A laccase-wiring redox hydrogel for efficient catalysis of O2 electroreduction

Article abstract of DOI:10.1021/jp055654e

Laccase was earlier wired to yield an O₂ electroreduction catalyst greatly outperforming platinum and its alloys. Here we describe the design, synthesis optimization of the composition, and characterization of the +0.55 V (AgAgCl) laccase-wiring redox hydrogel, with an apparent electron diffusion coefficient (D_app) of 7.6 × 10^-7 cm ² s^-1. The high D_app results in the tethering of redox centers to the polymer backbone through eight-atom-long spacer arms, which facilitate collisional electron transfer between proximal redox centers. The O₂ flux-limited, true-area-based current density was increased from the earlier reported 560 to 860 μA cm^-2. When the O ₂ diffusion to the 7-μm-diameter carbon fiber cathode was cylindrical, half of the O₂ flux-limited current was reached already at 0.62 V and 90% at 0.56 V vs Ag/AgCl, merely -0.08 and -0.14 V versus the 0.7 V (Ag/AgCl) reversible O₂/H₂O half-cell potential at pH 5.

Full text of DOI:10.1021/jp055654e

11180
J. Phys. Chem. B 2006, 110, 11180-11187
A Laccase-Wiring Redox Hydrogel for Efficient Catalysis of O2 Electroreduction
Nicolas Mano,* Valentine Soukharev, and Adam Heller
Department of Chemical Engineering and Texas Materials Institute, The UniVersity of Texas at Austin,
Austin, Texas 78712
ReceiVed: October 4, 2005; In Final Form: February 23, 2006
Laccase was earlier wired to yield an O
alloys. Here we describe the design, synthesis optimization of the composition, and characterization of the
0.55 V (AgAgCl) laccase-wiring redox hydrogel, with an apparent electron diffusion coefficient (Dapp) of
2
electroreduction catalyst greatly outperforming platinum and its
+
-
7
2
-1
7
.6 × 10 cm s . The high Dapp results in the tethering of redox centers to the polymer backbone through
eight-atom-long spacer arms, which facilitate collisional electron transfer between proximal redox centers.
The O flux-limited, true-area-based current density was increased from the earlier reported 560 to 860 µA
cm . When the O diffusion to the 7-µm-diameter carbon fiber cathode was cylindrical, half of the O flux-
2
-
2
2
2
limited current was reached already at 0.62 V and 90% at 0.56 V vs Ag/AgCl, merely -0.08 and -0.14 V
versus the 0.7 V (Ag/AgCl) reversible O
2
/H
2
O half-cell potential at pH 5.
Introduction
the polymer, decrease upon its cross-linking, and are reduced
by attractive Coulombic, hydrogen-bonding, dipolar, or induced
dipolar interactions between segments, known to raise the glass
transition temperature. The Dahms-Ruff theory describes
charge transport by electron hopping between freely diffusing
Redox hydrogels constitute the only electron-conducting
phases in which the permeation of water-soluble biological
reactants and products, including water-soluble biochemicals,
ions, and electrons, are all rapid. Their rapid permeation enables
the transport of electrons between electrodes and multiple layers
of redox enzymes. The three-dimensional hydrogels obviate the
need of orienting the enzyme molecule on electrode surfaces,
which is otherwise required to reduce the electron-transfer
distance and allow electron exchange between enzymes and
electrodes.1 The hydrogels electrically wire the reaction
centers of co-immobilized enzymes to electrodes, and absence
of leachable components from the wired enzyme electrodes
allows their use in the body, in flow cells, and in miniature,
compartmentless biofuel cells, such as the glucose-O2 cell,
where glucose is electrooxidized to gluconolactone, and O2 is
3
1,32
redox centers. Blauch and Saveant
developed a bounded
diffusion model, predicting Dapp for polymers when the dis-
placement of the redox centers is rapid and extensive. According
to the model
-5
2
2
(
(k (δ + 3λ ) C ))
ex RT
Dapp
)
(1)
6
where kex is the solution-phase self-exchange rate of the redox
species, δ is the characteristic electron-hopping distance, the
distance across which the tethered redox center can actually
move is λ, and CRT is the concentration of the redox species.
When the redox centers are well-attached to the polymer and
their physical diffusion does not contribute to electron tran-
electroreduced to water.6-9 The transport of electrons through
redox polymers is measured by their apparent electrons diffusion
coefficients, Dapp. The coefficient can be determined by
electrochemical impedance spectroscopy,
1
0,11
by measuring the
transient currents passing through the polymer coated electrodes
3
3,34
when the potential is stepped,1
2-20
or by measuring the steady-
sport
14,21-27
statecurrentsusinginterdigitatedmicroelectrodearrays(IDAs).
Electrons can diffuse in redox polymers by several mecha-
nisms, including percolation between immobile redox centers,
collision of mobile reduced and oxidized redox centers and, in
2
k δ C
ex
RT
Dapp
)
(2)
6
some polymers also by electron or hole conduction through a
conjugated backbone.1
,14,28,29
In the absence of conjugated
The equation can be regarded as the extreme limit of eq 1, with
Dphys ) 0 (where Dphys represents physical diffusion in the
absence of electron hopping). The reported apparent electron
diffusion coefficients in redox hydrogels in equilibrium with
backbone conduction and unless the polymer is so heavily
loaded with redox centers that the percolation is rapid, the
dominant cause of conduction is collisional electron transfer
between reduced and oxidized redox centers tethered to the
polymer backbone. Because the rate of electron transferring
collisions increases with the mobility of the tethered redox
centers, the greater their mobility, the faster the electrons
diffuse.30 The segmental mobilities increase upon solvation of
-12
-6
2 -1
aqueous solutions range between 10 and 10 cm s . Forster
and co-workers systematically investigated electron transport
3
5-38
in redox hydrogel films,
exemplified by poly(4-vinylpy-
ridine), with part of the pyridines coordinated with [Os(bpy) -
2
+
/2+ 16,17,39,40
-9
2 -1 37
Cl]
.
The Dapp, typically of ∼10 cm s
in H2-
3
5,39
*
Corresponding author. E-mail: mano@mail.utexas.edu.
SO4 and in 1 M NaCl,
depended on the nature of the
1
0.1021/jp055654e CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/18/2006

Laccase-Wiring Redox Hydrogel
J. Phys. Chem. B, Vol. 110, No. 23, 2006 11181
SCHEME 1: Synthetic Route to Polymer I
(
A) H
glycol, 140 °C, 24 h. (D) NaH (1.1 equiv)/CH
NaI, DMF, 80 °C, 24 h. (F) H NNH , EtOH, reflux, 24 h. (G) Ethylene glycol, 140 °C, 24 h. (H) Chloride resin/H
J) 6-Bromohexanoic acid/DMF, 90 °C, 24 h. (K) TSTU/N,N-diisopropylethylamine, DMF, 7-8 h. (L) N,N-Diisopropylethylamine, DMF, rt, 24 h.
M) Chloride resin/H O, 24 h. (N) Ultrafiltration.
2
O, 40-50 °C, 24 h. (B) NaH/methyl p-toluenesulfonate, DMF, 0 °C to room temperature, 4 h. (C) (NH
I (1.0 equiv), DMF, 0 °C to room temperature, overnight. (E) NaH/N-(6-bromohexyl)phthalimide/
O/Air, 24 h. (I) NH PF /H O.
4 2 6
) OsCl (0.5 equiv), ethylene
3
2
2
2
4
6
2
(
(
2
electrolyte and its concentration, on the temperature, and on
methylenebisacrylamide. In conjugated redox polymers based
on the complexation of poly(2-(2-bipyridyl)-bibenzimidazole)
with bis(2,2′-bipyridyl) Ru or bis(2,2′-bipyridyl) Os , Cam-
41
the density of redox centers. Sirkar and Pishko reported a Dapp
value as low as 2 × 10-12 cm s for a hydrogel based on the
2
-1
2+
2+
1
0,11
-8
2
-1
copolymer of poly(ethylene glycol) diacrylate and vinylferrocene
eron and co-workers
reported Dapp ≈ 10 cm s in
42
in a phosphate buffer solution. Komura et al. reported Dapp ∼
× 10 cm s for the conjugated nonredox polymer poly-
N,N′-bis(3-pyrrol-1-yl-propyl)-4,4′-bipyridinium) chloride in
acetonitrile with 0.1 M Et4NClO4. For the conjugated nonredox
polymers polypyrrole and poly(1-methyl-3-pyrrol-1-methyl py-
-
10
2 -1
2
-8
2 -1 44
(
ridinium), Mao reported Dapp ≈ 10 cm s . The two highest
4
3
-6 2 -1 -5 2 -1
aqueous 0.2 M NaCl. Bu and co-workers reported Dapp values
between 6 × 10 and 6 × 10 cm s for redox hydrogels
made by copolymerizing vinylferrocene, acrylamide, and N,N′-
Dapp values, 1.7 × 10 cm s and 6 × 10 cm s were
-
8
-7
2
-1
45,46
reported by Murray and co-workers,
respectively, for poly-
[Os(bpy)2(vpy)2] sandwiched between a Pt and a porous Au

11182 J. Phys. Chem. B, Vol. 110, No. 23, 2006
Mano et al.
SCHEME 2: Structure of
PVI-[Os(2,2′,6′,2′′-terpyridine)-
4-aminomethyl-4′-methyl-2,2′-bipyridine. The solution of
4-phthalimidylmethyl-4′-methyl-2,2′-bipyridine (0.3 g, 0.91
mmol) and hydrazine hydrate (0.3 mL, 6.12 mmol) in 40 mL
of EtOH was refluxed for 7 h and then cooled to room
temperature and poured into 100 mL of brine. The solution was
basified to pH 12 with 2 M NaOH aq and extracted with CH2-
Cl2 (50 mL × 3). The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The product was purified
with alumina column chromatography (CHCl3/MeOH/isopro-
pylamine ) 90/9/1) to yield pale-yellow oil (0.15 g, 82%).
(4,4′-dimethyl-2,2′-bipyridine)2]Cl2
+
1
MS: m/z 200 (M ). H NMR (CDCl3, δ): 2.31 (s, 3H), 2.70
s, 2H), 4.15 (s, 2H), 7.00 (m, 1H), 7.10 (m, 1H), 8.16 (br s,
(
1
H), 8.25 (br s, 1H), 8.51-8.57 (m, 2H).
Os(4,4′-dimethyl-2,2′-bipyridine)2(4-aminomethyl-4′-methyl-
,2′-bipyridine)](PF6)2. The complex [Os(4,4′-dimethyl-2,2′-
[
2
50
bipyridine)2Cl2] (0.38 g, 0.6 mmol) was dissolved in a mixture
of methanol (10 mL), DMF (10 mL), and water (10 mL). The
ligand 4-aminomethyl-4′-methyl-2,2′-bipyridine (0.12 g, 0.6
mmol) was added to the solution, which was refluxed under
nitrogen for 36 h. After cooling to room temperature and
filtration, 150 mL of water was added, and the solution was
poured slowly into a rapidly stirred NH4PF6 solution (5 g in
electrode bathed in dry N2 and for doped poly(benzimidazoben-
zophenanthroline).
Because the model of Blauch and Saveant (eq 1) predicted
that Dapp should scale in redox hydrogels with the square of
the length of the tethers binding the redox centers to the
backbones, we synthesized earlier an enzyme wire6,47 with 13-
atom-long flexible tethers binding the redox centers to the
backbone. Because the enzyme wired was glucose oxidase, the
tethered redox centers were dialkylated biimidazole complexes
1
3
00 mL H2O). The precipitate was collected and redissolved in
0 mL of acetonitrile and precipitated again from NH4PF6
solution (3 g in 150 mL H2O). The dark-green precipitate was
collected by filtration, washed with water (40 mL × 3) and
ether (40 mL × 3), and dried in vacuo at 40-50 °C for 24 h
(0.5 g, 80%).
2
+/3+
of Os
0
. Their redox potential was -0.2 V/ AgAgCl, only
.16 V positive to the redox potential of the FAD/FADH2
48
cofactor of GOx at pH 7.2. The long tethers increased the
frequency of effective electron-transferring collisions between
reduced and oxidized osmium centers in chloride and other
Poly(4-(N-(5-carboxypentyl)pyridinium)-co-4-Vinylpyridine)
was synthesized by a reported method, except that the poly-
(4-vinylpyridine) used had an average molecular weight of
4
7
-
6
2
-1 47,49
solutions, making Dapp as large as 6 × 10 cm s .
1
Here we describe the synthesis and characteristics of the
earlier reported laccase-wiring +0.55 V/AgAgCl redox hydrogel
with a relatively high Dapp in a pH 5 aqueous citrate buffer
solution. It contains eight-atom-long tethers between its redox
centers and its backbone. To probe the effect of the tether, we
compare its electrochemical characteristics with those of a +0.55
V/AgAgCl redox hydrogel with a short tether, PVI-[Os(2,2′,
50 000 instead of 160 000. HNMR (DMSO-d6, δ): 8.72 (br s,
2.0H), 8.23 (br s, 11.6H), 7.46 (br s, 2.0H), 6.56 (br s, 11.2H),
4.45 (br s, 2.0H), 1-2.3 (br m, 28.4H). The number of protons
given for each peak is relative, providing integration ratios
between the polymer protons, and shows that about 15% of
pyridine units was quaternized by 6-bromohexanoic acid.
Polymer I. To a dry DMF suspension of poly(4-(N-(5-
carboxypentyl)pyridinium)-co-4-vinylpyridine) (75 mg) O-(N-
succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate
(27 mg, 90 µmol) was added, the mixture was stirred for 15
min, and then N,N-diisopropylethylamine (14.5 µL, 155 µmol)
was added. After stirring the reaction mixture for 8 h, [Os(4,4′-
dimethyl-2,2′-bipyridine)2(4-aminomethyl-4′-methyl-2,2′-bipy-
6′,2′′-terpyridine)(4,4′-dimethyl-2,2′-bipyridine)2]Cl]2. We also
reconfirm that the bioelectrocatalyst outperforms platinum in
the catalysis of electroreduction of O2 to water.
Experimental Section
Synthesis of PVP-[Os(4,4′-dimethyl-2,2′-bipyridine)2(4-
aminomethyl-4′-methyl-2,2′-bipyridine)Cl]2
+/3+
ridine)](PF ) (130 mg, 124 µmol) was added slowly, followed
by 14.5 µL of N,N-diisopropylethylamine. The solution was
. 4-Phthalim-
6 2
idylmethyl-4′-methyl-2,2′-bipyridine. A mixture of 4,4′-methyl-
stirred for 24 h and then poured into 100 mL of EtOAc. The
solvent was removed by decanting, and the precipitate was
redissolved in 10 mL of CH3CN. To the solution was added
BioRad AG 2-X8 chloride exchange resin and 100 mL of water.
The resulting mixture was stirred for 24 h, filtered, and dialyzed
2
1
,2′-bipyridine (1.84 g, 10 mmol), N-bromosuccinimide (1.9 g,
0.7 mmol), and benzoyl peroxide (100 mg) in carbon tetra-
chloride (50 mL) was refluxed for 20 h. The reaction mixture
was cooled on ice for 1 h and filtered to remove the red
precipitate. The solvent was removed, and the residue containing
+
with H O by an ultramembrane filtration under 70 psi argon
4
-bromomethyl-4′-methyl-2,2′-bipyridine (MS: m/z 264 (M ))
2
pressure, using Millipore polyethersulfone membrane with a
as a major product was dissolved in 80 mL of N,N-dimethyl-
formamide. Potassium phthalimide (3 g, 16.2 mmol) was added,
and the reaction mixture was stirred at 40 °C for 1 h under
argon atmosphere. To the mixture was added 250 mL of distilled
water, and the mixture was extracted with dichloromethane (150
mL × 3). The organic layer was collected, dried over Na2SO4,
and concentrated. The residue was purified by running it through
neutral alumina using ethyl acetate/hexane (10 f 50%) as the
eluent to yield white crystalline solid (0.5 g, 15%). MS: m/z
molecular weight cutoff of 10 000. The dialyzed polymer was
lyophilized to yield a brown solid. Yield: 0.16 g. Elemental
analysis: C 70.6, H 7.3, N 12.1. Accordingly, the ratio between
free pyridine, carboxypentyl-pyridine, and pyridine with osmium
complex units of the polymer is approximately 43:5:2.
Other Chemicals and Materials. AG1 × 4 chloride resin
was purchased from Bio-Rad Laboratories, Hercules, CA. All
other chemical reagents were purchased from Aldrich Chemi-
cals, Co. Deionized water was used for the synthesis and
analysis. All of the electrochemical measurements were per-
formed in a citrate buffer solution (pH 5, 0.2 M). The solutions
+
1
3
(
8
30 (M ). H NMR (CDCl3, δ): 2.45 (3H, s), 4.7 (2H, s), 7.20
1H, d), 7.29 (1H, d), 7.49 (2H, m), 7.85(2H, m), 8.20 (1H, s),
.35 (1H, s), 8.60 (1H, d), 8.94 (1H, d).

Laccase-Wiring Redox Hydrogel
J. Phys. Chem. B, Vol. 110, No. 23, 2006 11183
were made with deionized water that was passed through a
purification train (Sybron Chemicals Inc, Pittsburgh, PA). Poly-
(ethylene glycol) (400) diglycidyl ether (PEGDGE) was pur-
chased from Polysciences Inc., Warrington, PA. The synthesis
of the redox polymer PVI-[Os(2,2′,6′,2′′-terpyridine)(4,4′-di-
methyl-2,2′-bipyridine)2]Cl2 (polymer II) was previously re-
ported.2
,51,52
1
Instrumentation and Analysis. The H NMR spectra were
recorded with a Varian Inova-500 spectrometer (500 MHz), and
elemental analyses were performed by Quantitative Technolo-
gies, Inc., Whitehouse, NJ. The electrochemical experiments
were performed by using a CH Instruments electrochemical
detector model CHI832 bipotentiostat and a dedicated computer.
Surfaces coverages of the modified electrodes were determined
-
1
by integration of 1 mV‚s scan rate voltammetric waves of
the osmium redox complexes and were based on the geometric
areas of the 3-mm-diameter glassy carbon electrodes or the 25-
µm-diameter platinum electrodes. The thicknesses of the dry
and swollen redox polymer films were obtained from backscat-
tered electron micrographs, obtained using an environmental
scanning electron microscope (ESEM).2
7,47
The temperature was controlled with an isothermal circulator
(Fisher Scientific, Pittsburgh, PA). The measurements were
carried out in a water-jacketed electrochemical cell at 37.5 °C
containing 50 mL of buffer. To maintain a fixed volume of
solution in the cell, the bubbled gases were presaturated with
water by passage through a bubbler, which also contained the
pH 5 citrate buffer. The potentials were measured versus a
commercial Ag/AgCl (3 M KCl) reference electrode. The
counterelectrode was a platinum wire (BAS, West Lafayette,
IN) and the 25-µm-diameter platinum microelectrode was from
Cypress Systems, Lawrence, KS. Ultrapure argon was purchased
from Matheson (Austin, TX).
Rotating Disk Electrodes. Vitreous carbon rotating disk
Figure 1. Cyclic voltammograms of polymers I (A) and II (B) on
electrodes (V-10 grade from Atomergic (Farmingdale, NY) of
12,13
3-mm-diameter glassy carbon electrodes under argon; 0.2 M citrate
3
mm diameter were prepared as reported.
They were
-
1
buffer pH 5, 37 °C, 20 mV‚s
.
polished sequentially with slurries of 5, 1, and 0.3 µm alumina
particles (Buehler, Lake Bluff, IL), sonicated, and rinsed with
ultrapure water. The cleaning process was repeated until no
voltammetric features were observed in the potential range of
interest, -0.4 to 0.4 V vs Ag/AgCl, in 50 mV/s scans in PBS.
The total loaded amount of polymer and cross-linker was
Results
Figure 1A shows the cyclic voltammograms of polymer I,
and Figure 1B shows the cyclic voltammograms of polymer II
under argon (3-mm-diameter glassy carbon electrode, pH 5 0.2
M citrate buffer, 37 °C). The voltammograms are characteristic
of polymer-bound osmium complexes with redox potentials of
-2
constant at 100 µg‚cm . After deposition and drying, the films
were cured for at least 18 h at room temperature before the
electrodes were used.
-
1
+
550 mV vs Ag/AgCl. At the 20 mV s scan rate, the
voltammogram of polymer I exhibited a symmetrical wave with
5 mV peak separation; the separation for polymer II was >110
mV.
Figure 2 shows the dependence of the apparent diffusion
Pt Microelectrodes (25-µm Diameter). The electrodes were
electrochemically cleaned by cycling in 0.1 M H2SO4 between
potentials adequate to oxidize, then reduce, the platinum
surface.49 Excessive cycling was avoided in order to minimize
the extent of surface roughening. A total of 15 mg/mL of the
polymer solution was deposited by evaporation on the electrode
surface. Upon hydratation, polymer I swells by a factor 2.4 and
polymer II by a factor 2.3.47 The polymer thicknesses were ∼36
µm for polymer I and ∼34.5 µm for polymer II for assumed
6
coefficients of polymer I (solid circles) and polymer II (open
circles) on the cross-linker concentration. Each value shown is
the average of five measurements. Upon increasing the cross-
linker concentration from 0 to 25 wt %, the apparent diffusion
coefficient of polymer I decreased by about 8-fold, from 7.6 ×
-7
-7
2
-1
1
0
to 1 × 10 cm s . The apparent coefficient diffusion
3
15,47
densities of 1 g/cm .
of polymer II did not change significantly with the concentration
-
9
-9
Carbon Fiber Electrodes. Prior to their coating, the 7-µm-
of the cross-linker, decreasing from 6.2 × 10 to 5.6 × 10
2
2 -1
diameter fibers (0.0044 cm ) were made hydrophilic by exposure
cm s .
Figure 3 shows the shifts of the peak at half-height, Ewhm
53
to 1 Torr O2 plasma for 3 min. The cathodic catalyst were
,
made by published procedures2
,8,54
One cathodic catalyst
upon increasing the cross-linker weight percent. For polymer
consisted of the cross-linked adduct of 45.6 wt % laccase, 47.2
wt % redox polymer I, and 7.2 wt % PEGDGE, and the other
of the 40 wt % laccase, 52.8 wt % redox polymer II, and 7.2
wt % PEGDGE.
I, Ewhm was 90 mV at ∼1 wt % cross-linker content, increasing
to ∼135 mV at 25 wt %. For polymer II, E
whm
was 120 mV at
low ∼1 wt % cross-linker content, increasing to ∼145 mV at
25 wt %.

11184 J. Phys. Chem. B, Vol. 110, No. 23, 2006
Mano et al.
Figure 4. Dependence of the current density on the laccase weight
percentage for the bioelectrocatalysts made with polymer I (solid circles)
and with polymer II (open circles).
Figure 2. Dependence of Dapp of polymer I (solid circles, left axis)
and polymer II (open circles, right axis) on the weight fraction of the
PEGDGE cross-linker; 25-µm-diameter platinum electrode. Other
conditions as in Figure 1.
Figure 5. Polarizations of 7-µm-diameter, 2-cm-long carbon fiber
cathodes modified with laccase wired by polymer I (with tether, curve
1
), and polymer II (which has no tethers between its redox centers and
its backbone, curve 2). The cathode was coated with a film containing
5.6 wt % laccase, 47.2 wt % redox polymer I, and 7.2 wt % PEGDGE;
Figure 3. Dependence of the peak width at half-height, Ewhm for
polymer I, (solid circles, right axis) and polymer II (open circles, left
axis) on the weight fraction of the cross-linker PEGDGE; 25-µm-
diameter platinum electrode. Other conditions as in Figure 1.
4
or containing of 40 wt % laccase, 52.8 wt % redox polymer II; or 7.2
wt % PEGDGE 45.6 wt % laccase, 47.2 wt % polymer I, and 7.2 wt
%
PEGDGE. 0.1 M pH 5 citrate buffer, quiescent solution, under air,
-
1
Figure 4 shows the optimal composition (polymer/laccase/
cross-linker) for the bioelectrocatalysts made with polymer I
solid circles) and polymer II (open circles) in a 0.2 M pH 5
37 °C. Scan rate 1 mV s . Polarization of a 6-µm-diameter, 2-cm-
long smooth solid platinum fiber cathode in 0.5 M H SO (curve 3).
Quiescent solution, under air, 37 °C. Scan rate 1 mV s
2
4
-
1
.
(
citrate buffer through the 0-60 wt % range. In the first group
of experiments, the cross-linker (PEGDGE) weight percentage
was fixed at 7 wt %, and the total loading of all film components
%
PEGDGE, and 40 wt % laccase, 52.8 wt % redox polymer
II, and 7.2 wt % PEGDGE .
-
2
was fixed at 0.8 mg cm . Between 5 and 40 wt %, the current
density increased with the weight percentage of laccase, reaching
The polarizations (potential versus the reversible potential
of the O2 /H2O half-cell in the same electrolyte) of a miniature
carbon fiber cathode (7 µm diameter, 2 cm long) modified with
either the bioelectrocatalyst made with polymer I (Figure 5,
curve 1) or polymer II (Figure 5, curve 2) in a quiescent pH 5
citrate buffer under air at 37.5 °C are compared in Figure 5.
With polymer I, oxygen is electroreduced already at -20 mV,
and a current density of 0.86 mA‚cm is reached at -0.2 V vs
Ag/AgCl. With polymer II, the current density at -0.2 V vs
Ag/AgCl is less, 0.58 mA‚cm . On the 6-µm-diameter platinum
electrode, oxygen is electroreduced on the fiber at -120 mV,
-
2
a current density of 0.6 mA‚cm at 40 wt % laccase for
-
2
polymer II and reaching a current density of 0.8 mA‚cm at
46 wt % laccase for polymer I. Above these weight percent-
∼
ages, the current density declined rapidly. Precipitation, at-
tributed to the formation of a neutral electrostatic adduct between
the cationic polymer and the enzyme (pH ) 4), was observed
above 60 wt % laccase. In experiments where the weight percent
of the cross-linker was varied and the laccase/redox polymer
weight ratio was kept constant, the optimal PEGDGE wt % was
found to be 7.2. The resulting optimal catalysts were composed
of 45.6 wt % laccase, 47.2 wt % redox polymer I, and 7.2 wt
-
2
-2
-
2
and a current density of 0.86 mA‚cm is reached at -0.8 V vs
Ag/AgCl (Figure 5, curve 3).

Laccase-Wiring Redox Hydrogel
J. Phys. Chem. B, Vol. 110, No. 23, 2006 11185
Discussion
transport process is radial. The time-independent steady-state
current, iss, is given by
Design and Synthesis of Polymer I. The design of the +0.55
V vs Ag/AgCl potential redox polymer I followed that for the
reported glucose oxidase “wiring” polymer.47 The osmium
complex, with two 4,4′-dimethyl-2,2′ -bipyridine ligands and
one 4-aminomethyl-4′-methyl-2,2′-bipyridine ligand, was reacted
to form amides, with N-(5-carboxypentyl) pyridinium functions
of poly(4-vinylpyridine), (PVP), introduced by partially quat-
ernizing PVP with 6-bromohexanoic acid. Unlike the earlier
wires of laccase or bilirubin oxidase, in which the redox
functions were coordinatively bound to ring nitrogens of
backbone imidazoles, polymer I had eight-atom-long, flexible
tethers between its Os complexes to its backbone. The redox
function of polymer I is the (4,4′-dimethyl-2,2′-bipyridine)2(4-
iss ) 4nFDappCRTr
(3)
where n is a number of electrons transferred, Dapp is the apparent
diffusion coefficient describing homogeneous charge transport
through the deposit, CRT is the effective concentration of Os2
centers within the deposit, and r is the radius of the microelec-
trode. For short times, or fast scan rates, the depletion layer is
significantly thinner than either the film thickness or the radius
of the microelectrode, making the diffusion linear, and peaking
voltammograms, reminiscent of dissolved redox species at
macroelectrodes, are observed. In this case, Dapp was derived
of the voltammograms through the Randles-Sevcik equation3
by plotting the voltammetric peak currents (ip) versus the square
+
8,59
2+/3+
aminomethyl-4′-methyl-2,2′-bipyridine) complex of Os
; the
1
/2
root of the scan rates ν
redox potential of the complex is +0.55 V vs Ag/AgCl. This
redox potential brought the redox potential of the hydrogel close
to the redox potential of the type-1 copper centers of laccase,
so that the electron transfer from the polymer to laccase was
potential-wise downhill, but driven by the smallest necessary
potential gradient, and thus maintaining the potential at which
O2 was reduced highly oxidizing.55 The flexible spacer tethering
the redox centers to the backbone was made eight atoms long,
allowing effective electron-transferring collisions between the
redox centers of the polymer and between polymer and enzyme
redox centers, the tethered centers wiping overlapping volumes
of the hydrogel. As a result, the apparent electron diffusion
coefficient was high relative to that of other redox hydro-
3/2
1/2
1/2
0
.4463 × nF × A × D
× CRT × ν
app
ip )
(4)
1
/2
(RT)
where n is the number of electrons transferred, F is the Faraday
constant, R is the gas constant, T is the temperature, A is the
area of the electrode, and CRT is the effective electroactive site
The slope of the plots, which were, as
expected, linear, provided the product (Dapp CRT). By com-
bining eq 3 and 4, the absolute value of DCT and CRT was
5
8,60
concentration.
1
/2
-12
determined. For polymer II, the product DappCRT is 9.5 × 10
-
1
-1
1/2
-7
(
(
0.5 mol‚cm
s
and the product Dapp CRT is 1.2 × 10
0.4 mol‚cm s-1/2. These yield a value of Dapp of 6.2 ×
-2
1
2,13,30
gels.
of the Os2
self-exchange rate56 and stability against ligand substitution
In the redox centers of Polymer I, full coordination
-9
2
-1
-7
1
0
( 0.5 cm s . For polymer I, Dapp is 7.6 × 10 ( 0.5
+/3+
by polydentate ligands provided the sought high
2
-1
cm s . Evidently, the eight-atom-long flexible tethers allowed
the redox centers to sweep large volume elements and increased
the rate of electron-transferring collisions between the polymer
segments. Although the centers were tethered, they were mobile
within the volumes defined by their long tethers. The 120-fold
app
57
encountered with monodentate ligands.
Electrochemical Characteristics. Comparison of the char-
acteristics of polymer I with those of polymer II having the
same redox potential (+550 mV vs Ag/AgCl) but only a short
tether (Figure 1B) shows that, with the long tether, the transport
of electrons is enhanced: while the voltammogram of polymer
increase in D could have been caused also by higher kex or
3
1,32
CRT (eq 1),
but the concentration of the redox centers was
about the same in the two polymers, and it was unlikely that
-
1
2+/3+
I exhibited at 20 mV s scan rate, a symmetrical wave, with
only 65 mV separation of the anodic and cathodic peaks, the
peak separation for polymer II was >120 mV.58 Comparison
of the peak separations suggested that Dapp of polymer I was
considerably higher than polymer II.
the kex of their Os
complexes varied by more than an order
of magnitude. We attribute, therefore, the observed 120-fold
increase in Dapp to the 11-fold increase in λ, the physical
displacement of the redox centers about their equilibrium
positions, upon the eight-atom extension of the tether.
Covalent and Coordinative Cross-linking. As a redox
polymer is increasingly cross-linked, whether electrostatically
or covalently, its swelling is reduced. The gel becomes
mechanically tough, and at the same time, as shown by Oh and
Apparent Electron Diffusion Coefficients. The apparent
electron diffusion coefficient of a redox hydrogel is a function
of (a) the structure of the redox polymer particularly, as seen,
of the mobility of the redox functions; (b) the nature and
diffusivity of the solution-phase anion, diffusing into the redox
polymer and neutralizing the added positive charge when the
polymer is oxidized and leaving the polymer when it is reduced;
and (c) the cross-linking of the polymer, which reduces the
mobility of its segments.
1
9
43
12,14
Faulkner, Bu, and Rajagopalan,
the segments of its
polymer become less mobile, and as a result, Dapp decreases.
As seen in Figure 3, Dapp of polymer I decreases faster upon
cross-linking than that of the already heavily electrostatically
cross-linked polymer II. Neither of the two polymers was
sheared off electrodes rotated for 20 h at 500 rpm in pH 5 citrate
buffer at 37 °C while cycled between -0.4 and +0.1 V versus
The apparent electron diffusion coefficients Dapp were
-
2
measured in a 0.2 M citrate buffer at 37 °C at 100 µg‚cm
-1
Ag/AgCl at 50 mV s : the heights of the voltammetric peaks
polymer loading. Details of the measurement procedures were
reported earlier. In brief, the technique exploits the difference
in the diffusion field at microelectrodes for different experi-
mental time scales, i.e., linear versus radial diffusion at short
versus long times. For long times, or slow scan rates, a steady-
state plateau current is observed that is independent of the scan
rate for 0.1 < υ < 10 mV s-1 because the thickness of the
layer of the reduced or oxidized species that is depleted is
comparable to the radius of the microelectrode, and the charge
decreased only by 4% for both. This is in contrast with our
previous results with a PVI-[Os(4,4′-diamino-2,2′-bipyridine)2-
+/2+
47
Cl]
redox hydrogel. The difference is attributed to the
coordinative cross-linking of the PVI-[Os(4,4′-diamino-2,2′-
+
/2+
bipyridine) Cl]
2
redox hydrogel by partial exchange of the
inner-sphere chloride of its osmium complex by imidazoles of
61
neighboring chains upon redox cycling. The added cross-
linking decreases the segmental mobility, the diffusivity of
electrons, and thereby thickness of electroactive film. Because

11186 J. Phys. Chem. B, Vol. 110, No. 23, 2006
Mano et al.
the polymers of this study do not have an inner-sphere chloride,
no coordinative cross-linking occurred, and the polymers were
more stable under redox cycling. The tether did not stabilize or
destabilize the redox hydrogel.
Acknowledgment. This study was supported by the Office
of Naval Research (N00014-02-1-0144) and by the Welch
Foundation. N.M. thanks The Oronzio de Nora Industrial
Electrochemistry Fellowship of The Electrochemical Society.
Composition. There are four electron-transfer steps in the
wired-laccase-catalyzed electrooxidation of O2. At steady state,
the current associated with each step is necessarily the same.
The currents flowing from the cathode to the polymer (1),
through the redox hydrogel (2), from the polymer to the laccase
molecules (3) and from laccase molecules to O2, are all identical.
The limit of each of these currents is defined by the polymer/
enzyme ratio. The limits of (3) and (4) increase upon increasing
the polymer/enzyme mass ratio, unless the electrostatic adduct
between the polycationic polymer and the polyanonic enzyme
precipitates. The polymer/enzyme mass ratio can be increased
if the specific activity of the enzyme is higher and less enzyme
is required in the optimal composition. If the apparent electron
diffusion coefficient of the polymer is higher and less polymer
mass is required, the ratio can be decreased. Increasing Dapp
thus allowed a decrease in the polymer/enzyme/cross linker ratio
from 1.32 to 1. As in other wired enzyme films, cross-linking
reduces Dapp because the more rigid the gel, the less the
segmental mobility and the frequency of effective collisions of
the redox functions on which Dapp depends. Because the eight-
atom-long tethers increased Dapp 120-fold, we were able to
increase the PEGDGE cross-linker weight fraction to 7.2 wt
References and Notes
(
1) Heller, A. J Phys. Chem. B 1992, 96, 3579.
(2) Katz, E.; Buckmann, A. F.; Willner, I. J. Am. Chem. Soc. 2001,
23, 10752.
3) Mano, N.; Kim, H.-H.; Zhang, Y.; Heller, A. J. Am. Chem. Soc.
002, 124, 6480.
4) Baldini, E.; Dall’Orto, V. C.; Danilowicz, C.; Rezzano, I.; Calvo,
E. J. Electroanalysis 2002, 14, 1157.
5) Katz, E.; Willner, I.; Kotlyar, A. B. J. Electroanal. Chem. 1999,
79, 64.
1
(
2
(
(
4
1
2
(6) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2002, 124, 12962.
(7) Mano, N.; Mao, F.; Heller, A. ChemBioChem 2004, 5, 1703.
(8) Soukharev, V. S.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2004,
26, 8368.
9) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2003, 125, 6588.
10) Cameron, C. G.; Pickup, P. G. J. Am. Chem. Soc. 1999, 121, 11773.
(
(
(11) Cameron, C. G.; Pittman, T. J.; Pickup, P. G. J. Phys. Chem. B
001, 105, 8838.
(12) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970.
(13) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5976.
(14) Rajagopalan, R.; Aoki, A.; Heller, A. J. Phys. Chem. 1996, 100,
3719.
(15) De Lumley-Woodyear, T.; Rocca, P.; Lindsay, J.; Dror, Y.;
Freeman, A.; Heller, A. Anal. Chem. 1995, 67, 1332.
(16) Forster, R. J.; Vos, J. G.; Lyons, M. E. G. J. Chem. Soc., Faraday
Trans. 1991, 87, 3769.
(17) Forster, R. J.; Vos, J. G.; Lyons, M. E. G. J. Chem. Soc., Faraday
Trans. 1991, 87, 3761.
%
, from the earlier 5 wt %,2,8 and form a leather-like, thick,
and well-adhering cathodic gel.
(
(
18) Forster, R. J.; Vos, J. G. J. Electrochem. Soc. 1992, 139, 1503.
19) Oh, S.-M.; Faulkner, L. R. J. Am. Chem. Soc. 1989, 111, 5613.
Electrooreduction of O2 to Water. The hydrated films of
cross-linked polymer I constitute the best wires of laccase to
date. Because the cathode was now close to being fully covered
by the electrocatalytic film, the O2 flux-limited current density
(20) Oh, S.-M.; Faulkner, L. R. J. Electroanal. Chem. 1989, 269, 77.
(
(
21) Goss, C. A.; Majda, M. J. Electroanal. Chem. 1991, 300, 377.
22) Chidsey, C. E.; Feldman, B. J.; Lundgren, C.; Murray, R. W. Anal.
Chem 1986, 58, 601.
23) Feldman, B. J.; Murray, R. W. Anal. Chem. 1986, 58, 5844.
-2
increased from the earlier reported 560 µA cm (Figure 5, curve
(
2
4
5
), reached with polymer I, (PVI-spacer-[Os(2,2′,6′,2′′-terpyridine-
(24) Feldman, B. J.; Murray, R. W. Inorg. Chem. 1987, 26, 1702.
(25) Dalton, E. F.; Surridge, N. A.; Jernigan, J. C.; Wilbourn, K. O.;
Facci, J. S.; Murray, R. W. Chem. Phys. 1990, 140, 143.
2+/3+
-2
,4′-dimethyl-2,2′-bipyridine)2Cl]
) to 860 µA cm (Figure
, curve 1). When the O2 diffusion to the 7-µm-diameter carbon
(
26) Nishihara, H.; Dalton, F.; Murray, R. W. Anal. Chem. 1991, 63,
2955.
(27) Aoki, A.; Rajagopalan, R.; Heller, A. J. Phys. Chem. 1995, 99,
102.
28) Aoki, A.; Heller, A. J. Phys. Chem. 1993, 97, 11014.
29) Heller, A. Acc. Chem. Res. 1990, 23, 128.
(30) Rajagopalan, R.; Heller, A. Electrical “wiring” of glucose oxidase
fiber cathode was cylindrical, half of the O2 flux-limited current
was reached already at 0.62 V and 90% at 0.56 V vs Ag/AgCl,
merely -0.08 and -0.14 V versus the 0.7 V (Ag/AgCl)
reversible O2/H2O half-cell potential at pH 5. To show that the
laccase-containing hydrogel of polymer I was superior to
platinum in electrocatalyzing the reduction of O2 to water, we
compared, at 37 °C, the polarizations (potential versus the
reversible potential of the O2 /H2O half-cell in the same
electrolyte) of the wired-laccase-coated 7-µm-diameter carbon
fiber (in pH 5 citrate buffer), and of a 6-µm-diameter platinum
fiber (in 0.5 M H2SO4) (Figure 5, curve 3). The Pt fiber reached
5
(
(
in electrons conducting hydrogels. In Molecular Electronics: A Chemistry
for the 21st Century Monograph; Jortner, J., Ratner, M., Eds.; Blackwell
Science Ltd, Cambridge, MA, 1997; p 241.
(31) Blauch, D. N.; Saveant, J.-M. J. Phys. Chem. 1993, 97, 6444.
(32) Blauch, D. N.; Saveant, J.-M. J. Am. Chem. Soc. 1992, 114, 3323.
(
33) Laviron, E. J. J. Electroanal. Chem. 1980, 112, 1.
(34) Andrieux, C. P.; Saveant, J.-M. J. Electroanal. Chem. 1980, 111,
3
77.
(35) Forster, R. J.; Kelly, A. J.; Vos, J. G.; Lyons, M. E. G. J.
Electroanal. Chem. 1989, 270, 365.
36) Forster, R. J.; Vos, J. G. J. Chem. Soc., Faraday Trans. 1991, 87,
863.
-
2
9
0% of the limiting 0.9 mA‚cm current density only at a
polarization of -0.40 V, while the wired-laccase-coated fiber
reached 90% of the limiting O2 electroreduction current density
at a polarization as small as -0.07 V.
(
1
(
(
(
(
(
(
37) Forster, R. J.; Vos, J. G. J. Electroanal. Chem. 1991, 314, 135.
38) Keane, L.; Hogan, C.; Forster, R. J. Langmuir 2002, 18, 4826.
39) Forster, R. J.; Vos, J. G. Electrochim. Acta 1992, 37, 159.
40) Forster, R. J.; Vos, J. G. Langmuir 1994, 10, 4330.
41) Sirkar, K.; Pishko, M. V. Anal. Chem. 1998, 70, 2888.
42) Komura, T.; Kijima, K.; Yamaguchi, T.; Takahashi, K. J. Elec-
Conclusion
The redox polymer described efficiently connects the laccase
reaction centers to electrodes. Its redox potential allows poising
of the O2 cathode just 0.15 V negative to the redox potential of
the laccase copper cluster at pH 5. Its eight-atom-long flexible
spacer arm increases the apparent coefficient of electron
troanal. Chem. 2000, 468, 166.
(43) Bu, H.-Z.; English, A. M.; Mikkelsen, S. R. J. Phys. Chem. B 1997,
101, 9593.
48
(44) Mao, H.; Pickup, P. G. J. Am. Chem. Soc. 1990, 112, 1776.
(45) Jernigan, J. C.; Murray, R. W. J. Am. Chem. Soc. 1987, 109, 1738.
(46) Wilbourn, K.; Murray, R. W. J. Phys. Chem. 1988, 92, 3642.
-
7
2
-1
diffusion to 7.6 × 10 cm s , and the coordination of the
2+/3+
Os
centers with three substituted bipyridines provides
(47) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2002, 125, 4951.
48) Stankovich, M. T.; Schopfer, L. M.; Massey, V. J. Biol. Chem.
978, 253, 4971.
49) Forster, R. J.; Walsh, D. A.; Mano, N.; Mao, F.; Heller, A. Langmuir
(
stability against ligand substitution. The reduced redox centers
deliver electrons to laccase so rapidly and at such a small
potential gradient that the electroreduction of O2 to water is
much better catalyzed than by the classical catalyst, platinum.
1
2
(
004, 20, 862.
(50) Kober, E. M. Inorg. Chem. 1988, 27, 4587.

Laccase-Wiring Redox Hydrogel
J. Phys. Chem. B, Vol. 110, No. 23, 2006 11187
(
51) Barton, S. C.; Kim, H.-H.; Binyamin, G.; Zhang, Y.; Heller, A. J.
Phys. Chem. B 2001, 105, 11917.
52) Barton, S. C.; Kim, H.-H.; Binyamin, G.; Zhang, Y.; Heller, A. J.
Am. Chem. Soc. 2001, 123, 5802.
(57) Binyamin, G.; Chen, T.; Heller, A. J. Electroanal. Chem. 2001,
500, 604.
(58) Kim, H.-H.; Mano, N.; Zhang, Y.; Heller, A. J. Electrochem. Soc.
2003, 150, A209.
(
(
53) Sayka, A.; Eberhart, J. G. Solid State Technol. 1989, 32, 69.
(59) Forster, R. J.; Vos, J. G. J. Inorg. Organomet. Polym. 1991, 1, 67.
(60) Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals
and Applications, 2nd ed.; John Wiley & Sons: New York, 2001.
(61) Gao, Z.; Binyamin, G.; Kim, H.-H.; Barton, S. C.; Zhang, Z.; Heller,
A. Angew. Chem., Int. Ed. 2002, 41, 810.
(54) Mano, N.; Fernandez, J. L.; Kim, Y.; Shin, W.; Bard, A. J.; Heller,
A. J. Am. Chem. Soc. 2003, 125, 15290.
(
(
55) Marcus, R. A. J. Phys. Chem. 1963, 67, 853.
56) Ryabov, A. D. AdV. Inorg. Chem. 2004, 55, 201.