Letter pubs.acs.org/Langmuir
Electroactive Nanobiomolecular Architectures of Laccase and Cytochrome c on Electrodes: Applying Silica Nanoparticles as Artificial Matrix Sven Christian Feifel,* Andreas Kapp, and Fred Lisdat* Biosystems Technology, Institute of Applied Sciences, Technical University of Applied Sciences, 15745 Wildau, Germany S Supporting Information *
ABSTRACT: Fully electroactive multilayer architectures combining the redox protein cytochrome c and the enzyme laccase by the use of silica nanoparticles as artificial matrix have been constructed on gold electrodes capable of direct dioxygen reduction. Laccase form Trametes versicolor and cytochrome c from horse heart were electrostatically coimmobilized by alternate deposition with interlayers of silica nanoparticles in a multilayer fashion. The layer formation has been monitored by quartz crystal microbalance. The electrochemical properties and performance of the nanobiomolecular entities were investigated by cyclic voltammetry, indicating, that a multistep electron transfer cascade, from the electrode via cytochrome c in the layered system toward the enzyme laccase, and here to molecular dioxygen was achieved. The response of the novel architecture is based on direct electron exchange between immobilized proteins and can be tuned by the assembly process.
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sequential reduction of cyt c, laccase, and dioxygen.12 However, in this case, only one layer was in contact with the multilayer system, and laccase has not been embedded in the assembly. Additionally, several investigations have been directed to the immobilization and electron transfer of laccase on carbon materials13−18 or gold19−22 in a monolayer state or by crosscoupling reactions for the development of bioconjugated materials.23−25 Moreover, the combination of nanoparticles with biomolecules on electrodes is a matter of particular interest since several examples with direct electron transfer have been found.26,27 In a different direction, the application of silica nanoparticles (SiNPs) can be seen here. They have been used in biosensors due to the high biocompatibility and the high surface area of the nanosized particles resulting in highly active surface loading of proteins and thus to a high performance of the biosensor.26,28 However, so far, laccase has not been embedded with another redox protein like cyt c in a nonconducting artificial matrix composed of carboxy-modified nanosized SiNPs and assembled in multiple layers to study the electron transfer chain in such a nanobiomolecular environment.
INTRODUCTION Reactions between metalloproteins have been a subject of interest because of their biological importance. Respiratory and photosynthetic chains are composed of a series of specific and directional electron transfer reactions that include several proteins.1 Although electron transfer between inherent redox partners is naturally important, the reactions between metalloproteins, which are not intrinsic redox partners, have also gained significant interest since they allow the construction of new functional systems. In line with this, the reactions between the heme proteins, cytochromes, and the blue copper proteins, azurin, stellacanin, plastocyanin,2−4 and laccase,5 have been examined kinetically. Laccases belong to the class of blue multicopper enzymes responsible for the four-electron reduction of molecular dioxygen to water without formation of reactive intermediates, and therefore they are of great interest as catalysts in biofuel cells and for biosensor applications.6 Much of the research on laccase (Lac) has focused on defining the structure of its complex active site and the four-electron-reduction process at the trinuclear center.7 Further investigations targeted the interaction of laccase with other proteins in solution,8,9 as, e.g., cytochrome c (cyt c)5 a small heme redox protein. Although Lac and cyt c are not natural reaction partners, it was shown that Lac can accept cyt c as an electron donor delivering electrons for oxygen reduction.5,9 The driving force for the oxidation of cyt c is due to the difference in the redox potential of the two proteins.10,11 Later on, laccase has been immobilized on top of a polyelectrolyte/cyt c multilayer film, and it has been proofed that the appearance of a catalytic current is consistent with © 2014 American Chemical Society
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EXPERIMENTAL SECTION
Fabrication of Nanobiomolecular Laccase·Cyt c/SiNPs Assemblies: Modified Electrodes. The bare gold-wire electrodes are cleaned by incubation in piranha solution (3:1 H2SO4/H2O2) three times for 10 min. The electrodes are washed with Millipore water after Received: February 3, 2014 Revised: May 6, 2014 Published: May 7, 2014 5363
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the cleaning steps. For the construction of nanobiomolecular layers, the electrodes are modified by incubation for 48 h in 5 mM 3:1 solution of mercaptoundecanol (MU)/mercaptoundecanoic acid (MUA). Laccase (Lac), cyt c, and SiNPs are immobilized by chemophysical adsorption. The cyt c monolayers have been prepared by adsorption in a solution of cyt c (30 μM) in potassium phosphate buffer (5 mM, pH 7) on gold wire electrodes previously modified with a solution of MUA/MU (1:3) in ethanol (5 mM) for 48 h.2 For the following nanobiomolecular assembly consisting of cyt c, laccase, and SiNPs, a premixed protein solution (cyt c, Lac) was made in potassium phosphate buffer (5 mM, pH 7), which contained cyt c (20 μM) and Lac (1 μM) in 20:1 ratio. The subsequent assembly of [SiNPs/Lac·cyt c] nanobiomolecular layers has been performed by alternating incubation steps of a cyt c monolayer electrode in premixed-Lac·cyt c (1:20) and SiNPs (5.0 mg·mL−1) solutions for 10 min per step. Each of the 10 min-long adsorption steps in solutions of SiNPs (5.0 mg· mL−1) and Lac·cyt c (1:20) was followed by rinsing the electrodes with 5 mM potassium phosphate buffer pH 7 (to remove loosely bound material). The incubation procedures were repeated until the desired number of layers was reached.
Figure 1. Schematic depiction of the structure and proposed electron transfer cascade of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac] multilayer electrode. The cyt c monolayer is prepared on a mixed thiol layer (MUA/MU). The multiple layers are composed of [SiNPs/cyt c·Lac]n (n = 1, 2, 3, 4, 5, 6 bilayers).
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RESULTS AND DISCUSSION In order to integrate electroactive laccase in a nanobiomolecular architecture on electrodes we constructed multilayer systems composed of laccase and cyt c hosted by an artificial silica nanoparticle-based matrix. We selected cyt c, Lac, and SiNPs on the basis of following considerations: First SiNPs can be synthesized in a variety of sizes, and characterized by a narrow size distribution; Second, silica is a dielectric material, which does not adsorb light or conduct electrons; as inert host, it already enabled the assembly of biological materials (e.g., cyt c, CDH) while keeping its natural function.29,30 Finally, the pI values of carboxy-modified silica colloids, Lac, and cyt c are acidic and basic, respectively, and therefore, sufficient Coulomb interaction can be expected between the SiNPs (−), Lac (−) and the oppositely charged cyt c (+) at neutral pH, which allows layer formation by electrostatic interactions. Therefore, Lac, cyt c, and nonconducting carboxy-modified SiNPs were assembled on a cyt c monolayer by alternating incubation steps in solutions of SiNPs and a defined mixture of Lac/cyt c. A schematic representation of the suprananobiomolecular entity and the proposed electron transfer steps in this system are shown in Figure 1. As a starting point for the formation of the protein assembly, we used a monolayer electrode consisting of cyt c adsorbed on a mixed self-assembled alkanthiol layer of mercaptoundecanoic acid (MUA) and mercaptoundecanol (MU). The electrostatic interactions between the negatively charged electrode and the positively charged protein (cyt c) permits the immobilization of a first layer of cyt c without blocking its mobility in a surface fixed manner and thus providing a good communication not only with the electrode but also with the proteins assembled on top. The arrangement makes use of the different net-charges of the proteins. As laccase from Trametes versicolor possess a low negative net-charge (pI 4.3) and cytochrome c from horse heart a high positive one (pI 10.0) at pH 7, this can be used to form precomplexes of cyt c/Lac in a defined mixture, with an excess of cyt c to interact with the negative charges on the SiNPs surface. The formation of the entity with cyt c, Lac, and SiNPs on a gold surface was confirmed by quartz crystal microbalance (QCM) experiments, which indicate a defined binding of the components to the surface (see Supporting Information, Figure S1). By definition, an arrangement composed of two immobilization steps (SiNP, Lac/cyt c) is termed a bilayer.
To investigate whether laccase coimmobilized in a SiNPs matrix in multiple layers on electrodes is able to reduce molecular dioxygen to water, the voltammetric response of a multilayer electrode consisting of 6-bilayers of SiNPs/Lac·cyt c was recorded in the presence and absence of dioxygen. From Figure 2, an increase in the catalytic reduction current in the presence of air saturated solution can be seen. The
Figure 2. Cyclic voltammograms of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac]6 multilayer electrode in nitrogen (in black) and air (in red) saturated buffer (20 mM phosphate-citrate buffer pH 4.5). Scan rate 3 mV/s, surface area of the electrode A = 0.0471 cm2. Reduction current is assessed at a potential of −200 mV.
dioxygen reduction process starts at a potential where cyt c is reduced. This demonstrates that the immobilized Lac molecules in the multilayer architecture are active and can be electrochemically addressed because they accept the immobilized cyt c molecules in the different protein stacks as electron donor. In certain circumstances cyt c may also be able to reduce molecular dioxygen albeit only to a small extent, depending on the given environment of cyt c. To evaluate a possible contribution of cyt c molecules in our Lac/cyt c-architecture for dioxygen reduction, a multilayer consisting only of cyt c and 5364
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SiNPs has been constructed and investigated in air-saturated buffer by CV (Figure 3).
Figure 4. Cyclic voltammograms of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac]1−6 multilayer electrodes in air saturated buffer (scan rate 3 mV/ s, surface area of the electrode A = 0.0471 cm2, 20 mM phosphatecitrate buffer pH 4.5). Catalytic reduction currents are assessed at a potential of −200 mV. 1−6 denotes the number of bilayers on the electrode. Figure 3. Cyclic voltammogram of a Au-MUA/MU-cyt c-[SiNPs/cyt c]6 multilayer electrode in air saturated buffer (scan rate 3 mV/s, surface area of the electrode A = 0.0471 cm2, 20 mM phosphate-citrate buffer pH 4.5). Inset: comparison of the reduction currents of a [SiNPs/cyt c]6 and [SiNPs/cyt c·Lac]4 multilayer electrode.
Table 1. Catalytic Currents of Laccase and Cyt c Concentrations Are Given for Nanobiomolecular Architectures of 1−6 Bilayersa
The control experiment clearly demonstrates that the contribution of cyt c to the bioelectrocatalysis is only marginal for this system (Figure 3). For a six-bilayer assembly of pour SiNPs/cyt c layers, only a current of maximum 8 nA (at a potential of −200 mV vs. Ag/AgCl) can be observed compared to about 62 nA for a SiNPs/cyt c·Lac system; this proves the proposed role of laccase in the layered architecture to be the main player for catalytic dioxgen reduction. To gain insight of addressable Lac molecules in the layered entity electrodes with different numbers of SiNPs/cyt c·Lac layers have been examined by CV (Figure 4). Subsequently, the catalytic reduction current of the electrodes was investigated by determining the difference in the reduction current in the presence and absence of dioxygen. On the basis of the recorded CV data, it can be shown that, by raising the number of layers, the catalytic current increases almost proportionally (Table 1). This means that by an increase of catalytic sites (amount of laccase), the bioelectrocatalytic conversion of dioxygen can be enhanced. It is also worth mentioning that, not only does the catalytic current rise with the number of layers, but also the amount of electrochemically addressable cyt c molecules in the system. This is of significant importance since cyt c is not only acting as an electron donor but also as electron transfer molecule based on self-exchange (Figure 5). From the experiments it can be also concluded that the concept of a layered deposition of active proteins was successful. The verifiable increase of the catalytic current and the amount of electroactive cyt c with the growing number of deposited layers provides evidence for an efficient signal chain through the entire nanobiomolecular system for the reduction of dioxygen. It also implicates that the laccase molecules deposited in different stacks are in electrical contact with the electrode via cyt c. Furthermore, it can be stated that the multilayer arrangement is useful to tune the sensitivity by the amount of assembled biocatalyst (Lac).
nanobiomolecular protein electrodes
number of bilayers (n)
cyt c monolayer [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n
1 2 3 4 5 6
cytochrome c concentration (pmol/cm2) v = 100 mV/s 13 25 30 39 51 56 67
± ± ± ± ± ± ±
1 1 2 3 1 4 3
catalytic current (nA) for air saturation at −200 mV vs Ag/AgCl v = 3 mV/s
current densities (nA/cm2) v = 3 mV/s
± ± ± ± ± ±
339 ± 63 509 ± 21 658 ± 42 955 ± 63 1082 ± 42 1316 ± 105
16 24 31 45 51 62
3 1 2 3 2 5
a
Cyt c concentrations are determined by calculating the peak area in CV (without substrate). Catalytic reduction currents are measured in air-saturated buffer.
Therefore, significant progress has been accomplished compared to a previously described system12 where only one layer of laccase was in contact with one outer layer of a cyt c/ PASA multilayer and has not been integrated into the entity. The nonconducting SiNPs within the nanobiomolecular assembly act as a framework in which the redox protein cyt c and the enzyme Lac are embedded and held together by electrostatic interactions in such a way that cyt c and Lac are still in close proximity to each other so that direct ET between the enzyme and the redox protein and between the redox proteins is feasible. Multilayer formations without SiNPs could not be observed, which in turn demonstrates that they are needed as scaffold for the construction of electroactive protein assemblies. In a further step of characterization, the stability of the prepared multilayer architecture has been investigated. The findings reflected a rather good stability of the multilayer structure during the measurement over a day. No leakage of the proteins has been found during a day. If the multilayer 5365
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OUTLOOK In conclusion, we have shown that the construction of an electroactive nanobiomolecular architecture by the use of laccase as biocatalyst, cyt c as an electron-shuttle, and SiNPs acting as an artificial scaffold is feasible. The construction of the entity is based on a self-assembly process, whereby carboxymodified SiNPs have been used to integrate laccase in a fully electroactive layered assembly with a high amount of cyt c. The nanobiomolecular entity displays a specific catalytic activity for the reduction of molecular dioxygen. Cyt c operates as a solid-state electron donor in the multilayered architecture, transferring electrons effectively from the electrode by DET and IET toward the enzyme. The system is also an example for direct electron exchange between different proteins in a surfacefixed state. It needs to be emphasized that this architecture does not demand diffusional redox mediators, which makes this entity highly valuable for the development of third-generation biosensors. This approach is expected to have a considerable impact on the development of further nanosized biprotein systems where specific biocatalysts are supposed to react with heme-containing proteins. In addition, the protein multicomponent films are interesting as novel biologically active materials. It shows that new functionalities can be prepared by arranging electroactive biomolecules in such a way that efficient communication and defined signal generation can take place.
Figure 5. Cyclic voltammograms of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac]1−6 multilayer electrodes. The increase of the addressable cyt c is shown with increasing number of layers (1−6), compared to a monolayer (M) electrode (scan rate 100 mV/s, surface area of the electrode A = 0.0471 cm2, 5 mM potassium phosphate buffer, pH 7).
electrodes have been stored at 4 °C for 1 week, a decrease (≤40%) in the catalytic activity can be observed. For the electron transfer pathway we propose that the catalytic current results from reduction of cyt c, which is enhanced by subsequent oxidation by neighboring laccase molecules. Dioxygen reduction near the potential of cyt c conversion verifies the proposed functionality of the protein architecture. Thus, a signal chain can be concluded, which starts at the electrode, where cyt c is reduced and the electrons are transferred via cyt c−cyt c interprotein ET to the T1 center of laccase and from there to the trinuclear copper-cluster at which the electrons are used for molecular O2 reduction to produce water (Figure 1). To evaluate the sensitivity of the system, the multilayered electrodes have been analyzed by cyclic voltammetry at different dioxygen concentrations (see Supporting Information, Figure S2). The collected CV data show a relatively linear dependence of the reduction current on the dioxygen content in solution. Additionally, the nanobiomolecular architecture is able to follow the enhanced dioxygen conversion at the laccase even for higher dioxygen concentrations, which suggests that the overall current is not limited by the reactions between the protein molecules or cyt c and the electrode. As already mentioned above, the enzyme and the redox protein have different functions within the architecture. Therefore, the relative amount of Lac and cyt c in each layer should have a profound effect on dioxygen reduction. When the cyt c/Lac ratio is decreased from 20:1 to 10:1, we observe a significant decrease in the catalytic response (data not shown). Therefore, although the number of recognition sites for dioxygen is increased (larger amount of Lac), the delivery of electrons to the enzyme becomes limiting, and thus the activity is diminished. Since the electrons need to be transferred from cyt c to cyt c by self-exchange through the layered architecture and from cyt c to Lac to ensure dioxygen reduction, the proposed role of cyt c as a solid-state electron donor is again supported. When high concentrations of Lac are present, the electron pathway from cyt c to cyt c seems to be restricted.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures, Chemicals, QCM experiments. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(S.C.F) E-mail: [email protected]. Phone: +49(0)3375-508158. Fax: +49(0)3375-508-458. *(F.L) E-mail: fl[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the DFG, LI706I7-1. We are grateful to D. Schäfer for technical assistance in the laboratory.
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ABBREVIATIONS Cyt c, cytochrome c; SiNPs, silica nanoparticles; Lac, laccase; DET, direct electron transfer; ET, electron transfer; IET, interprotein electron transfer
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REFERENCES
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(3) Wherland, S.; Pecht, I. Protein−protein electron transfer: A Marcus theory analysis of reactions between c-type cytochromes and blue copper proteins. Biochemistry 1978, 17, 2585−2591. (4) Dronov, R. V.; Kurth, D. G.; Scheller, F. W.; Lisdat, F. Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold. Electroanalysis 2007, 19, 1642−1646. (5) Sakurai, T. Kinetics of electron transfer between cytochrome c and laccase. Biochemistry 1992, 31, 9844−9847. (6) Shleev, S.; Tkac, J.; Christenson, A.; Ruzgas, T.; Yaropolov, A. I.; Whittaker, J. W.; Gorton, L. Direct electron transfer between coppercontaining proteins and electrodes. Biosens. Bioelectron. 2005, 20, 2517. (7) Reinhammer, B. Purification and properties of laccase and stellacyanin from Rhus vernicifera. Biochim. Biophys. Acta 1970, 205, 35−47. (8) Zhao, D.; Wang, Y.; Zhao, M. Bioelectrochemistry of laccase. Prog. Chem. 2011, 23, 1224−1236. (9) Jin, W.; Wollenberger, U.; Bier, F. F.; Makover, A.; Scheller, F. W. Electron transfer between cytochrome c and copper enzymes. Bioelectrochem. Bioenerg. 1996, 39, 221. (10) Bourbonnais, R.; Leech, D.; Paice, M. G. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim. Biophys. Acta 1998, 1379, 381. (11) Correia dos Santos, M. M.; Paes de Sousa, P. M.; Simoes Goncalves, M. L.; Lopes, H.; Moura, I.; Moura, J. J. G. Electrochemical studies on c-type cytochromes at microelectrodes. J. Electroanal. Chem. 1999, 464, 76. (12) Balkenhol, Th.; Adelt, S.; Dronov, R.; Lisdat, F. Oxygenreducing electrodes based on layer-by-layer assemblies of cytochrome c and laccase. Electrochem. Commun. 2008, 10, 914−917. (13) Bourourou, M.; Elouarzaki, K.; Lalaoui, N.; Agnes, C.; Le Goff, A.; Holzinger, M.; Maaref, Ab.; Cosnier, S. Supramolecular immobilization of laccase on carbon nanotube electrodes functionalized with (methylpyrenylaminomethyl)anthraquinone for direct electron reduction of oxygen. Chemistry 2013, 19 (28), 9371−9375. (14) Deng, L.; Shang, L.; Wang, Y. Z.; Wang, T.; Chen, H. J.; Dong, S. J. Multilayer structured carbon nanotubes/poly-L-lysin/laccase composite cathode for glucose/O2 biofuel cell. Electrochem. Commun. 2008, 10, 1012−1015. (15) Zheng, W.; Zho, H. M.; Zheng, Y. F.; Wang, N. A comparative study on electrochemistry of laccase at two kinds of carbon nanotubes and its application for biofuel cell. Chem. Phys. Lett. 2008, 457, 381− 385. (16) Lalaoui, N.; Elouarzaki, K.; Le Goff, A.; Holzinger, M.; Cosnier, S. Efficient direct oxygen reduction by laccases attached and oriented on pyrene-functionalized polypyrrole/carbon nanotube electrodes. Chem. Commun. 2013, 49, 9281−9283. (17) Kapustin, A. V.; Tarasevich, M. R.; Chirkov, Y. G.; Bogdanovskaya, V. A. Active layer of an oxygen electrode based on nanocomposite disperse carbon carrier plus laccase material. Russian J. Electrochem. 2004, 40, 909−916. (18) Tarasevich, M. R.; Bogdanovskaya, V. A.; Kapustin, A. V. Nanocomposite material laccase/dispersed carbon carrier for oxygen electrode. Electrochem. Commun. 2003, 5, 491−496. (19) Salaj-Kosla, U.; Poller, S.; Schuhmann, W.; Shleev, S.; Magner, E. Direct electron transfer of Trametes hirsuta laccase adsorbed at unmodified nanoporous gold electrodes. Bioelectrochemisry 2013, 91, 15−20. (20) Frasconi, M.; Boer, H.; Koivula, A.; Mazzei, F. Electrochemical evaluation of electron transfer kinetics of high and low redox potential laccases on gold electrode surface. Electrochim. Acta 2010, 56, 817− 827. (21) Ressine, A.; Vaz-Dominquez, C.; Fernandez, V. M.; De Lacey, A. L.; Laurell, T.; Ruzgas, T.; Shleev, S. Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on goldmodified nano/microstructured silicon. Biosens. Bioelectron. 2010, 25, 1001−1007. (22) Santucci, R.; Ferri, T.; Morpurgo, L.; Savini, I.; Avigliano, L. Unmediated heterogeneous electron transfer of ascorbate oxidase and laccase at a gold electrode. Biochem. J. 1998, 332, 611−615.
(23) Galliker, P.; Hommes, G.; Schlosser, D.; Corvini, P. F.-X.; Shahgaldian, P. Laccase-modified silica nanoparticles efficiently catalyze the transformation of phenolic compounds. J. Colloid Interface Sci. 2010, 349 (1), 98−105. (24) Cabana, H.; Ahamed, A.; Leduc, R. Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan. Bioresour. Technol. 2011, 102, 1656− 1662. (25) Corvini, P. F. X.; Shahgaldian, P. Laccase−nanoparticle conjugates for the elimination of micropollutants (endocrine disrupting chemicals) from wastewater in bioreactors. Rev. Environ. Sci. Bio-Technol. 2010, 9, 23−27. (26) He, P.; Hu, N. Electrocatalytic properties of heme proteins in layer-by-layer films assembled with SiO2 nanoparticles. Elelctroanalysis 2003, 16, 1122−1131. (27) Katz, E.; Willner, I. Integrated nanoparticle−biomolecule hybrid systems: Synthesis, properties and applications. Angew. Chem., Int. Ed. 2004, 43, 6042−6108. (28) Sun, Y.; Yan, F.; Yang, W.; Sun, C. Multilayered construction of glucose oxidase and silica nanoparticles on Au electrodes based on layer-by-layer covalent attachment. Biomaterials 2006, 27 (21), 4042− 4049. (29) Feifel, S. C.; Lisdat, F. Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers. J. Nanobiotechnol. 2011, 9, 59. (30) Feifel, S. C.; Ludwig, R.; Gorton, L.; Lisdat, F. Catalytically active silica nanoparticle-based supramolecular architectures of two proteins − Cellobiose dehydrogenase and cytochrome c on electrodes. Langmuir 2012, 28, 9189−9194.
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Electroactive Nanobiomolecular Architectures of Laccase and Cytochrome c on Electrodes: Applying Silica Nanoparticles as Artificial Matrix Sven Christian Feifel,* Andreas Kapp, and Fred Lisdat* Biosystems Technology, Institute of Applied Sciences, Technical University of Applied Sciences, 15745 Wildau, Germany S Supporting Information *
ABSTRACT: Fully electroactive multilayer architectures combining the redox protein cytochrome c and the enzyme laccase by the use of silica nanoparticles as artificial matrix have been constructed on gold electrodes capable of direct dioxygen reduction. Laccase form Trametes versicolor and cytochrome c from horse heart were electrostatically coimmobilized by alternate deposition with interlayers of silica nanoparticles in a multilayer fashion. The layer formation has been monitored by quartz crystal microbalance. The electrochemical properties and performance of the nanobiomolecular entities were investigated by cyclic voltammetry, indicating, that a multistep electron transfer cascade, from the electrode via cytochrome c in the layered system toward the enzyme laccase, and here to molecular dioxygen was achieved. The response of the novel architecture is based on direct electron exchange between immobilized proteins and can be tuned by the assembly process.
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sequential reduction of cyt c, laccase, and dioxygen.12 However, in this case, only one layer was in contact with the multilayer system, and laccase has not been embedded in the assembly. Additionally, several investigations have been directed to the immobilization and electron transfer of laccase on carbon materials13−18 or gold19−22 in a monolayer state or by crosscoupling reactions for the development of bioconjugated materials.23−25 Moreover, the combination of nanoparticles with biomolecules on electrodes is a matter of particular interest since several examples with direct electron transfer have been found.26,27 In a different direction, the application of silica nanoparticles (SiNPs) can be seen here. They have been used in biosensors due to the high biocompatibility and the high surface area of the nanosized particles resulting in highly active surface loading of proteins and thus to a high performance of the biosensor.26,28 However, so far, laccase has not been embedded with another redox protein like cyt c in a nonconducting artificial matrix composed of carboxy-modified nanosized SiNPs and assembled in multiple layers to study the electron transfer chain in such a nanobiomolecular environment.
INTRODUCTION Reactions between metalloproteins have been a subject of interest because of their biological importance. Respiratory and photosynthetic chains are composed of a series of specific and directional electron transfer reactions that include several proteins.1 Although electron transfer between inherent redox partners is naturally important, the reactions between metalloproteins, which are not intrinsic redox partners, have also gained significant interest since they allow the construction of new functional systems. In line with this, the reactions between the heme proteins, cytochromes, and the blue copper proteins, azurin, stellacanin, plastocyanin,2−4 and laccase,5 have been examined kinetically. Laccases belong to the class of blue multicopper enzymes responsible for the four-electron reduction of molecular dioxygen to water without formation of reactive intermediates, and therefore they are of great interest as catalysts in biofuel cells and for biosensor applications.6 Much of the research on laccase (Lac) has focused on defining the structure of its complex active site and the four-electron-reduction process at the trinuclear center.7 Further investigations targeted the interaction of laccase with other proteins in solution,8,9 as, e.g., cytochrome c (cyt c)5 a small heme redox protein. Although Lac and cyt c are not natural reaction partners, it was shown that Lac can accept cyt c as an electron donor delivering electrons for oxygen reduction.5,9 The driving force for the oxidation of cyt c is due to the difference in the redox potential of the two proteins.10,11 Later on, laccase has been immobilized on top of a polyelectrolyte/cyt c multilayer film, and it has been proofed that the appearance of a catalytic current is consistent with © 2014 American Chemical Society
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EXPERIMENTAL SECTION
Fabrication of Nanobiomolecular Laccase·Cyt c/SiNPs Assemblies: Modified Electrodes. The bare gold-wire electrodes are cleaned by incubation in piranha solution (3:1 H2SO4/H2O2) three times for 10 min. The electrodes are washed with Millipore water after Received: February 3, 2014 Revised: May 6, 2014 Published: May 7, 2014 5363
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the cleaning steps. For the construction of nanobiomolecular layers, the electrodes are modified by incubation for 48 h in 5 mM 3:1 solution of mercaptoundecanol (MU)/mercaptoundecanoic acid (MUA). Laccase (Lac), cyt c, and SiNPs are immobilized by chemophysical adsorption. The cyt c monolayers have been prepared by adsorption in a solution of cyt c (30 μM) in potassium phosphate buffer (5 mM, pH 7) on gold wire electrodes previously modified with a solution of MUA/MU (1:3) in ethanol (5 mM) for 48 h.2 For the following nanobiomolecular assembly consisting of cyt c, laccase, and SiNPs, a premixed protein solution (cyt c, Lac) was made in potassium phosphate buffer (5 mM, pH 7), which contained cyt c (20 μM) and Lac (1 μM) in 20:1 ratio. The subsequent assembly of [SiNPs/Lac·cyt c] nanobiomolecular layers has been performed by alternating incubation steps of a cyt c monolayer electrode in premixed-Lac·cyt c (1:20) and SiNPs (5.0 mg·mL−1) solutions for 10 min per step. Each of the 10 min-long adsorption steps in solutions of SiNPs (5.0 mg· mL−1) and Lac·cyt c (1:20) was followed by rinsing the electrodes with 5 mM potassium phosphate buffer pH 7 (to remove loosely bound material). The incubation procedures were repeated until the desired number of layers was reached.
Figure 1. Schematic depiction of the structure and proposed electron transfer cascade of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac] multilayer electrode. The cyt c monolayer is prepared on a mixed thiol layer (MUA/MU). The multiple layers are composed of [SiNPs/cyt c·Lac]n (n = 1, 2, 3, 4, 5, 6 bilayers).
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RESULTS AND DISCUSSION In order to integrate electroactive laccase in a nanobiomolecular architecture on electrodes we constructed multilayer systems composed of laccase and cyt c hosted by an artificial silica nanoparticle-based matrix. We selected cyt c, Lac, and SiNPs on the basis of following considerations: First SiNPs can be synthesized in a variety of sizes, and characterized by a narrow size distribution; Second, silica is a dielectric material, which does not adsorb light or conduct electrons; as inert host, it already enabled the assembly of biological materials (e.g., cyt c, CDH) while keeping its natural function.29,30 Finally, the pI values of carboxy-modified silica colloids, Lac, and cyt c are acidic and basic, respectively, and therefore, sufficient Coulomb interaction can be expected between the SiNPs (−), Lac (−) and the oppositely charged cyt c (+) at neutral pH, which allows layer formation by electrostatic interactions. Therefore, Lac, cyt c, and nonconducting carboxy-modified SiNPs were assembled on a cyt c monolayer by alternating incubation steps in solutions of SiNPs and a defined mixture of Lac/cyt c. A schematic representation of the suprananobiomolecular entity and the proposed electron transfer steps in this system are shown in Figure 1. As a starting point for the formation of the protein assembly, we used a monolayer electrode consisting of cyt c adsorbed on a mixed self-assembled alkanthiol layer of mercaptoundecanoic acid (MUA) and mercaptoundecanol (MU). The electrostatic interactions between the negatively charged electrode and the positively charged protein (cyt c) permits the immobilization of a first layer of cyt c without blocking its mobility in a surface fixed manner and thus providing a good communication not only with the electrode but also with the proteins assembled on top. The arrangement makes use of the different net-charges of the proteins. As laccase from Trametes versicolor possess a low negative net-charge (pI 4.3) and cytochrome c from horse heart a high positive one (pI 10.0) at pH 7, this can be used to form precomplexes of cyt c/Lac in a defined mixture, with an excess of cyt c to interact with the negative charges on the SiNPs surface. The formation of the entity with cyt c, Lac, and SiNPs on a gold surface was confirmed by quartz crystal microbalance (QCM) experiments, which indicate a defined binding of the components to the surface (see Supporting Information, Figure S1). By definition, an arrangement composed of two immobilization steps (SiNP, Lac/cyt c) is termed a bilayer.
To investigate whether laccase coimmobilized in a SiNPs matrix in multiple layers on electrodes is able to reduce molecular dioxygen to water, the voltammetric response of a multilayer electrode consisting of 6-bilayers of SiNPs/Lac·cyt c was recorded in the presence and absence of dioxygen. From Figure 2, an increase in the catalytic reduction current in the presence of air saturated solution can be seen. The
Figure 2. Cyclic voltammograms of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac]6 multilayer electrode in nitrogen (in black) and air (in red) saturated buffer (20 mM phosphate-citrate buffer pH 4.5). Scan rate 3 mV/s, surface area of the electrode A = 0.0471 cm2. Reduction current is assessed at a potential of −200 mV.
dioxygen reduction process starts at a potential where cyt c is reduced. This demonstrates that the immobilized Lac molecules in the multilayer architecture are active and can be electrochemically addressed because they accept the immobilized cyt c molecules in the different protein stacks as electron donor. In certain circumstances cyt c may also be able to reduce molecular dioxygen albeit only to a small extent, depending on the given environment of cyt c. To evaluate a possible contribution of cyt c molecules in our Lac/cyt c-architecture for dioxygen reduction, a multilayer consisting only of cyt c and 5364
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SiNPs has been constructed and investigated in air-saturated buffer by CV (Figure 3).
Figure 4. Cyclic voltammograms of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac]1−6 multilayer electrodes in air saturated buffer (scan rate 3 mV/ s, surface area of the electrode A = 0.0471 cm2, 20 mM phosphatecitrate buffer pH 4.5). Catalytic reduction currents are assessed at a potential of −200 mV. 1−6 denotes the number of bilayers on the electrode. Figure 3. Cyclic voltammogram of a Au-MUA/MU-cyt c-[SiNPs/cyt c]6 multilayer electrode in air saturated buffer (scan rate 3 mV/s, surface area of the electrode A = 0.0471 cm2, 20 mM phosphate-citrate buffer pH 4.5). Inset: comparison of the reduction currents of a [SiNPs/cyt c]6 and [SiNPs/cyt c·Lac]4 multilayer electrode.
Table 1. Catalytic Currents of Laccase and Cyt c Concentrations Are Given for Nanobiomolecular Architectures of 1−6 Bilayersa
The control experiment clearly demonstrates that the contribution of cyt c to the bioelectrocatalysis is only marginal for this system (Figure 3). For a six-bilayer assembly of pour SiNPs/cyt c layers, only a current of maximum 8 nA (at a potential of −200 mV vs. Ag/AgCl) can be observed compared to about 62 nA for a SiNPs/cyt c·Lac system; this proves the proposed role of laccase in the layered architecture to be the main player for catalytic dioxgen reduction. To gain insight of addressable Lac molecules in the layered entity electrodes with different numbers of SiNPs/cyt c·Lac layers have been examined by CV (Figure 4). Subsequently, the catalytic reduction current of the electrodes was investigated by determining the difference in the reduction current in the presence and absence of dioxygen. On the basis of the recorded CV data, it can be shown that, by raising the number of layers, the catalytic current increases almost proportionally (Table 1). This means that by an increase of catalytic sites (amount of laccase), the bioelectrocatalytic conversion of dioxygen can be enhanced. It is also worth mentioning that, not only does the catalytic current rise with the number of layers, but also the amount of electrochemically addressable cyt c molecules in the system. This is of significant importance since cyt c is not only acting as an electron donor but also as electron transfer molecule based on self-exchange (Figure 5). From the experiments it can be also concluded that the concept of a layered deposition of active proteins was successful. The verifiable increase of the catalytic current and the amount of electroactive cyt c with the growing number of deposited layers provides evidence for an efficient signal chain through the entire nanobiomolecular system for the reduction of dioxygen. It also implicates that the laccase molecules deposited in different stacks are in electrical contact with the electrode via cyt c. Furthermore, it can be stated that the multilayer arrangement is useful to tune the sensitivity by the amount of assembled biocatalyst (Lac).
nanobiomolecular protein electrodes
number of bilayers (n)
cyt c monolayer [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n [SiNP/cyt c·Lac]n
1 2 3 4 5 6
cytochrome c concentration (pmol/cm2) v = 100 mV/s 13 25 30 39 51 56 67
± ± ± ± ± ± ±
1 1 2 3 1 4 3
catalytic current (nA) for air saturation at −200 mV vs Ag/AgCl v = 3 mV/s
current densities (nA/cm2) v = 3 mV/s
± ± ± ± ± ±
339 ± 63 509 ± 21 658 ± 42 955 ± 63 1082 ± 42 1316 ± 105
16 24 31 45 51 62
3 1 2 3 2 5
a
Cyt c concentrations are determined by calculating the peak area in CV (without substrate). Catalytic reduction currents are measured in air-saturated buffer.
Therefore, significant progress has been accomplished compared to a previously described system12 where only one layer of laccase was in contact with one outer layer of a cyt c/ PASA multilayer and has not been integrated into the entity. The nonconducting SiNPs within the nanobiomolecular assembly act as a framework in which the redox protein cyt c and the enzyme Lac are embedded and held together by electrostatic interactions in such a way that cyt c and Lac are still in close proximity to each other so that direct ET between the enzyme and the redox protein and between the redox proteins is feasible. Multilayer formations without SiNPs could not be observed, which in turn demonstrates that they are needed as scaffold for the construction of electroactive protein assemblies. In a further step of characterization, the stability of the prepared multilayer architecture has been investigated. The findings reflected a rather good stability of the multilayer structure during the measurement over a day. No leakage of the proteins has been found during a day. If the multilayer 5365
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OUTLOOK In conclusion, we have shown that the construction of an electroactive nanobiomolecular architecture by the use of laccase as biocatalyst, cyt c as an electron-shuttle, and SiNPs acting as an artificial scaffold is feasible. The construction of the entity is based on a self-assembly process, whereby carboxymodified SiNPs have been used to integrate laccase in a fully electroactive layered assembly with a high amount of cyt c. The nanobiomolecular entity displays a specific catalytic activity for the reduction of molecular dioxygen. Cyt c operates as a solid-state electron donor in the multilayered architecture, transferring electrons effectively from the electrode by DET and IET toward the enzyme. The system is also an example for direct electron exchange between different proteins in a surfacefixed state. It needs to be emphasized that this architecture does not demand diffusional redox mediators, which makes this entity highly valuable for the development of third-generation biosensors. This approach is expected to have a considerable impact on the development of further nanosized biprotein systems where specific biocatalysts are supposed to react with heme-containing proteins. In addition, the protein multicomponent films are interesting as novel biologically active materials. It shows that new functionalities can be prepared by arranging electroactive biomolecules in such a way that efficient communication and defined signal generation can take place.
Figure 5. Cyclic voltammograms of a Au-MUA/MU-cyt c-[SiNPs/cyt c·Lac]1−6 multilayer electrodes. The increase of the addressable cyt c is shown with increasing number of layers (1−6), compared to a monolayer (M) electrode (scan rate 100 mV/s, surface area of the electrode A = 0.0471 cm2, 5 mM potassium phosphate buffer, pH 7).
electrodes have been stored at 4 °C for 1 week, a decrease (≤40%) in the catalytic activity can be observed. For the electron transfer pathway we propose that the catalytic current results from reduction of cyt c, which is enhanced by subsequent oxidation by neighboring laccase molecules. Dioxygen reduction near the potential of cyt c conversion verifies the proposed functionality of the protein architecture. Thus, a signal chain can be concluded, which starts at the electrode, where cyt c is reduced and the electrons are transferred via cyt c−cyt c interprotein ET to the T1 center of laccase and from there to the trinuclear copper-cluster at which the electrons are used for molecular O2 reduction to produce water (Figure 1). To evaluate the sensitivity of the system, the multilayered electrodes have been analyzed by cyclic voltammetry at different dioxygen concentrations (see Supporting Information, Figure S2). The collected CV data show a relatively linear dependence of the reduction current on the dioxygen content in solution. Additionally, the nanobiomolecular architecture is able to follow the enhanced dioxygen conversion at the laccase even for higher dioxygen concentrations, which suggests that the overall current is not limited by the reactions between the protein molecules or cyt c and the electrode. As already mentioned above, the enzyme and the redox protein have different functions within the architecture. Therefore, the relative amount of Lac and cyt c in each layer should have a profound effect on dioxygen reduction. When the cyt c/Lac ratio is decreased from 20:1 to 10:1, we observe a significant decrease in the catalytic response (data not shown). Therefore, although the number of recognition sites for dioxygen is increased (larger amount of Lac), the delivery of electrons to the enzyme becomes limiting, and thus the activity is diminished. Since the electrons need to be transferred from cyt c to cyt c by self-exchange through the layered architecture and from cyt c to Lac to ensure dioxygen reduction, the proposed role of cyt c as a solid-state electron donor is again supported. When high concentrations of Lac are present, the electron pathway from cyt c to cyt c seems to be restricted.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures, Chemicals, QCM experiments. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(S.C.F) E-mail: [email protected]. Phone: +49(0)3375-508158. Fax: +49(0)3375-508-458. *(F.L) E-mail: fl[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the DFG, LI706I7-1. We are grateful to D. Schäfer for technical assistance in the laboratory.
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ABBREVIATIONS Cyt c, cytochrome c; SiNPs, silica nanoparticles; Lac, laccase; DET, direct electron transfer; ET, electron transfer; IET, interprotein electron transfer
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REFERENCES
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