2017, Vol.46, No.12

Horseradish peroxidase apoprotein (apoHRP) was immobilized on a heme-modified polythiophene film, which was electropolymerized on an indium tin oxide surface to yield immobilized and reconstituted HRP. The HRP-immobilized electrode exhibits a 6-fold enhanced electrochemical response toward hydrogen peroxide reduction relative to having the protein randomly immobilized on the polythiophene electrode. This indicates that the insertion of the heme moiety into the heme pocket of HRP leads to an increase in the electrocatalytic current.

Well-organized electron-transfer interfaces between redox-active proteins and conductive materials have been a critical target for the development of biosensors and biodevices.1 In particular, redox-active hemoproteins are expected to be useful for the construction of functional protein-immobilized electrode materials due to their diverse functions, which include electron transfer, catalysis, and sensing. Hemoprotein-immobilized electrodes have thus been studied for their electrochemical behavior2 and as components of functional systems, such as biocatalysts,3 biosensors,4 and biofuel cells.5

Methodologies that provide a means for efficient electrochemical communication have been explored by immobilization of proteins via noncovalent interactions, such as electrostatic interactions with self-assembled monolayers on electrodes,6 affinity interactions with tag peptides,7 and specific protein–ligand interactions.8 Hemoproteins generally retain their redox-active heme cofactors by noncovalent interactions and at least one metal–protein coordination. This unique feature enables oriented anchoring of a hemoprotein via the heme–heme pocket interaction.9

We have recently reported the development of a process for programmed assembly of hemoproteins on metal electrodes and the surfaces of nanoparticles based on the specific heme–heme pocket interaction.10 To increase the electrochemical communication between the redox-active site of the immobilized proteins and the electrode, a fabrication technique for immobilizing sufficient quantities of proteins with favorable orientations is required. In this paper, we demonstrate the immobilization of an apo-form of horseradish peroxidase (HRP), apoHRP, on the surface of a polythiophene (PT) electrode via the heme–heme pocket interaction (Figure 1). Conductive polymers including polythiophene have been studied in the field of solar cells, field effect transistors, and actuators. Abundance of applications is facilitated by the ease of tailoring properties such as processability, functionality, electronic properties, and electrical stability.11 Polythiophene is easily fabricated by electropolymerization on the electrode, and are thus attractive electrode materials for promoting bioelectrocatalysis.12 We thus apply the immobilization of hemoproteins by heme-anchoring on the polythiophene-modified electrode. The characterization and electrochemical properties of the indium tin oxide (ITO) electrode containing oriented HRP immobilized on the surface of the electropolymerized polythiophene electrode are described.

A bare ITO electrode was first treated with 2,2′:5′,2′′-terthiophene-5-phosphonic acid to form a self-assembled monolayer to perform surface-initiated polymerization in the presence of 3-thiopheneacetic acid N-succinimidyl ester (3TAS) (Figure 2a). This method allows us to prepare the electropolymerized film that has improved adhesion properties and greater electrical contact.13 A constant voltage of 2.1 V (vs. Ag|AgCl) was applied until the total electric charge reached 25 mC during the electropolymerization by a radical coupling to obtain a homogeneous poly(3TAS) film that adheres tightly to the surface of the ITO electrode.14 A potential of 0 V for 30 s was then applied as a dedoping treatment to the resultant film of poly(3TAS). The peak current density for p-doping on poly(3TAS) was observed in the potential range of 0.5–1.5 V (vs. Ag|AgCl) in the cyclic voltammetry (CV) measurement, indicating successful preparation of a polythiophene layer (Figure 2b). The resultant poly(3TAS) layer was also characterized by attenuated total reflection (ATR) FT-IR, which can analyze the µm-depth range. Signals assigned to the ν(C=O) vibration of a succinimide ester were observed at 1813, 1783, and 1732 cm−1, which correspond to the signals of the monomer of 3TAS. This indicates that the succinimide ester moiety in the monomer is retained within the poly(3TAS) film of PT/ITO (Figure S1).

The cross-section and surface of the PT/ITO electrode were analyzed by SEM measurements. The images of the poly(3TAS) layer show the thickness of ca. 200 nm (Figure S2). The surface of the poly(3TAS) layer was also analyzed by AFM measurements and the average roughness of the surface was found to be 16.3 ± 0.6 nm (Figure S3). This evidence also indicates that the layer grows homogenously and is tightly attached to the ITO surface.

The electropolymerized layer of poly(3TAS) that contains active ester moieties in PT/ITO was next soaked in a DMSO solution of heme 1 containing an amino group on the one of the propionate side chains (Figure 1). This process yields the PT/ITO electrode covalently modified with the heme (heme-PT/ITO). The color of the film changes from pale yellow to orange after coupling of the heme moiety (Figure 3). The formation of an amide bond within the layer was also analyzed by ATR FT-IR (Figure 3). The absorption bands originating from the ν(C=O) vibration of succinimide ester disappeared and new absorption bands assigned to ν(C=O) vibration of an amide bonds were found at 1650 and 1659 cm−1. This indicates that the coupling modification of the heme proceeds smoothly to provide heme-PT/ITO.

The surface of the modified electrode was also analyzed by Fe2p and N1s peaks in X-ray photoelectron spectroscopy (XPS) (Figure 4). Although the PT/ITO electrode does not give any peaks in the region of Fe2p1/2 and Fe2p3/2 as expected, the spectrum for the heme-PT/ITO electrode shows clear peaks in those regions (Figures 4a and 4b). The two weak peaks Fe2p3/2 and Fe2p1/2 at 710.2 and 723.7 eV, respectively, are assigned to the Fe atom of the heme. The N1s peak at 402.1 eV is assigned to the N atom of the succinimide group of PT/ITO (Figure 4c). By contrast, heme-PT/ITO shows three N1s peaks: The peak at 400.7 eV assigned to the N atoms of the amide group, and the peaks at 398.5 and 399.7 eV assigned to the N atoms of the heme (Figure 4d). The XPS analysis indicates that the heme groups are linked to the poly(3TAS) layer on the heme-PT/ITO electrode.

Immobilization of the redox-active heme moieties in heme-PT/ITO electrode was analyzed by cyclic voltammetry. The CV indicates that the redox-active heme does not diffuse in solution and is immobilized within the layer of poly(3TAS) (Figure S4). The FeII/FeIII redox couple was observed at −260 mV (vs. Ag|AgCl). This value is similar to that observed for the heme-immobilized electrode in our previous studies (Figure 5a).10b In addition, the linear correlation between the current and the sweep rate indicates that the redox-active heme does not diffuse in solution and is immobilized within the layer of poly(3TAS) (Figure S4).

The heme-modified electrode (heme-PT/ITO) was next immersed in the protein solution of apoHRP for 12 h at 4 °C to immobilized apoHRP onto the electrode via heme–heme pocket interaction. The apoHRP proteins unbound or non-specifically bound on the electrode were thoroughly washed away to provide the apoHRP-immobilized electrode ([email protected]/ITO). The incorporation of the heme moiety into the apoHRP matrix was evaluated by CV experiments (Figure 5b). The previous studies reported that the successful incorporation of the heme moiety immobilized on the electrode surface into the apoprotein reduces the peak current,9a9c,9f since the heme within the protein matrix has slower electron-transfer kinetics due the increase in the electron-transfer distance relative to the free heme on the surface. The peak current decreases significantly upon the incorporation of apoHRP in [email protected]/ITO, whereas the shift of the redox couple of the heme cofactor was not observed. This clearly indicates the incorporation of the heme moiety into apoHRP via the heme–heme pocket interaction. Since the incorporation of the heme moiety did not proceed completely, we decreased the surface concentration of 1 to increase the immobilization efficiency.

The incorporation of the heme moiety linked to the PT electrode surface into the heme pocket was also evaluated by examining the electrocatalytic reduction of H2O2 by [email protected]/ITO. We observed a clear electrocatalytic current, which significantly increases with increasing concentrations of H2O2 (Figure S5). In addition, the electrocatalytic currents of [email protected]/ITO are greater than the electrocatalytic currents provided by heme-PT/ITO and bare ITO, indicating that the insertion of the heme moiety into the heme pocket of HRP leads to an increase of the electrocatalytic current. If the immobilized ferric HRP would directly react with H2O2 and then generates Compound I as an active species, we could observe the catalytic current of around 700 mV (Ag/AgCl). However, we observe the catalytic current of around −200 mV (Ag/AgCl), which corresponds to the Fe(II)/Fe(III) redox couple, suggesting that the immobilized ferric HRP is first reduced to ferrous state and electrocatalytic reduction of H2O2 proceeds by the immobilized ferrous HRP.3a

To further enhance the electrocatalytic current in [email protected]/ITO, we surveyed the optimum concentration of the heme moiety on the surface of the PT layer. Since the incorporation of the heme moiety would not proceed completely, we decreased the surface concentration of the heme to increase the immobilization efficiency.15 The number of immobilized HRP would decrease when the surface coverage of heme is too high. Because the apoprotein cannot easily access the linked heme. We thus prepared heme-PT/ITO electrodes, which have different heme concentrations, by mixing the heme with methylamine as a diluent during the coupling reaction (Figure S7, Table S1) and the corresponding [email protected]/ITO electrodes were prepared (Figure S8). Eventually, we observed a significant increase in the electrocatalytic current when the heme concentration is decreased by a factor of ten, indicating that the incorporation efficiency of the heme is critical for the performance of the HRP-modified electrode (Figures 6 and S9).

After the optimization of the surface concentration of the heme, the electrocatalytic current in [email protected]/ITO was found to be ca. 6-fold larger than the electrocatalytic current of heme-PT/ITO (Figure 7). In addition, we prepared a PT/ITO electrode with holoHRP covalently linked to the surface with random orientations. The similar concentration of HRP solution was used when HRP was immobilized on the electrode surface. The electrocatalytic current of [email protected]/ITO also has approximately a 6-fold greater electrocatalytic current relative to that of the HRP-linked PT/ITO electrode (holoHRP-PT/ITO), in which the proteins are immobilized with random orientations in [email protected]/ITO (Figure 1). Therefore, the reconstituted HRP, which is directed toward the surface of the polythiophene layer in [email protected]/ITO, would enable efficient electrochemical communication between the redox-active heme and the electrode. The surface concentration of the reconstituted HRP is also higher in the case of [email protected]/ITO.

The apoprotein of horseradish peroxidase was immobilized on the surface of polythiophene-modified electrodes via a specific noncovalent heme–heme pocket interaction. Although the recent successful work using HRP and carbon nanotube reports a high current density of ca. 2.0 mA cm−2, the HRP-immobilized polythiophene electrode using the reconstitution method shows also high current density in the electrochemical reduction of H2O2.16 The present work demonstrates that the polythiophene electrode with the anchored heme moieties serves as a useful platform for the fabrication of an electrode with hemoproteins oriented with their heme moieties close to the electrochemically active surface, thereby providing an increased electrochemical response.

This work was supported by JSPS KAKENHI Grant Numbers JP25102527 and JP15H00746 in Innovative Areas “New Polymeric Materials Based on Element-Blocks” and JP17H05370 in Innovative Areas “Coordination Asymmetry” to A.O., JSPS KAKENHI Grant Number JP15H05804 in Innovative Areas “Precisely Designed Catalysts with Customized Scaffolding” to T.H. This program was supported by the Strategic International Collaborative Research Program (SICORP), JST. We acknowledge Dr. Taro Uematsu and Prof. Susumu Kuwabata at Department of Applied Chemistry, Graduate School of Engineering, Osaka University for SEM measurements.

Supporting Information is available on http://dx.doi.org/10.1246/cl.170837.

Dedicated to the late Professor Yoshihiko Ito on the occasion of the 10th anniversary of his sudden death.