Bioelectrocatalytic reduction of O2 at a supramolecularly associated laccase electrode

Article abstract of DOI:10.1039/c3nj01143g

In order to improve the bioelectrocatalytic reduction of dioxygen, a method for the supramolecular immobilization of Trametes versicolor laccase is reported. The immobilization support consisted of a self-assembled monolayer (SAM) of mercaptoundecanoic acid tyrosine amide on gold electrodes. To mediate the electron transfer between the enzyme and the electrode surface, ferrocenehexanethiol was introduced into the SAM as a second component. The resulting system was studied by cyclic voltammetry. Reduction of dioxygen started at 0.80 V, with a reduction peak at 0.52 V and a decrease in its intensity was observed when the solution was deaerated. The achieved current density for dioxygen reduction (23 μA cm^-2, open to air) suggests a high surface density of active enzyme prone to bioelectrocatalytic activity. This system offers new insights into the supramolecular design of biofuel cells. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014.

Full text of DOI:10.1039/c3nj01143g

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Bioelectrocatalytic reduction of O at a
2
supramolecularly associated laccase electrode†
Cite this: New J. Chem., 2014,
3
8, 386
Manuel Antuch, Darıo G. Abradelo and Roberto Cao*
´
In order to improve the bioelectrocatalytic reduction of dioxygen, a method for the supramolecular
immobilization of Trametes versicolor laccase is reported. The immobilization support consisted of a self-
assembled monolayer (SAM) of mercaptoundecanoic acid tyrosine amide on gold electrodes. To mediate
the electron transfer between the enzyme and the electrode surface, ferrocenehexanethiol was introduced
into the SAM as a second component. The resulting system was studied by cyclic voltammetry. Reduction
of dioxygen started at 0.80 V, with a reduction peak at 0.52 V and a decrease in its intensity was observed
Received (in Porto Alegre, Brazil)
1st September 2013,
Accepted 31st October 2013
2
À2
when the solution was deaerated. The achieved current density for dioxygen reduction (23 mA cm , open
DOI: 10.1039/c3nj01143g
to air) suggests a high surface density of active enzyme prone to bioelectrocatalytic activity. This system
offers new insights into the supramolecular design of biofuel cells.
www.rsc.org/njc
7
,20
covalent binding or adsorption.
Immobilization processes
Introduction
should guarantee the appropriate orientation of the electron-
The search for renewable low energy sources for biomedical receptor T1 site towards the surface of the electrode in order to
2
1
devices is very active. Efforts have been made to develop achieve an adequate electrochemical communication with it.
1
4,19
enzymatic biofuel cells, which take advantage of the natural As the T1 site of laccase is rich in aromatic aminoacids,
efficiency of oxidoreductase enzymes to oxidize or reduce easily it may be assumed that surfaces modified with tyrosine (Tyr)
1
–6
accessible substrates.
in biofuel cells. In particular, dioxygen reducing enzymes, such when a supramolecular procedure is used.
as laccases, have attracted great attention due to their ability to reported the immobilization of laccase on gold nanoparticles
Several enzymes have been employed should be able to achieve an adequate orientation of the enzyme
1
7,22
We have recently
6
,7
decrease the activation barrier of dioxygen reduction.
Laccases are copper containing oxidoreductase enzymes that means of multivalent supramolecular interactions.
couple the oxidation of a wide family of aromatic substrates to On the other hand, the presence of additional redox species
modified with a SAM of a thiolated derivative of Tyr, solely by
2
3
6
–8
the concomitant reduction of dioxygen to water. In general, is frequently necessary to mediate the electron transfer process
they contain four copper atoms as the prosthetic group. The between the redox enzyme and the supporting electrode.
6
oxidation of the substrate takes place at the type 1 (T1) copper, Many species have been used for this purpose, such as carbon
2
4
25
from which the electrons are transferred to a trinuclear cluster nanotubes, certain phenolic dyes or osmium complexes.
containing type 2 (T2) and type 3 (T3) copper atoms, where the Redox mediation of immobilized laccase with ferrocene has
7
,9
reduction of dioxygen occurs. Laccases have been investi- been less studied, even when it offers a stable electrochemical
1
0
11
12
6,26
gated from structural, spectroscopic and electrochemical
points of view. It has been found that the T1, T2 and T3 copper
sites have standard potentials within the range of 0.69–1.00 V, modified layer-by-layer with a mixed SAM of mercaptoundecanoic
signal.
Herein we describe the reduction of dioxygen at gold electrodes
3
,9
0
.36 V, and 0.57–1.00 V (vs. Ag/AgCl), respectively. Thus, the acid tyrosine amide (MUATyr) and ferrocenehexanethiol (FcH)
onset of the dioxygen reduction reaction is always near the (Fig. 1). Laccase from Trametes versicolor, Lac (EC 1.10.3.2), was
1
3–19
potential of the T1 site.
supramolecularly immobilized on the mixed SAM. In such a
Several approaches have been used for the immobilization of system, the terminal Tyr moieties should have directed the
laccases such as entrapment, encapsulation, self-immobilization,
Laboratorio de Bioinorg a´ nica, Universidad de La Habana, Zapata y G, Vedado,
La Habana, 10400 Cuba. E-mail: caov@fq.uh.cu; Fax: +53 7 8733502;
Tel: +53 7 8792145
†
Electronic supplementary information (ESI) available: Further electrochemical
considerations. See DOI: 10.1039/c3nj01143g
Fig. 1 Components of the mixed SAM.
3
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orientation of the T1 site of laccase towards the electrode observed in order to guarantee the formation of a Au{111}
surface while the ferrocene derivative promoted the dioxygen surface. The electrode was then cleaned with HCl (0.1 M) and
reduction.
thoroughly rinsed with doubly distilled water (3 mS). The SAM of
À3
MUATyr was obtained by immersion of a gold sheet in a 10
M
methanolic solution for 24 h. The SAM of FcH was obtained by
immersion of gold electrodes in a 10 M ethanolic solution
À2
Experimental section
for 20 h. The mixed SAM of MUATyr and FcH was obtained
by 20 h immersion of gold electrodes into an equimolar
ethanolic solution of both components (total thiol concen-
tration 10 M). The SAMs obtained were thoroughly rinsed
with methanol, ethanol and double distilled water to remove
residual reactants.
Materials
All reagents and solvents employed were commercially available
high-grade purity materials (Aldrich Chemicals) and used as
supplied without further purification.
À2
Spectroscopy
1
13
H and C NMR spectra were recorded on a Variant Gemini 400 Immobilization of laccase
spectrometer. UV-Vis spectra were recorded on an Ultrospec III
Pharmacia-LKB). The X-ray photoelectron spectra (XPS) were
Laccase was immobilized onto the SAM of MUATyr by incuba-
tion in a 1 mg mL laccase buffered solution (pH 5) for 2 h.
(
À1
obtained using a VG Escalab 250 iXL ESCA instrument (VG
Scientific). The measurements were carried out using mono-
chromatic Al-Ka radiation (1486.92 eV). Photoelectrons were
collected from a takeoff angle of 901 relative to the sample
surface. The measurement was obtained in a constant analyzer
energy mode (CAE) with 100 eV pass energy for survey spectra
The enzymatic activity was determined in a buffered aqueous
solution (pH 5) containing ABTS (5 mM, 100 mL). Absorbance
28,29
values were acquired every 15 s at 420 nm.
For the electro-
chemical measurements, laccase was immobilized in the same
fashion onto the mixed SAM of FcH and MUATyr.
and 20 eV pass energy for high-resolution spectra. The binding Cyclic voltammetry
scale was referenced by setting Au4f7/2 BE at 84.0 eV. Mass spectra
All glassware was cleaned using ‘‘piranha’’ solution followed by
were obtained using a ToF-SIMS spectrometer of Ion-TOF GmbH
Germany. The sample was bombarded with a pulsed gallium ion
beam (25 keV) at 451 incidence. The secondary ions generated
were extracted with a 10 kV voltage, and their time-of-flight from
the sample to the detector was measured using a reflectron mass
spectrometer.
thoroughly rinsing with double distilled water. Cyclic voltam-
metry was performed using a commercial EG&G Model 384B
Polarographic Analyzer in a three electrode cell configuration.
A gold bead, Pt, and Ag/AgCl (KCl saturated) were used as the
working, counter, and reference electrodes, respectively. Na SO4
2
(
0.1 M) was employed as the supporting electrolyte for all
measurements, while KOH (0.5 M) was used for all the reductive
desorption experiments. Purging of dissolved O was achieved by
bubbling via an external source.
Synthesis of MUATyr
2
A mixture of 11-mercaptoundecanoic acid (100 mg, 0.458 mmol)
and N-hydroxysuccinimide (52.7 mg, 0.458 mmol) was stirred at
À5 1C in THF (60 mL) for 15 min. A solution of dicyclohexyl-
carbodiimide (94.3 mg, 0.458 mmol) in THF (5 mL) was added
N
2
Results and discussion
dropwise for 45 min. The mixture was stirred for another 24 h Spectroscopic characterization of the SAM of MUATyr and
and filtered. An additional solution of Tyr (83 mg, 0.458 mmol) in laccase supramolecularly immobilized on it
ethanol was prepared by the addition of NaOH (0.5 M) dropwise.
The formation of a SAM of MUATyr on gold sheets was confirmed
Both solutions were mixed and stirred for 24 h. The solvent was
by ToF-SIMS (Fig. 2a). The spectrum showed peaks corresponding
evaporated under vacuum; the white product was washed
+
+
to Au-MUATyr (591 u.m.a.) and MUATyr (394 u.m.a.) frag-
ments. Such a SAM served as a host for the supramolecular
several times with a hydroalcoholic solution and dried in air.
1
Yield 160.1 mg (86.6%). H NMR (400 MHz, D
2
O, 25 1C):
a
b
d = 2.50–0.90, (w, 20H), 3.12 (s, H ), 2.40 (s, H ), 4.54 (s, H),
.77 (dd, 4H); C NMR (125 MHz, DMSO-d
1
3
6
6
, 25 1C) d = 30–23
(10C), 34, 39, 41, 49, 116, 125, 130, 153, 168, 170.
Preparation of the SAMs
4
Gold sheets were cleaned consecutively with NaBH and a
dilute nitric acid (1 : 1 v/v) solution for 3 h and then thoroughly
washed with doubly distilled water prior to the immobiliza-
2
7,28
tion.
The gold electrode was prepared by annealing the tip
of a gold wire (1 mm diameter, 5 cm long, 99.99%), which had
previously been cleaned overnight with nitric acid (68%). The
Fig. 2 (a) ToF-SIMS of the SAM of Au-MUATyr. Arrows indicate the frag-
ments at 394 (MUATyr ) and 591 u.m.a. (Au-MUATyr ). (b) XPS spectra of the
formed gold bead was electrochemically pretreated in HClO
0.1 M) at 2.3 V until gold(III)oxide formation (red color) was SAM of MUATyr on gold sheets with (up) and without (down) laccase.
4
+
+
(
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immobilization of the enzyme, which took place spontaneously a small wave separation independently of the scan rate
within 2 h of immersion. Direct evidence for the immobiliza- (Fig. S1, ESI†).
tion process of laccase onto the SAM of MUATyr (Au-MUATyr-
Lac) was observed in the XPS spectrum in the N 1s region redox waves (DEfwhm = 280 mV, Fig. S1 and S2, ESI†) which
Fig. 2b) as the number of nitrogen atoms increased from 3.3 to indicated strong interactions between the terminal ferrocene
.9% (semiquantitative evaluation), caused by the presence of moieties. Moreover, the SAM of FcH presented high peak
In contrast to the latter, SAM of pure FcH exhibited broad
(
7
3
0
À1
the amino acid residues of laccase. The enzymatic activity separation (E
of the SAM of MUATyr-Lac was determined by the oxidation neous dependence on the scan rate, indicating quasireversible
a
À E
c
= 440 mV at 100 mV s ) with a simulta-
kinetics of ABTS and normalized against the area of the gold (i.e. slow) electron transfer.
À6
À2
sheets (5.19 Â 10 U cm ).
The difference found in the DEfwhm values of the pure and
mixed SAM of FcH suggests that FcH and MUATyr are distri-
Electrochemical behaviour of the mixed SAM of FcH and
MUATyr
35
buted homogeneously on the gold surface. The increase in
the rate of electron transfer of the ferrocene moieties, due to
The ferrocene moiety of FcH (Fig. 1) was chosen to enhance the the presence of MUATyr, was also desirable since FcH was
electron transfer process between the electrode and the immo- included in the SAM to promote a fast electron transfer with the
bilized enzyme as its equilibrium formal potential is lower than enzyme.
2
2,31
that reported for the T1 site of all laccases.
The electrochemical behaviour of the mixed SAM of MUATyr
Biocatalytic reduction of dioxygen
and FcH was studied by cyclic voltammetry (CV). The chemi- The electrochemical properties of electrodes modified with
cally reversible redox waves of FcH and the irreversible tyrosine FcH, MUATyr and supramolecularly associated laccase were
oxidation signal were observed at 0.42 V and 0.84 V, respectively studied. The electrochemical cell was initially deaerated with a
(Fig. 3a, dashed). The anodic shift of about 100 mV in the flow of nitrogen and the potential was swept in the anodic
ferrocene signal is in agreement with previous reports of direction up to 0.55 V in order to avoid the irreversible oxida-
3
2
36
SAM containing embedded ferrocene derivatives. The electro- tion of the Tyr moiety. Under these conditions it was possible
chemical confirmation of the formation of this SAM was to detect only the chemically reversible response of FcH. After
assessed by the linear dependence of the intensity of the anodic additional scans with the electrochemical system open to air,
3
3
and cathodic waves on the applied scan rate (Fig. S1, ESI†).
the cathodic current increased and a second wave at 0.52 V was
In the reductive desorption experiment, peaks corresponding recorded, corresponding to the presence of inflowing dioxygen
À11
À2
À2
37,38
to FcH at À1.0 V (G = 9.5 Â 10
mol cm ) and MUATyr at (Fig. 3b).
À11
À1.2 V (G = 8.5 Â 10
mol cm ) confirmed the presence of
After each deaeration of the cell, the same tendency was
the mixed monolayer (Fig. S2, ESI†), with a ratio very similar observed, confirming the dependence of this new electro-
1 : 1.18) to the equimolar proportion used in the formation of chemical signal on the concentration of dioxygen. Moreover,
the mixed SAM. when the potential was swept up to 1.00 V, not only the signals of
(
Additional information on the formed mixed SAM regarding FcH and MUATyr were detected, but also that of the reduction of
the interaction of terminal ferrocene species with MUATyr was dioxygen (Fig. 3a, solid). It has to be highlighted that the current
À2
obtained. The obtained width at the half maximum of the density of the dioxygen reduction reaction ( j = 23 mA cm , air
oxidation and reduction waves (DEfwhm) of 80 mV for FcH saturated) was even more intense than that of FcH, suggesting a
suggested an almost ideal (90.6/n mV) electrochemical beha- high surface density of catalytic sites (active laccase) with the
3
4
viour for this species. In this mixed SAM, FcH presented appropriate orientation of the enzyme (Fig. 4). The same electro-
a reversible (i.e. fast) electron transfer (E À E = 40 mV) with chemical determination, but with a SAM that only contained
a
c
Fig. 3 Cyclic voltammograms of (a) the mixed SAM of FcH and MUATyr (dashed) and the mixed SAM of FcH, MUATyr and immobilized laccase as a
second monolayer (solid) where the oxygen reduction is observed as a broad signal with its maximum at 0.52 V. (b) Increase in oxygen reduction as it
À1
diffuses to the modified electrode containing laccase. All scans were performed in the anodic direction; scan rate = 100 mV s
.
3
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