Long tethers binding redox centers to polymer backbones enhance electron transport in enzyme "wiring" hydrogels

Article abstract of DOI:10.1021/ja029510e

A redox hydrogel with an apparent electron diffusion coefficient (D_app) of (5.8 ± 0.5) × 10^-6 cm² s^-1 is described. The order of magnitude increase in D_app relative to previously studied redox hydrogels results from the tethering of redox centers to the backbone of the cross-linked redox polymer backbone through 13 atom spacer arms. The long and flexible tethers allow the redox centers to sweep electrons from large-volume elements and to collect electrons of glucose oxidase efficiently. The spacer arms make the collection of electrons from glucose oxidase so efficient that glucose is electrooxidized already at -0.36 V versus Ag/AgCl, the reversible potential of the redox potential of the FAD/FADH₂ centers of the enzyme at pH 7.2. The limiting current density of 1.15 mA cm^-2 is reached at a potential as low as -0.1 V versus Ag/AgCl. The novel redox center of the polymer is a tris-dialkylated N,N′-biimidazole Os^2+/3+ complex. Its redox potential, -0.195 V versus Ag/AgCl, is 0.8 V reducing relative to that of Os(bpy)^2+/3+, its 2,2′-bipyridine analogue.

Full text of DOI:10.1021/ja029510e

Published on Web 03/28/2003
Long Tethers Binding Redox Centers to Polymer Backbones
Enhance Electron Transport in Enzyme “Wiring” Hydrogels
Fei Mao,† Nicolas Mano,‡ and Adam Heller*.‡
Contribution from TheraSense Inc., 1360 South Loop Road, Alameda, California 94502, and
Department of Chemical Engineering and Texas Materials Institute,
The UniVersity of Texas at Austin, Austin, Texas 78712
Received November 27, 2002; Revised Manuscript Received February 24, 2003; E-mail: Email: heller@che.utexas.edu
Abstract: A redox hydrogel with an apparent electron diffusion coefficient (Dapp) of (5.8 ( 0.5) × 10-6 cm2
s-1 is described. The order of magnitude increase in Dapp relative to previously studied redox hydrogels
results from the tethering of redox centers to the backbone of the cross-linked redox polymer backbone
through 13 atom spacer arms. The long and flexible tethers allow the redox centers to sweep electrons
from large-volume elements and to collect electrons of glucose oxidase efficiently. The spacer arms make
the collection of electrons from glucose oxidase so efficient that glucose is electrooxidized already at -0.36
V versus Ag/AgCl, the reversible potential of the redox potential of the FAD/FADH2 centers of the enzyme
at pH 7.2. The limiting current density of 1.15 mA cm-2 is reached at a potential as low as -0.1 V versus
Ag/AgCl. The novel redox center of the polymer is a tris-dialkylated N,N′-biimidazole Os2+/3+ complex. Its
redox potential, -0.195 V versus Ag/AgCl, is 0.8 V reducing relative to that of Os(bpy)2+/3+, its 2,2′-bipyridine
analogue.
polymers with Dapp values as low as 10-12 cm2 s-1 and as large
as 10-6 cm2 s-1. The coefficient can be determined by
electrochemical impedance spectroscopy,8,9 by measuring the
transient currents passing through the polymer-coated electrodes
when the potential is stepped4,10-19 or by measuring the steady-
state currents using interdigitated microelectrode arrays
(IDAs).4,20-26 While the IDA-measured Dapp is independent of
the diffusion coefficient of the mobile counterions, it depends
on the microscopic counterion displacement associated with the
reduction or oxidation of a particular redox center,27,28 the
subject of theoretical studies of Dahms,29 Laviron,30 Andrieux,31
Introduction
The conduction of electrons in redox polymers has been the
subject of studies spanning three decades.1,2 Electrons are
transported in redox polymers by hopping between immobile
redox centers, by collisions of mobile reduced and oxidized
redox centers, and in some through conjugated and conductive
backbones.3-6 In the absence of conjugation and unless the
polymer is so heavily loaded with redox centers that hopping
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 the segmental mobility of the polymer,
the faster the electrons diffuse.7 The segmental mobilities
increase upon solvation of the polymer, decrease upon its cross-
linking, and are reduced by attractive Coulombic, hydrogen-
bonding, dipolar, or induced-dipolar interactions between seg-
ments, which raise the glass transition temperatures of polymers.
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11779.
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The transport of electrons through redox polymers is meas-
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Variations in the controlling parameters have led to redox
.
† TheraSense In.
‡ The University of Texas at Austin.
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J. AM. CHEM. SOC. 2003, 125, 4951-4957
4951

A R T I C L E S
Mao et al.
Ruff,32,33 and Buck.34,35 In conjugated redox polymers based
on the complexation of poly(2-(2-bipyridyl)bibenzimidazole)
with bis(2,2′-bipyridyl) Ru2+ or bis(2,2′-bipyridyl) Os2+, Cam-
eron and co-workers reported Dapp ≈ 10-8 cm2 s-1 in acetonitrile
with 0.1 M Et4NClO4.8,9 For the conjugated nonredox polymers
polypyrrole and poly(1-methyl-3-pyrrol-1-methylpyridinium),
10-10 cm2 s-1 for the conjugated nonredox polymer poly(N,N′-
bis(3-pyrrol-1-yl-propyl)-4,4′-bipyridinium) chloride in aqueous
0.2 M NaCl. Bu and co-workers57 reported Dapp values between
6 × 10-8 and 6 × 10-7 cm2 s-1 for redox hydrogels made by
copolymerizing vinylferrocene, acrylamide, and N,N′-methyl-
enebisacrylamide.
Mao and Pickup reported Dapp ≈ 10-8 cm2 s-1 36
.
The two
Blauch and Saveant58,59 developed a bounded diffusion model
to predict Dapp for polymers when the displacement of the redox
centers is rapid and extensive. According to the model
highest Dapp values, 1.7 × 10-6 and 6 × 10-5 cm2 s-1 were
reported by Murray and co-workers,37,38 respectively, for poly-
[Os(bpy)2(vpy)2] sandwiched between a Pt and a porous Au
electrode bathed in dry N2 and for doped poly(benzimidazo-
benzophenanthroline).
Dapp ) 1/6kex(δ2 + 3λ2)CRT
(1)
Redox hydrogels constitute the only electron-conducting
matrixes in which both the permeation of water-soluble biologi-
cal reactants and products and the transport of electrons are
rapid. They are particularly relevant to electrochemical bio-
sensors and biofuel cells,6,39-48 in which redox hydrogels
electrically “wire” reaction centers of coimmobilized enzymes
to electrodes. The absence of leachable components from the
“wired” enzyme electrodes allows their use in the body, in flow
cells, and in miniature, compartmentless biofuel cells, exempli-
fied by the glucose-O2 cell, in which glucose is electrooxidized
to gluconolactone, while O2 is electroreduced to water. The
reported values of Dapp in redox hydrogels in equilibrium with
where kex is the solution-phase self-exchange rate of the redox
species, δ is the characteristic electron hopping distance, λ is
the distance across which the tethered redox center can actually
move, and CRT is the concentration of the redox species. The
model suggested that Dapp could scale in redox hydrogels4,7 with
the square of the length of the tethers. Here we describe the
synthesis, characteristics, and application of a redox hydrogel
with a particularly high Dapp. Its redox polymer differs from
earlier redox polymers by having 13-atom-long tethers between
the redox centers and the polymer backbone. When the hydrogel
swells, the tethers and their terminal cationic redox center
become mobile, sweeping large volume elements. They collect
electrons from redox centers of coimmobilized glucose oxidase
efficiently already at highly reducing potentials.
aqueous solutions range between 10-12 and 10-7 cm2 s-1 40,44
.
Forster and co-workers systematically investigated electron
transport in redox hydrogel films,49-52 exemplified by poly(4-
vinylpyridine) with part of the pyridines coordinated with
Experimental Section
Synthesis of PVP-[Os(N,N′-dialkylated-2,2′-biimidazole)3]2+/3+
.
[Os(bpy)2Cl]+/2+ 15,16,53,54
.
D
app, typically of ∼10-9 cm2 s-1 51
in H2SO4 and in 1 M NaCl,49,53 depended on the nature of the
electrolyte and its concentration, the temperature, and the loading
of redox centers. Sirkar and Pishko55 reported a Dapp value as
low as 2 × 10-12 cm2 s-1 for a hydrogel based on the copolymer
of poly(ethylene glycol) diacrylate and vinylferrocene in a
phosphate buffer solution. Komura et al.56 reported Dapp∼2 ×
The synthesis of polymer I is outlined in Scheme 1 and its details are
provided as Supporting Information. 2,2′-Biimidazole60 was bismeth-
ylated in DMF with methyl iodide in the presence of sodium hydride,
and the resulting N,N′-dimethyl-2,2′-biimidazole (DMB) was purified
by sublimation. To introduce the 13-atom-long flexible tether, the
primary amine of the N-(6-aminohexyl)-N′-methyl-2,2′-biimidazole
(AMB) ligand was condensed with the alkylidene carboxylate attached
to the backbone of the polymer, as described below. AMB was
synthesized in three steps: In the first, 2,2′-biimidazole was methylated
with 1 equiv of methyl iodide, the reaction yielding a mixture of mono-
and dimethylbiimidazole and unreacted 2,2′-biimidazole. The desired
monomethylbiimidazole, was, however, the major product and was
readily isolated by recrystallization from EtOAc/hexane. In the second
step, the monomethylbiimidazole was alkylated with N-(6-bromohexyl)-
phthalimide plus sodium hydride, with sodium iodide activating the
bromoalkyl compound. In the third step, the ligand, in which the primary
amine was still protected, was deprotected by hydrazine in refluxing
ethanol. After silica gel column chromatography with NH4OH/CH3CN,
the yield of AMB was 50%.
The primary amine-containing osmium complex was synthesized in
a one-pot reaction. Heating of 2 equiv of DMB and 1 equiv of
NH4OsCl6 (or K2OsCl6) in ethylene glycol under nitrogen yielded the
[OsIII(DMB)2Cl2]Cl intermediate, which, without isolation, was directly
reacted with 1 equiv of AMB to give the desired complex [OsII(DMB)2-
(AMB)](Cl)2. The complex was readily air oxidized, its green color
changing to blue as the [OsIII(DMB)2(AMB)](Cl)2 formed. To convert
the dichloride to DMF-soluble [OsIII(DMB)2(AMB)](PF6)3, the aqueous
solution of [OsII(DMB)2(AMB)](Cl)2 was stirred with beads of the
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Heller, A. J. Am. Chem. Soc. 2001, 123, 8630-8631.
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8848.
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124, 6480-6486.
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9
4952 J. AM. CHEM. SOC. VOL. 125, NO. 16, 2003

Electron Transport in Enzyme “Wiring” Hydrogels
A R T I C L E S
Scheme 1. Synthetic Route to Polymer I, the Chloride of PVP-[Os(N,N′-dialkylated-2,2′-biimidazole)3]2+/3+a
a Reagents and Conditions: (a) H2O, 40-50 °C, 24 h; (b) NaH/methyl p-toleunesulfonate, DMF, 0 °C to room temperature, 4 h; (c) 0.5 equiv of (NH4)2OsCl6,
ethylene glycol, 140 °C, 24 h; (d) 1.1 equiv of NaH/1.0 equiv of CH3I, DMF, 0 °C to room temperautre, overnight; (e) NaH/N-(6-bromohexyl)phthalimide/
NaI, DMF, 80 °C, 24 h; (f) H2NNH2, EtOH, reflux, 24 h; (g) ethylene glycol, 140 °C, 24 h; (h) chloride resin/H2O/air, 24 h; (i) NH4PF6/H2O; (j) 6-bromohexanoic
acid/DMF, 90 °C, 24 h; (k) TSTU/N,N-diisopropylethylamine, DMF, 7∼8 h; (l) N,N-diisopropylethylamine, DMF, room temperature, 24 h; (m) chloride
resin/H2O, 24 h; (n) ultrafiltration.
chloride form of Bio-Rad AG1 × 4 anion-exchange resin, air-oxidized,
and then stirred with an excess of NH4PF6 to precipitate the air- and
humidity-stable [OsIII(DMB)2(AMB)](PF6)3 in high yield.
the quantitative quaternization were confirmed by 1H NMR. The
reactive N-hydroxysuccinimidyl ester of the (4-(N-(5-carboxypentyl)-
pyridinium)-co-4-vinylpyridine) polymer was formed in DMF by
reacting the polymer with O-(N-succinimidyl)-N,N,N′,N′-tetramethyl-
uronium tetrafluoroborate (TSTU) and a base. The NHS ester of the
Poly(4-vinylpyridine) (PVP; MW ∼160 000) was quaternized with
1 equiv of 6-bromohexanoic/∼6 mers of vinylpyridine. The ratio and
9
J. AM. CHEM. SOC. VOL. 125, NO. 16, 2003 4953

A R T I C L E S
Mao et al.
Chart 1. Structure of Polymer II,
+/2+
2
PVI-[Os(4,4′-diamino-2,2′bipyridine) Cl]
Figure 1. Cyclic voltammograms of adsorbed, non-cross-linked thin films
of polymers I (heavy line) and II (fine line) on a 3-mm-diameter glassy
carbon electrode under argon: 0.1 M NaCl, 20 mM phosphate buffer pH
7, 37 °C, 20 mV/s. Potentials versus that of the Ag/AgCl (3M KCl) reference
electrode.
polymer was not isolated but directly reacted at room temperature while
still in the DMF solution with a 1.3-fold excess of OsIII(DMB)2(AMB)]-
-
(PF6)3 to form the PF6 salt of the desired blue redox polycation I.
-
The PF6 salt of the polymer was soluble in polar organic solvents
such as CH3CN and DMF but was insoluble in water. To convert it to
The thicknesses of the dry and swollen redox polymer films were
obtained from backscattered electron micrographs, produced by an
environmental scanning electron microscope (ESEM).26 The imaged
film consisted of 91 wt % polymer and 9 wt % cross-linker. After the
film was deposited on the polished side of silicon wafers, it was cured
at room temperature for >24 h. The wafer was then cleaved to allow
cross sectional imaging of the polymers film.
Electrodes. Vitreous carbon rotating disk electrodes (V-10 grade
from Atomergic (Farmingdale, NY) of 3-mm diameter were prepared
as previously reported.12,13 They were 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 between the
potentials 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 constant
at 130 µg/cm2, with 2 µL of a 4.3 mg/mL polymer/PEDGE solution
deposited on the surface. After deposition and drying, the films were
cured for at least 18 h at room temperature before the electrodes were
used.
Carbon Fiber Electrodes. Prior to their coating, the 7-µm-diameter
fibers (0.0044 cm2) were made hydrophilic by exposure to 1 Torr O2
plasma for 3 min.61 The anodic catalyst solution was made as follows:
100 µL of 40 mg/mL GOx in 0.1 M NaHCO3 was oxidized by 50 µL
of 7 mg/mL NaIO4 in the dark for 1 h, and then 2 µL of the periodate-
oxidized GOx was mixed with 8 µL of a 10 mg/mL concentration of
the chloride of polymer II or of polymer I and a 0.5-µL droplet of 2.5
mg/mL PEGDGE. A 5-µL aliquot of this solution was applied to the
fiber. The resulting electrocatalyst consisted of the cross-linked adduct
of 39.6 wt % GOx, 59.5 wt % redox polymer, and 0.9 wt % PEDGE.
the water soluble polymer, the PF6 was Cl- exchanged using the
-
chloride of the AG1 × 4 resin. The polymer was characterized by
measuring its Os, C, and N content and its NMR spectrum. These
showed that 70% of the N-(5-carboxypentyl)pyridinium functions
reacted to form the amide with the osmium complex. The (tethered
complex)/pyridine/(carboxypentylpyridinium) ratio was 10.6:85:4.4.
Other Chemicals and Materials. Ultrapure argon was purchased
from Matheson (Austin, TX). K2OsCl6 was purchased from Alpha
Chemicals, N-(6-bromohexyl)phthalimide from Lancaster, and AG1 ×
4 chloride resin from Bio-Rad Laboratories (Hercules, CA). Glucose
oxidase from Aspergillus niger was purchased from Fluka (Milwaukee,
MI). The other chemicals were purchased from Aldrich Chemicals (St.
Louis, MO). The electrochemical measurements were performed in pH
7.4 20 mM phosphate-buffered 0.15 M NaCl (PBS). The solutions were
made with water deionized in a purification train (Sybron Chemicals
Inc, Pittsburgh, PA). Poly(ethylene glycol) (400) diglycidyl ether
(PEGDGE) was purchased from Polysciences Inc., Warrington, PA.
The synthesis of redox polymer II PVI-[Os(4,4′-diamino-2,2′-
bipyridine)2Cl]+/2+, shown in Chart 1, was previously reported.44
Instrumentation and Analysis. 1H NMR spectra were obtained on
a JEOL 400 or a Nicolet/GE NT300 NMR spectrometer by Acorn
NMR, Inc. (Livermore, CA) and elemental analyses were performed
by Quantitative Technologies, Inc. (Whitehouse, NJ). Both cyclic
voltammetry and double-step chronoamperometry were performed using
a bipotentiostat (CH-Instruments, Electrochemical Detector model
CHI832) and a dedicated computer. In the chronoamperometric
experiments, the potential was stepped between -0.4 and +1.2 V versus
Ag/AgCl and 20 measurements were averaged. The contribution from
the capacitive background was estimated by stepping the potential from
+0.1 to 1.2 V, where no Faradic reaction occurred. After extrapolation,
this capacitive component was subtracted from the total current
measured for the Faradic reaction. Surface coverages of the modified
electrodes were determined by integration of the voltammetric waves
of the redox polymer films at 1 mV/s, assuming that the geometrical
area of the polished 3-mm-diameter glassy carbon electrodes was their
true area.
Results and Discussion
Polymer I, PVP-[Os(N,N′-dialkylated-2,2′-bi-imidazole)3]2+/3+
,
with a 13-atom-long flexible tether, had a novel redox function,
a tris-N,N′-dialkylated 2,2′-biimidazole complex of Os2+/3+. Its
redox potential, -0.195 V versus Ag/AgCl in a 0.1 M chloride
solution (Figure 1) was 0.8 V negative of the familiar analogous
tris-2,2′-bipyridine complex of Os2+/3+, Os(bpy)32+/3+. The
reference PVI-[Os(diamino-bipyridine)2Cl]+/2+, polymer II,
without a tether between its redox function and the backbone,
had a similar redox potential of -0.185 V versus Ag/AgCl.
The measurements were carried out in a water-jacketed electro-
chemical cell containing 50 mL of PBS at 37.5 °C. To maintain a fixed
volume of solution, the bubbled gases were presaturated with water.
The potentials were measured versus a Ag/AgCl (3 M KCl) reference
electrode. The counter electrode was a platinum wire (BAS, West
Lafayette, IN).
(61) Sayka, A.; Eberhart, J. G. Solid State Technol 1989, 32, 69-70.
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4954 J. AM. CHEM. SOC. VOL. 125, NO. 16, 2003

Electron Transport in Enzyme “Wiring” Hydrogels
A R T I C L E S
Figure 1 shows the cyclic voltammograms of adsorbed but
not cross-linked thin films of polymer I (heavy line) and polymer
II (fine line) under argon in pH 7, 0.1 M NaCl, 20 mM
phosphate buffer at 37 °C. The voltammograms are characteristic
of polymer-bound osmium complexes. At 1 mV/s scan rate,
polymer I exhibited a symmetrical wave, with only slight (5
mV) separation of the oxidation and reduction peaks, while
polymer II exhibited a separation of 40 mV. At 20 mV/s scan
rate, the voltammogram of polymer I exhibited a symmetrical
wave with 40-mV peak separation, the separation for polymer
II was >100 mV. Comparison of the peak separations suggested
that Dapp of polymer I was considerably higher than that of
polymer II.
The Dapp of redox hydrogels depends on the mobility of their
tethered redox function; the solution-phase anion, diffusing into
and out of the redox polymer when the polymer is oxidized
and reduced; and the cross-linking of their polymer, which
1/2
reduces the mobility of the segments. The products Dapp
C
RT
Figure 2. Dependence of Dapp polymers II (open circles, right axis) and
polymer I (solid circles, left axis) on the weight fraction of the PEGDGE
cross-linker. The results obtained were identical when obtained by cyclic
voltammetry using the Randles-Sevcik equation and by double-potential
stepped chronoamperometry with a large-amplitude potential step applied
to avoid effect of the iR drop across the polymer film, by plotting i versus
t1/2 using the Cottrell equation. Conditions as in Figure 1.
were obtained from the cyclic voltammograms by the Randles-
Sevcik equation (eq 2),52,62 by plotting the voltammetric peak
ip ) 0.4463nF3/2ADapp C ν1/2/(RT)1/2
(2)
1/2
RT
currents (ip) versus the square root of the scan rates (ν1/2). In
eq 2, n is the number of electrons transferred, F is the Faraday’s
constant, R is the gas constant, T is the temperature, A is the
area of the electrode, and CRT is the effective electroactive site
concentration.44,63 CRT in the hydrated film was calculated from
the weight of polymer loaded per unit area and from its swelling,
determined by cross sectional ESEM imaging. The images in
Figure 6A and B are cross sectional electron micrographs of
dry and hydrated films, respectively, of the cross-linked polymer
I. As seen, the polymer swells upon hydration in PBS to 3.4
times its initial thickness (∼60 µm dry vs ∼200 µm hydrated).
As seen in the images of Figure 7, polymer II swells upon its
hydration much less, only by 60% (∼70 µm dry vs ∼120 µm
hydrated). The polymer loadings on all electrodes were fixed
at 130 µg/mL, an amount that would yield a 1.3-µm-thick dry
and a 4.4-µm-thick hydrated film for polymer I and a 1.3-µm-
thick dry and a 2.2-µm-thick hydrated film for polymer II, for
an assumed density of 1 g/cm3.14 The resulting estimate of Dapp
is (5.8 ( 0.5) × 10-6 cm2 s-1 for polymer I and (3.4 ( 0.4)
10-9 cm2 s-1 for polymer II. Dapp of polymer I was measured
also by double-potential stepped chronoamperometry,63 with a
large-amplitude potential step applied to avoid the effect of the
iR drop across the polymer film,1,64,65 by plotting i versus t1/2
per the Cottrell equation (eq 3).63 The value obtained was
Figure 2 shows the dependence of the Dapp of polymers I
(solid circles) and II (open circles) on the weight percent of
the cross-linker. Each value reported is the average for four
double-step chronoamperometric and cyclic voltammetric meas-
urements. Upon increasing the weight percent of the cross-linker
from 0 to 25 wt %, the apparent diffusion coefficient of polymer
I decreased ∼19-fold, from 5.8 × 10-6 cm2 s-1 to 0.3 × 10-6
cm2 s-1. The apparent diffusion coefficient of polymer II did
not change significantly with the weight percent of the cross-
linker decreasing only from 3.4 × 10-9 cm2 s-1 to 2.3 × 10-9
cm2 s-1. Figure 3 shows the shifts of the peak potential Ep (open
circles, right axis) and peak width at half-height, Ewhm, (solid
circles, left axis) upon increasing the weight percent of the cross-
linker. For polymer I (Figure 3A), Ewhm was 90 mV at low
(∼2 wt %) cross-linker content, increasing to ∼200 mV at 25
wt %, with Ep increasing from -0.195 to -0.110 V vs Ag/
AgCl. Figure 3B shows that Ewhm for polymer II was 120 mV
at ∼2 wt % cross-linker, increasing to ∼150 mV at 25 wt %,
while Ep shifted from -0.195 to -0.155 V vs Ag/AgCl.
Swelling was reduced as the cross-linking of the redox polymer
increased. Consistent with the observations of Oh and Faulkner,18
Bu,57 and Rajagopalan4 as the segments became less mobile,
Dapp decreased.
The stability of the integrals of the voltammetric waves of
the cross-linked films of polymers I and II is shown in Figure
4. The films contained 9 wt % of the cross-linker and were
cured for 24 h at room temperature. In their stability test, the
electrodes were rotated for 20 h at 300 rpm in the pH 7.2, 0.1
M NaCl 20 mM phosphate buffer at 37 °C, while their potential
was cycled between -0.4 and +0.1 V versus Ag/AgCl at 50
mV/s. The heights of the voltammetric peaks decreased by 4%
for polymer I (solid circles) and by 12% for polymer II (open
circles). Neither the peak potential (∆EP) nor the width of the
peak at half-height (Ewhm) changed for either polymer. As seen
in the figure, the integral of polymer I was considerably more
stable than that of polymer II. The difference is attributed to
the coordinative cross-linking of polymer II: upon redox cycling
i ) nFADapp C π-1/2 1/2
t
(3)
1/2
RT
essentially identical with that obtained by the first method, (5.2
( 0.3) × 10-6 cm2 s-1. The observed 1700-fold difference in
Dapp is attributed to a 41-fold increase in λ, the physical dis-
placement of the redox centers about their equilibrium positions
(eq 1) upon the 13-atom extension of the length their tether.
(62) Forster, R. J.; Vos, J. G. J. Inorg. Organomet. Polym. 1991, 1, 67-86.
(63) Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals and
Applications; 2nd ed.; John Wiley and Sons: New York, 2001.
(64) Murray, R. W. Molecular Design of Electrode Surfaces; Wiley: New York,
1992.
(65) Andrieux, C. P.; Haas, O.; Saveant, J.-M. J. Am. Chem. Soc. 1986, 108,
8175-8182.
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A R T I C L E S
Mao et al.
Figure 4. Percent decrease in the integrated voltammetric waves of cross-
linked (9 wt % cross-linker) films of polymers I (solid circles) and II (open
circles) upon cycling the applied potential for 20 h between -0.4 and +0.1
V vs Ag/AgCl at 50 mV/s. The loading of the polymers was 130 µg/cm2.
Electrodes rotating at 300 rpm, pH 7.2, 0.1 M NaCl, 20 mM phosphate
buffer; 37 °C.
Figure 3. Dependence of the peak potential Ep (open circles, right axis)
and the peak width at half-height, Ewhm (solid circles, left axis), on the
weight fraction of the cross-linker PEGDGE: (A) polymer I; (B) polymer
II. Conditions as in Figure 1.
Figure 5. Polarization of 7-µm-diameter, 2-cm-long carbon fiber anode
modified with PVI-[Os(4,4′-diamino-2,2′-bipyridine)2Cl]+/2+ (fine line) and
with PVP-[Os(N,N′-dialkylated-2,2′biimidazole)3]2+/3+ (heavy line). Quies-
cent solution, under air, 37.5 °C, PBS buffer, 15 mM glucose, 1 mV/s.
the inner-sphere chloride of the osmium complex of polymer
II is exchanged by imidazoles of neighboring chains.66 The
added cross-linking decreases the segmental mobility, the
diffusivity of electrons, and thereby the thickness of the
electroactive film. Because the complex of polymer I does not
have an inner-sphere chloride, it is not coordinatively cross-
linked and is more stable under redox cycling.
The polarizations of miniature carbon fiber anodes (7 µm
diameter, 2 cm long) modified with 39.6 wt % glucose oxidase,
0.9 wt % PEDGE, and 59.5 wt % concentration of either
polymer II (fine line) or polymer I (heavy line) in a quiescent
pH 7.2, 0.1 M NaCl, 20 mM phosphate, 15 mM glucose solution
under air at 37.5 °C are compared in Figure 5. Glucose was
electrooxidized on the fiber with polymer I at - 0.1 V versus
Ag/AgCl at a current density of 1.15 mA/cm2 and the threshold
for glucose electrooxidation was -0.36 V versus Ag/AgCl. On
the fiber with polymer II, the current density at -0.1 V versus
Ag/AgCl was only 0.15 mA/cm2. The hydrated films of cross-
linked polymer I constitute the best electron-conducting hy-
drogels and the best “wires” of glucose oxidase to date. Even
though the redox potential of the hydrogel formed upon cross-
linking polymer I with PEGDGE is highly reducing, -195 mV
versus Ag/AgCl, the polymer efficiently oxidizes the FADH2
centers of glucose oxidase. The Figure 5 polarization curves of
7-µm-diameter, 2-cm-long carbon fiber anodes made by wiring
GOx with polymers I and II show that the limiting current
density of glucose electrooxidation is reached with both
polymers already at - 0.1 V versus Ag/AgCl, only 0.26 V
positive the redox potential of the FAD/FADH2 cofactor in GOx
at pH 7.2.67 The limiting current density of electrodes made
with polymer II is, however, only 0.15 mA/cm2, well below
the 1.15 mA/cm2 limiting current density of the electrode made
(66) Gao, Z.; Binyamin, G.; Kim, H.-H.; Barton, S. C.; Zhang, Z.; Heller, A.
Angew. Chem., Int. Ed. 2002, 41, 810-813.
(67) Stankovich, M. T.; Schopfer, L. M.; Massey, V. J. Biol. Chem. 1978, 253,
4971-4979.
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Electron Transport in Enzyme “Wiring” Hydrogels
A R T I C L E S
Figure 6. Cross sectional scanning electron micrographs of films of cross-
linked polymer I on a polished silicon wafer. The images were obtained
using backscattered electron detection: (A) image of the dry film; (B) the
same film after its hydration. 9 wt % PEDGE. Scale 250 µm, Magnification
×200. Pressure 4.9 T. The dark zones on the left are silicon; the redox
polymer films are lighter.
Figure 7. Cross sectional scanning electron micrographs of films of cross-
linked polymer II on a polished silicon wafer. The images were obtained
using backscattered electron detection: (A) image of the dry film; (B) the
same film after its hydration. 9 wt % PEDGE. Scale 200 µm. Magnification
× 200. Pressure 4.9 T. The dark zones on the left are silicon; the redox
polymer films are lighter.
V versus Ag/AgCl, the redox potential of the enzyme’s redox
centers.67 A 1.15 mA/cm2 limiting current density of glucose
electrooxidation is reached at a potential as reducing as -0.1
V versus Ag/AgCl. In combination with a reported cathode,
enabling the rapid four-electron electroreduction of molecular
oxygen to water under physiological conditions,42 this glucose
anode forms the basis for a compartment-less, miniature glucose/
O2 biofuel cell.40,68
with polymer I. Electrodes made with polymer I are the only
ones on which the electrooxidation of glucose starts already at
the redox potential of the FAD/FADH2 centers of GOx, -360
mV versus Ag/AgCl67 at pH 7.2. (Figure 5) Thus, the long
tethers not only increase the apparent diffusivity of electrons
but also facilitate electron transfer from the reduced (FADH2)
reaction centers of glucose oxidase to the redox polymer.
Conclusion
Acknowledgment. We thank Dr. Hyug-Han Kim for the
synthesis of polymer II and Prof. Michael A. Schmerling for
assistance with the ESEM. Gary Sandhu effectively assisted
F.M. in the synthesis of polymer I. The study was supported
by the Welch Foundation and by the Office of Naval Research
(Grant N00014-02-1-0144).
Supporting Information Available: Additional information
as noted in text. This material is available free of charge via
the Internet at http://pubs.acs.org.
The redox potential of the novel tris(N, N′-dialkylated 2,2′-
biimidazole) Os2+/3+ complex, -0.195 V versus Ag/AgCl, is
0.8 V reducing relative to its familiar 2,2′bipyridine analogue,
2+/3+
Os(bpy)32+/3+. Like Os(bpy)3
and its derivatives, the
dialkylated bi-imidazole complex is a fast redox couple. Its
tethering to the backbone of a polymer through a 13-atom-long
flexible spacer and cross-linking and hydration of the resulting
polymer yields a hydrogel in which electrons diffuse 1 order
of magnitude more rapidly [Dapp ) (5.8 ( 0.5) × 10-6 cm2
s-1] than in previously reported hydrogels. When the hydrogel
“wires” the FAD/FADH2 centers of glucose oxidase to an
electrode, glucose is electrooxidized at pH 7.2 already at -0.36
JA029510E
(68) Mano, N.; Mao, F.; Shin, W.; Chen, T.; Heller, A. Chem. Commun. 2003,
518-519.
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J. AM. CHEM. SOC. VOL. 125, NO. 16, 2003 4957