Letter pubs.acs.org/Langmuir
Catalytically Active Silica Nanoparticle-Based Supramolecular Architectures of Two Proteins − Cellobiose Dehydrogenase and Cytochrome c on Electrodes Sven C. Feifel,*,† Roland Ludwig,‡ Lo Gorton,§ and Fred Lisdat*,† †
Biosystems Technology, University of Applied Sciences, 15745 Wildau, Germany Food Biotechnology Laboratory, University of Natural Resources and Life Sciences, 1190 Vienna, Austria § Department of Biochemistry and Structural Biology, Lund University, SE-22100 Lund, Sweden ‡
S Supporting Information *
ABSTRACT: Artificial nanobiomolecular architectures that follow natural examples in protein assembly become more and more important from basic and applied points of view. Our study describes the investigation on cellobiose dehydrogenase (CDH), cytochrome c (cyt c), and silica nanoparticles (SiNP's) for the construction of fully catalytically active supramolecular architectures on electrodes. We report on intraprotein, interprotein, and direct electron-transfer reaction cascades of cellobiose dehydrogenase and cytochrome c immobilized in multiple supramolecular layers. Carboxy-modified SiNP's are used to provide an artificial matrix, which enables protein arrangement in an electroactive form. Direct and interprotein electron transfer has been established for a two-protein system with CDH and cyt c in a layered architecture for the first time. We also highlight that the glycosylation of CDH and the silica nanoparticle size play key roles in the mode of operation in such a complex system. The response of the specific substrate, here lactose, can be tuned by the number of immobilized nanobiomolecular layers.
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INTRODUCTION The electrical communication of redox enzymes with electrode surfaces for direct electron-transfer reactions (DET) is fundamental to the development of amperometric biosensors and biofuel cells.1−3 Effective electrical communication of redox centers with electrodes can lead to high current outputs at low substrate concentrations, thus resulting in sensitive sensors and enabling their miniaturization. The rate of DET between a redox enzyme and an electrode is dependent on the location of the redox center in the enzyme, the molecular environment of the redox center, and the orientation of the enzyme on the electrode surface.4,5 Cellobiose dehydrogenase (CDH) is an interesting enzyme for biosensor applications because of efficient sugar conversion without the need for oxygen and its ability to facilitate efficient DET reactions with electrodes.6 Therefore, CDH-based lactose biosensors can be an excellent alternative to previous lactose biosensors reported in the literature or commercially available.7−12 CDH is an extracellular hemoflavoenzyme that is produced by a number of wooddegrading phytopathogenic fungi.13,14 CDH from the basidiomycete Trametes villosa is a 90 kDa monomer (11 to 12 wt % glycosylated) with an isoelectric point at pH 4.3 consisting of two separate domains, one sugar-oxidizing (dehydrogenase) domain containing FAD as a cofactor and the other domain harboring a cytochrome b-type heme.15,16 The FAD domain is responsible for the catalytic oxidation of a sugar substrate, such as cellobiose or lactose, and it strictly discriminates the © 2012 American Chemical Society
monosaccharide from the catalytic reaction. Recently, it was found that CDH can accept cytochrome c (cyt c) as a reaction partner to deliver the electrons from the oxidation reaction.17 Additionally, we could show that CDH can also react with surface-bound cyt c.18 A few attempts to arrange CDH on electrode surfaces have been successful,19,20 but for a biosensor with high sensitivity, there is a need to increase the functional density, which can be achieved by going beyond the monolayer arrangement. Therefore, it is necessary to construct stable supramolecular architectures in which the embedded proteins can effectively exchange electrons with the electrode. Various approaches have been developed to increase the sensitivity of protein electrodes. The immobilization of proteins into multistructured systems via layer-by-layer deposition has become one of the favorite methods. Such similar assemblies have recently been shown to allow the incorporation of enzymes and establish communication with the electrode, thus allowing the construction of different analytical signal chains.21−24 Moreover, the combination of nanoparticles with biomolecules on electrodes is a matter of particular interest because several examples with DET have been reported.25,26 The fact that nanostructures are in the same size domain as proteins makes them particularly suitable Received: March 28, 2012 Revised: May 29, 2012 Published: June 4, 2012 9189
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for their interaction with biomolecules. Accordingly, nanotechnology involves the assembly of small molecules into complex architectures for improved function by controlling the location of each component in a 3-D space. The integration of nanotechnology and miniaturized devices with novel biochemical detection methodologies can lead to very sensitive and fast assays for the detection of desired species. On the basis of this background knowledge, we construct new functional supramolecular architectures by arranging CDH and cyt c in such a way that interprotein electron exchange and direct electron transfer become feasible. For this purpose, differently sized nonconducting silica nanoparticles (SiNP's) have been prepared and modified with a carboxylic function to ensure electrostatic interaction with the proteins and their use as an artificial matrix. Additionally, we highlight that the glycosylation of the enzyme (CDH) dramatically influences the catalytic activity and the adsorption behavior. Furthermore, it is shown that electrons can be transported through and within the supramolecular layers toward the electrode. As a result, an artificial multiprotein complex with electron-transfer cascades is formed that follows natural examples of a defined signal transfer in complex protein structures.
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Letter
RESULTS AND DISCUSSION
In this investigation, we confine the CDH-cyt c interprotein reaction to a surface and couple it with the DET of cyt c to the transducing electrode. The supramolecular architectures with embedded CDH, cyt c, and SiNP's as an artificial matrix are assembled on a cyt c monolayer electrode by alternating incubation steps in solutions of SiNP and a CDH·cyt c mixture for coimmobilization. The monolayer electrode consists of cyt c adsorbed on a mixed thiol layer of mercaptoundecanoic acid (MUA) and mercaptoundecanol (MU). A schematic representation of the complex artificial onionlike architecture is shown in Figure 1.
MATERIALS AND METHODS
Fabrication of Nanobiomolecular Assemblies: Modified Electrodes. Gold-wire electrodes are cleaned by three incubations in piranha solution (3:1 H2SO4/H2O2) for 10 min. The electrodes are washed with Millipore water after the cleaning steps. For the construction of nanobiomolecular layers, the electrodes are modified by incubation for 48 h in a 5 mM 3:1 solution of mercapto-undecanol/ mercaptoundecanoic acid. CDH, cyt c, and SiNP's are immobilized by simple chemophysical adsorption. The cyt c monolayers are prepared by incubation of the electrodes in 30 μM cyt c in 5 mM potassium phosphate buffer at pH 7 for 2 h.27 For the nanobiomolecular assembly, a premixed protein solution was made in potassium phosphate buffer (5 mM, pH 7) that contained cyt c (20 μM) and CDH (2 μM) in a 10:1 ratio. The assembly of CDH·cyt c/SiNP's nanobiomolecular layers has been performed by alternating incubations of the cyt c monolayer electrode in CDH·cyt c (1:10) and SiNP (5.0 mg·mL−1) solutions for 10 min per step. Each of the 10-minute-long adsorption steps of SiNP's (5.0 mg·mL−1) and a premixed CDH·cyt c (1:10) solution was followed by rinsing the electrodes with 5 mM potassium phosphate buffer at pH 7. The incubation procedures were repeated until the desired number of layers was reached. Electrochemistry. All electrochemical measurements were carried out in a custom-made 1 mL cell using an Ag/AgCl/1 M KCl reference (Biometra, Germany) and Pt-wire counter electrode. The working electrodes were modified gold wires (diameter 0.5 mm) obtained from Goodfellow (Bad Nauheim, Germany) that were modified according to the procedures described above. Cyclic voltammetric (CV) experiments were carried out with a CH Instruments CHI 660D device (Austin, TX, USA). Scan rates were varied between 0.005 and 50 V·s−1, but a scan rate of 5 mV·s−1 was normally used to record catalytic currents; 100 mV·s−1 was applied for the determination of the cyt c concentration and to get a further impression of the limitations on electron transfer for cyt c, where the scan rate has been varied between 0.1 and 50 V·s−1. The potential range has been chosen to be between −0.15 and +0.45 V versus Ag/AgCl/1 M KCl. A 20 mM phosphate−citrate buffer at pH 4.5 was used for all CV experiments. All measurements were performed at room temperature, 25 °C. Data analysis has been performed using CHI 660D (Austin, TX, USA) software.
Figure 1. Schematic representation of a supramolecular [SiNP/ CDH·cyt c] architecture prepared on a cyt c monolayer electrode (M). The cyt c monolayer is assembled on a mixed thiol layer (MU/MUA). The layer structure is [SiNP's/CDH·cyt c]n (n = 1, 2, 3, 4).
In a first attempt, we use native CDH to construct supramolecular electrodes of SiNP/CDH·cyt c with one, three, and four bilayers. By the time the [SiNP/CDH·cyt c]n multistructured electrodes are exposed to a solution with lactose (5 mM) and cyclic voltammetry is performed, a clearly visible oxidation current can be observed (Figure 2). Control experiments in which only one protein, either native CDH or cyt c, is immobilized in the nanobiomolecular system display no catalytic current, which in turn demonstrates that both protein components are necessary for the successful generation of a catalytic current. On the basis of the preassigned approach, we demonstrate that the construction of a functional nanosupramolecular entity with coimmobilized CDH/cyt c in an artificial SiNP-based matrix is possible (the low pI (4.3) of Trametes villosa CDH and the high pI (10.0) of cyt c favor coimmobilization by electrostatic interactions between the proteins). Unfortunately, the obtained catalytic currents for the native CDH-based electrodes are not very large. For the four-bilayer architecture of [SiNP/CDH·cyt c]4, we get a catalytic current of merely 4 nA, and for electrodes with one and three bilayers, we get 0.5 and 2 nA, respectively. In the absence of the enzyme substrate, the electrochemical behavior is determined by the cyt c conversion allowing only the determination of the cyt c concentration on the electrode surface for each layered entity (Table 1). Glycosylation has been discussed as one major drawback in the interaction of enzymes and redox proteins with electrodes.28−30 For this reason, we assume that this is also valid for the interaction of different proteins in nanosized supra9190
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Figure 2. Cyclic voltammograms of Au-MUA/MU-cyt c-[SiNP-CDH·cyt c]n nanobiomolecular electrodes. (a) Native CDH-based multilayer (n = 4) with and without lactose (5 mM). (b) Native CDH-based multilayers (n = 1, 3, 4) with lactose. Scan rate 5 mV/s, 20 mM phosphate-citrate buffer, pH 4.5.
molecular arrangements, particular when the enzyme reaction occurs in the immobilized state. Accordingly, on the basis of the experiments with native CDH, the construction of nanobiomolecular assemblies with deglycosylated CDH (dCDH) is investigated. A freshly prepared four-bilayer SiNP/dCDH·cyt c electrode is used, and cyclic voltammetry performed to explore the catalytic activity of dCDH-based architecture. Impressively, we see here an enormous increase in catalytic current, compared to that of native CDH-based electrodes (Figure 3). To gain more insight into the electron-transfer process of these nanobiomolecular systems with deglycosylated CDH, the dependence of the catalytic efficiency on the number of supramolecular entities is investigated. Thus, electrodes with different numbers of [SiNP/dCDH·cyt c]n layers are prepared, and cyclic voltammetry is performed. By increasing the number of nanobiomolecular assemblies, the catalytic current and the amount of electrochemically detectable cyt c within the system increases almost proportionally (Figure 3 and Table 1). Remarkably, a 7-fold enhancement in the catalytic current
Table 1. Catalytic Currents (of Native and Deglycosylated CDH) and Cyt c Concentrations in Protein Architectures Built from One to Four Bilayersa multilayer electrodes CDH·cyt c/ SiNP CDH·cyt c/ SiNP CDH·cyt c/ SiNP dCDH·cyt c/ SiNP dCDH·cyt c/ SiNP dCDH·cyt c/ SiNP dCDH·cyt c/ SiNP
number of bilayers 1
cyt c conc. (pmol/cm2) 11
catalytic current E = 0.45 V (nA) 0.5
2
17
2
4
31
4
1
16
4
2
27
9
3
39
18
4
66
28
a
Cyt c concentrations are determined by calculating the peak area in CV in the absence of lactose. Catalytic currents are measured after the addition of 5 mM lactose.
Figure 3. Cyclic voltammograms of Au-MUA/MU-cyt c-[SiNP-CDH·cyt c]n nanobiomolecular electrodes. (a) Catalytic currents of dCDH-based multilayer electrodes (n = 1, 2, 3, 4) in the presence of lactose (5 mM) at a scan rate of 5 mV/s. (Inset) Four-bilayer electrode (c) without and (d) with lactose. (b) Increase in the cyt c concentration with the number of bilayers (1, 2, 3, 4) measured at a scan rate of 100 mV/s. CV is performed in 20 mM phosphate−citrate buffer at pH 4.5. 9191
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Figure 4. Dependence of the catalytic current on lactose concentration shown for a Au-MUA/MU-cyt c-[SiNP-cyt c·dCDH]4 nanobiomolecular electrode. (a) Increase in catalytic current with respect to the lactose content in solution (10 μM−20 mM). (b) Linear dependence of the oxidation current on the lactose content in solution in the range of 10 μM−1 mM. Catalytic currents are determined by CV (scan rate 5 mV/s, 20 mM phosphate−citrate, pH 4.5).
Because the electron-transfer kinetics of the protein interaction in the immobilized state differs from the behavior in solution, the kinetic analysis cannot be carried out on the basis of the rate constants for the corresponding reactions in solution. To evaluate the rate-limiting step in the artificial electron-transfer chain, the dCDH-based nanobiomolecular assembly is analyzed with cyclic voltammetry at different lactose concentrations (Figure 4). The deliverables of the experiments show a distinct linear dependence of the oxidation current on the lactose content in solution in the range of 10 μM−1 mM (scan rate 5 mV s−1). Given that the system can follow the enhanced lactose conversion at the dCDH, it suggests that the overall current is limited by the catalytic oxidation in the FAD domain of the enzyme rather than by the subsequent intraprotein and interprotein reaction steps of cyt c−CDH and cyt c−cyt c or the direct electron transfer of cyt c and the electrode. However, at high scan rates the catalytic current disappears, which in turn denotes that the protein−protein electron transfer becomes rate-limiting in our silica nanoparticle-based biomolecular architecture. From a continuous examination of how the interactions of both proteins can be facilitated by the artificial matrix (SiNP's), we built up supramolecular systems with carboxy-terminated SiNP's of different sizes (5, 15, 20, and 40 nm). The size of the SiNP's is analyzed by dynamic light scattering and TEM measurements (details in the Supporting Information). From cyclic voltammetric experiments of electrodes with the same number of nanobiomolecular layers (four), a significant disparity in their behavior is found. The highest amount of electroactive cyt c and the largest catalytic current is observed for supramolecular architectures assembled with 20 nm SiNP's (Table 2). There is quite a significant correlation observed among the amount of cyt c deposited in different layers on the electrode, the catalytic currents, and the SiNP size used for the construction of the supramolecular architectures. The data suggests that increasing the size of the SiNP's (from 5 to 20 nm) results in a greater amount of electroactive redox protein within the layered system. This is connected to a larger amount of enzyme (dCDH) in the assembly and thus a larger catalytic current. However, there appears to be a limit to this phenomenon in which the protein adsorption and catalytic current decrease if the size of the SiNP's exceeds 20 nm.
(28 nA) is found for a four-bilayer assembly of [SiNP/ dCDH·cyt c]4 compared to that for a one-bilayer electrode. The distinct increase in the catalytic current and the amount of electro-active cyt c with the growing number of deposited biomolecular layers provides evidence for efficient interprotein electron transfer through the system. In a reverse conclusion, this means that by increasing the number of catalytic sites (amount of dCDH) within the system the specific bioelectrocatalytic conversion of lactose can be enhanced. This circumstance, however, also implies that all CDH molecules deposited in different layers are in electrical contact with the electrode. Because a large portion of the enzyme molecules are immobilized rather far away from the electrode surface, we conclude that the catalytic oxidation reaction at CDH provides the electrons for the catalytic current, which are in turn transferred by cyt c−cyt c interprotein electron transfer toward the electrode. This notion is supported by the fact that assemblies of dCDH and SiNP's alone do not show any catalytic lactose oxidation current. The different electrontransfer steps can be summarized in a simple way as follows: electrode ↔ cyt c ↔ cyt c ↔ [cyt b ↔ FAD/CDH] ↔ lactose. The nonconducting silica nanoparticles within the nanobiomolecular assembly act as a framework in which redox protein cyt c and enzyme CDH are embedded and held together by electrostatic interactions in such a way that cyt c and CDH are still in close proximity to each other so that direct electron transfer between proteins is feasible. Supramolecular layer formation without SiNP's could not be observed, which in turn demonstrates that they are needed as a scaffold for the assembly of catalytically active protein layers. In addition, SiNP's most likely facilitate an enhancement of the cyt c−cyt c self-reduction rate. This may be due to the increased probability of productive collisions in the densely packed assembly because the negatively charged SiNP's in parallel increase the dielectric constant of the medium surrounding cyt c and reduce the heme edge-to-edge distance by interacting with the positively charged Lys and His residues at cyt c, which are important for establishing the optimum distance. This notion is also in agreement with earlier reports on long-distance electron transfer in cyt c multilayers.31,32 For applications of such nanosupramolecular architectures, this means that the sensitivity toward the enzyme substrate (here, lactose) can be tuned to a large extent by the number of deposited protein layers without being limited by mediator diffusion or leakage. 9192
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have also shown that assembly formation with a large amount of protein strongly depends on the particle size of the silica nanoparticle used. The carboxy-modified SiNP's provide a substantial artificial matrix, which enables a biomolecular arrangement in a fully active form. This improvement is very likely due to the fact that silica nanoparticles increase the probability for productive collisions in the densely packed assembly. In addition, they increase the dielectric constant of the medium and reduce the heme-to-heme distance. This approach is expected to have a considerable impact on the development of further nanoparticle-based supramolecular architectures and also represents a significant advance in modeling biological electron-transfer processes: with cyt c or other small redox proteins as the electron shuttle, further potential reaction partners might be used in nanosized biomolecular arrangements for the development of artificial signal chains mimicking natural examples. We anticipate that this concept will stimulate progress in the development of more complex multiprotein electrode designs and biomimetic signal cascades that take advantage of direct communication between proteins in a surface-fixed state. Additionally, we believe that downsizing the enzyme dimensions or the use of truncated enzymes may offer new possibilities to circumvent poor electrical contact among enzymes, reaction partners, and electrode surfaces.
Table 2. Influence of the SiNP Size on the Amount of Electroactive cyt c and Catalytic Current of the Nanobiomolecular Assemblies with dCDH and cyt ca multilayer electrodes
SiNP's size (nm)
number of bilayers
cyt c conc. (pmol/cm2)
dCDH·cyt c/ SiNP dCDH·cyt c/ SiNP dCDH·cyt c/ SiNP dCDH·cyt c/ SiNP
40
4
17
4
20
4
66
28
15
4
31
9
5
4
26
8
catalytic current E = 0.45 V (nA)
a
Cyt c concentrations are determined by calculating the peak area in CV in the absence of lactose. Catalytic currents are measured after the addition of 5 mM lactose.
Therefore, a certain balance in the interactive forces seems to be necessary for a stable onionlike formation on one side and the rotational flexibility of cyt c and CDH within the system for efficient interprotein electron exchange on the other side. As a terminal investigation, quartz crystal microbalance (QCM) experiments have been carried out to verify that by alternating silica nanoparticle and protein (CDH, cyt c) deposition a defined nanostructured assembly is formed. The experiments reveal a clear adsorption behavior for each alternating deposition step of the dCDH·cyt c or SiNP solution, respectively. In the case of the native CDH-based systems, the binding behavior is not as defined as for the dCDH-based assembly and less mass accumulation can be found (four-bilayer architecture with native CDH Δf = 230 Hz, with dCDH Δf = 355 Hz, where Δf is the change in frequency, see the Supporting Information). This, however, is in accordance with the observation made during the electrochemical experiments, where less electroactive cyt c and a lower catalytic current is detected for native CDH-based arrangements. On this account, it can be stated that both native and deglycosylated CDH are successfully immobilized into nanostructured, biomolecular architectures, which are fully bioelectrocatalytically active. The discrepancy between the two CDHs in catalytic current and enzyme/protein loading seems to originate from the glycosylation pattern on the protein surface; therefore, dCDH is favorable for the construction of multiple-layer supramolecular entities. Glycosylation contributes to a reduced immobilization of native CDH in the artificial protein architecture, which is possibly due to weaker electrostatic forces between native CDH and cyt c rather than between dCDH and cyt c. In addition, the glycosylation pattern of native CDH causes larger electron-transfer distances between the heme centers of cyt c and the cyt b-like heme of CDH and may also influence the rotational flexibility of the nanobiomolecular arrangement.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures, synthesis and modification of silica nanoparticles, characterization of SiNP's via TEM, DLS, zeta potential, and FT-IR, and details of the QCM experiments. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(S.C.F.) E-mail:
[email protected]. (F.L.) E-mail: fl
[email protected]. Phone: +49(0)3375-508-137. Fax: +49(0)3375-508458. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the BMBF (PNT51513) and The Swedish Research Council. We are grateful to A. Kapp and D. Schäfer for technical assistance in the laboratory.
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REFERENCES
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CONCLUSIONS Our results indicate that on the basis of an artificial nanoparticle matrix the immobilization of cellobiose dehydrogenase in supramolecular architectures is possible and that the deglycosylated CDH-based electrodes exhibit much higher current densities than the assemblies with native CDH for the same number of nanobiomolecular layers. IET and DET have been established for a two-protein system with CDH in a nanosized supramolecular architecture for the first time. We 9193
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