffusion of superoxide through the increasingly thick protein/polyelectrolyte assembly on the electrode. A 6-layer electrode provided the highest sensitivity compared to the monolayer system. Obviously, with this arrangement a compromise between the accesibilty from the electrode side (electron transfer) and the solution side (reaction with the analyte) was reached. The sensor response to superoxide was investigated in the concentration range between 0.4 and 1.5 µmol/L. The sensor could follow the different radical levels in solution. The sensitivity can be given as 0.398 A/cm2 mol, which is 5 times higher than the one found for a cyt. c monolayer electrode. Stability of the Multilayer Electrodes. A few amperometric measurements at constant XOD activity showed no decrease in signal. However, since the multilayer assembly is stabilized only by electrostatic interactions and not a covalent bond, as is the case for the monolayer electrode, overnight storage or a higher number of superoxide measurements inevitably resulted in a loss of protein from the electrode. Thus, several attempts were made to improve the stability of the system. The addition of cross-linking agents such as EDC and glutaraldehyde caused a massive change of electrochemical behavior as described above. These drastic influences, particularly in the rate of electron transfer, disqualify such an attempt of stabilization for sensor application.
A more successful strategy was developed by exposing the electrodes to a higher temperature after the multilayer deposition and prior to their sensor application. Electrodes which were incubated in a buffer solution at 45 °C for 30 min showed no loss of protein after overnight storage or 10 successive amperometric measurements at +150 mV (see Figure 8), while proving equally capable at superoxide measuring. Higher temperatures resulted also in stable systems but with a drastic loss in electroactivity and electron-transfer rate. The effect of temperature treatment at 45 °C may cause structural rearrangements, which provides a better interaction of both building blocks of the assembly, but still allows for a rotational flexibility of the cyt. c and thus appears as an optimum for the construction of a multilayer-based sensor. CONCLUSIONS In this work, a new type of superoxide sensor electrode has been developed. The study of previous monolayer cyt. c electrodes showed that the sensitivity of the sensor was directly proportional to the amount of protein immobilized on the sensor surface. Therefore, multilayer structures of cyt. c and PASA were designed to increase the surface density of electroactive cyt. csthe recognition element for superoxide radicals. These multilayer electrodes showed an increase in electrochemically active protein with every layer of cyt. c deposited. With a 10-layer electrode, the content of cyt. c immobilized was found to be more than an order of magnitude higher than that in a monolayer system. The formal potential remained unchanged as
compared to the monolayer electrode, while the half peak width increased and the electron-transfer rate constant was lower. Several experiments suggest that the electron pathway through the layers to the electrode surface is mainly based on proteinprotein electron transfer. The multilayer assembly of cyt. c and PASA on modified gold electrodes was applied to measurements of superoxide radicals. The electrodes yielded a significant increase in sensitivity to the radical as compared to monolayer electrodes. Besides an efficient electron transfer of a large number of protein molecules (high surface density of electroactive cyt. c), also accessibility of the protein molecules by the short-lived analyte was found to be determining the electrodes’s response to the radical. Thermal treatment of the multilayer assembly offered sufficient electrode stability for serial sensor applications and storage. Thus, an electrode was constructed which can follow the superoxide concentration in a sensitive, selective, and stable manner. ACKNOWLEDGMENT The authors would like to thank the Bundesministerium fu¨ r Bildung und Forschung Germany (0311487A) and the Ministerium fu¨ r Forschung, Wissenschaft und Bildung Brandenburg (24#259804*327) for financial support.
Received for review February 16, 2004. Accepted May 13, 2004 AC049738F
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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