investigation of novel mediators for a glucose biosensor based on metal picolinate complexes

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Bioelectrochemistry 6

Investigation of novel mediators for a glucose biosensor

based on metal picolinate complexes

Susan Warren, Timothy McCormac, Eithne DempseyT

Electrochemical Technology Research Centre, Dept. Applied Science, Institute of Technology Tallaght, Tallaght, Dublin 24, Ireland

Received 22 March 2004; received in revised form 2 June 2004; accepted 5 July 2004

Available online 25 February 2005

Abstract

The metal complexes [Os(byp)2(pic)]+ and [Ru(byp)2(pic)]

+ where byp is 2,2V-bipyridine and HPic is o-picolinic acid were synthesised

and characterised using spectroscopic and electrochemical techniques. These complexes were then evaluated as mediators for a glucose

oxidase (GOx)-based biosensor. Results demonstrate the electrocatalytic behaviour of both metal couples towards regeneration of the

flavoprotein GOx (FADH2) group, when co-immobilised with glucose oxidase. Surface immobilisation was achieved by potential cycling in

aqueous solutions of the metal complexes at a glucose oxidase (GOx)/Nafion modified electrode. This proved successful in terms of catalytic

efficiency and stability of redox sites. Kinetic parameters associated with both enzymatic and mediator reactions were estimated and the

stability/performance properties of the sensor were tested.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Mediator; Biosensor; Electrocatalysis

1. Introduction

Biosensors continue to attract considerable attention as

potential replacements for a range of analytical techniques

due to their unique properties. The fabrication of a glucose

sensor is an important area of research due to the fact that

determination of blood and urine glucose levels in a rapid,

convenient and precise manner is necessary for the

diagnosis and management of diabetes mellitus [1,2].

The most widely studied group of enzymes for electro-

chemical sensors are the oxidases, which under aerobic

conditions, allow hydrogen peroxide from the reduction of

dioxygen to be monitored amperometrically during the

enzymatic reaction sequence. As the responses of oxygen/

hydrogen peroxide-based sensors are affected by fluctua-

tions in the concentration of dissolved oxygen, electron

mediators [3], e.g., quinones [4,5], ferrocenes [6,7],

viologens [8] and Ru [9,10], and Os complexes [11,12]

have been employed to eliminate this effect and to

1567-5394/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bioelechem.2004.07.005

T Corresponding author. Tel.: +353 1 404 28 62; fax: +353 404 27 00.

E-mail address: [email protected] (E. Dempsey).

decrease the applied potential with which to follow the

enzyme catalysed reaction. However, all mediators used in

bioelectrochemistry and biosensors are also general elec-

trocatalysts and therefore compromise the selectivity of

these devices.

Transition-metal complexes have proven to be suitable

redox mediators, which offer adaptability in terms of the

possibility of varying the ligand shell of the central metal,

hence modulating the redox potential [10]. There is

considerable interest in the chemistry of ruthenium and

osmium due to the versatile electron transfer properties

exhibited by their complexes. The variation in the

coordination environment around the metal plays a key

role in modulating the redox properties of the complexes.

In this work, we have replaced the Cl ligand of both

Ru(bpy)2Cl2 and Os(bpy)2Cl2 with picolinic acid. The

picolinate ion binds metal ions as bidentate N,O donors

forming a five-membered chelate ring [13] and, to date,

such complexes have received relatively little attention.

Mediated electrocatalysis is a powerful tool for electron

transfer measurements of redox active biomolecules. The

rate at which the mediator exchanges electrons with an

7 (2005) 23–35

S. Warren et al. / Bioelectrochemistry 67 (2005) 23–3524

enzyme is a consideration in bioelectrochemistry and a

critical factor in the design of biosensors [14]. Analysis of

the dependence of the electrocatalytic currents on mediator,

enzyme and substrate concentrations can lead to isolation of

the dynamics of specific reactions steps [15,16].

An important step in sensor design and optimisation is

the characterisation of the diffusion and kinetic parameters

of the immobilisation matrix. In many such electrocatalytic

sensor systems, charge transport between redox molecules

in a polymer matrix plays an essential role in their function.

The redox molecules employed here are incorporated into a

Nafion (sulphonated perfluoroalkyl polyanion polymer)

membrane by electrostatic attraction during potential

cycling. The mobility of the redox centre determines the

mechanism of charge transport, i.e., either physical diffusion

of the molecules, charge hopping or a combination of both

processes [17].

The main aims of this study are to synthesise the metal

complexes [Os(byp)2(pic)]+ and [Ru(byp)2(pic)]

+ (organic

and aqueous salts), to characterise the products by

spectroscopic and electrochemical means and to perform

an examination of their mediation properties with respect

to glucose oxidase. We describe a stable and reproducible

method of immobilising the redox species within an

anionic polymer/enzyme layer and study the characteristics

of the film, which allows regeneration the FAD site of

glucose oxidase. Some kinetic and analytical performance

parameters of the reagentless sensor are reported and to the

best of our knowledge this is the first such report which

examines the electrocatalytic/mediation capabilities of

these compounds.

Fig. 1. H1 NMR assignment of [Ru(bpy)2Pic]PF6.

2. Experimental

2.1. Materials

RuCl3d xH2O (Aldrich), N98% 2-picolinic acid (Fluka),

HPLC Grade Methanol (Aldrich), 95+% ammonium

hexafluorophosphate–NH4PF6 (Aldrich), DOWEX 1�4,

50–100-mesh ion-exchange resin (Aldrich), potassium

hexachloroosmate–K2OsCl6 (Aldrich), 99+% ethylene

glycol (Aldrich) and tetrabutylammonium perchlorate–

TBAP (Sigma) were used as received. HPLC grade

acetonitrile–CH3CN (Aldrich) (for electrochemical experi-

ments) was dried using 4-A molecular sieves, 8–12-mesh

(Aldrich). HPLC grade dichloromethane (Labscan), deu-

terated dichloromethane-d2 99.5 at.% D, deuterated

methyl-d3 alcohol-d 99.8 at.% D (SpectranalR), phos-

phate-buffered saline tablets (PBS) (Aldrich), sodium

dihydrogen phosphate ACS (Merck), potassium chloride–

KCl (Fluka), glucose oxidase from Aspergillus niger 200

units/mg (Fluka) and 25% aq. glutaraldehyde (Sigma), 5%

Nafion (Fluka) were used as received. d-(+)-Glucose

anhydrous was obtained from Fluka, solutions of which

were made 24 h before use to allow for mutarotation.

2.2. Synthesis of complexes

Ru(bpy)2Cl2d 2H2O and Os(bpy)2Cl2 were synthesised as

described previously by Sullivan et al. [18] and Habermfller

et al. [19], respectively. Preparation of [Ru(bpy)2(pic)]PF6was based on a previously described method [20] with the

following variations.

Ru(bpy)2Cl2d 2H2O (0.2 mmol/0.100 g) and 2-picolinic

acid (0.6 mmol/0.074 g) was refluxed in 50% aq. methanol

(166 cm3) in the dark under a nitrogen atmosphere for 12 h.

To ensure completion, the reaction was monitored using

cyclic voltammetry and TLC. The TLC system used was

EtOAc/MeOH/AcOH/H2O in the ratio of 15:5:1:1.

Upon completion of the reaction, the solution changed

from a dark purple to a deep red colour. The solution was

cooled and the volume was reduced to circa 10 cm3 prior to

the dropwise addition of a saturated aqueous solution of

NH4PF6. The precipitated PF6 salt of the complex was then

filtered off and dried under vacuum at 60 8C, 200 mbar

overnight. Percentage yield of product obtained was 90%/

0.123 g.

H1 NMR: (300 MHz, dichloromethane-d2) (Fig. 1):

bipyridine ring A and X, d: 8.95 (dd, H1A), 8.15 (H2A),

8.05 (H3A) 7.25 (H4A), 7.45 (H1X), 7.35 (H2X), 7.85 (H3X),

8.35 (H4X). bpy rings B and Y, d: 7.85 (H1B), 7.60 (H2B),

7.85 (H3B), 8.30 (H4B), 7.6 (H1Y), 7.25 (H2Y) 7.85 (H3Y),

8.3 (H4Y). Picolinic acid ring C, d: 7.85 (H1C), 7.45 (H2C),

8.05 (H3C), 8.35 (H4C).

C13 NMR: d:159.3, 158.3, 157.7, 153.4, 152.0, 151.3,150.2, 137.6, 137.0, 137.0, 136.6, 128.7, 127.9, 127.6,

127.5, 127.4, 126.7, 126.7, 124.3, 123.8, 123.6, 123.4.

Synthesis of [Os(bpy)2(pic)]PF6 involved dissolving

Os(bpy)2Cl2 (0.35 mmol/200 mg) and 2-picolinic acid

(0.70 mmol/86.2 mg) in 25-cm3 ethylene glycol and

refluxing in the dark, under a nitrogen atmosphere for 3 h.

The reaction was monitored using cyclic voltammetry to

ensure completion. This was indicated when the potential of

the Os(II)/(III) redox process increased from �0.2 to +0.2 V

vs. Ag/Ag+. The solution was cooled, the same volume of

water added to the flask and then filtered. A saturated

S. Warren et al. / Bioelectrochemistry 67 (2005) 23–35 25

solution of NH4PF6 was added to the solution and the

resulting precipitate was filtered off and washed with cold

water. The solid was then dried in a vacuum oven at 60 8C,200 mbar overnight. Percentage yield of product obtained

was 80%/0.228 g.

H1 NMR: bipyridine ring A and X, d: 7.75 (dd, H1A),

8.08 (H2A), 7.75 (H3A), 7.05 (H4A), 7.3 (H1X), 7.3 (H2X),

7.75 (H3X), 8.24 (H4X). bpy rings B and Y, y: 7.75 (H1B),

7.05 (H2B), 7.75 (H3B), 8.37 (H4B), 7.75 (H1Y), 7.35 (H2Y),

7.75 (H3Y), 7.06 (H4Y). Picolinic acid ring C, d: 7.75 (H1C),

7.3 (H2C), 7.75 (H3C), 8.24 (H4C).

C13 NMR: d: 151.2, 150.2, 150.1, 149.2. 135.4, 134.9,134.9, 134.7, 134.0, 126.5, 125.8, 125.3, 125.1, 124.4,

123.7, 122.1, 121.6, 120.8, 120.5.

2.2.1. Conversion from [M(bpy)2Pic]PF6 to [M(bpy)2Pic]Cl

(M=Ru/Os)

Cations were isolated as hexafluorophosphate salts

(Scheme 1) and converted to aqueous soluble chloride

salts using the method employed previously by Gregg and

Heller [12] using Os(bpy)2Cl-PVP redox polymers. The

DOWEX ion-exchange resin was pre-treated before use by

washing sequentially with 20 cm3 volumes of 2 M HCl

and 2 M NaOH, while rinsing with 20 cm3 of deionised

water in between acid and base. This was repeated three

times, after which the resin was rinsed with water until the

eluent was pH neutral. A total of 0.12 mmol of the organic

salt of each metal complex was dissolved in acetonitrile (4

cm3) and diluted with deionised water (10 cm3). The ion-

Scheme 1. Synthetic scheme and struc

exchange resin (1.040 g) was added to this solution and

stirred in the dark for 2 h. The resin was then filtered off,

rinsed with minimal amounts of cold water and the solvent

removed under vacuum to produce a dark red powder. The

product was then dried in a vacuum oven at 60 8C, 200mbar overnight.

2.3. Procedures

2.3.1. Biosensor preparation

Ten milligrams of glucose oxidase was mixed with 40

mg of bovine serum albumin in 1 cm3 of 0.1 M KCl/PBS

(freshly prepared solution). A total of 100 Al of this enzyme

solution was mixed with 20 Al of 2.5% glutaraldehyde and

30 Al 5% Nafion. Twenty microliters of this mixture was

then manually deposited onto the surface of a clean glassy

carbon electrode and allowed to crosslink for approximately

1 h at room temperature. The enzyme electrode was then

stored in phosphate buffer at 4 8C overnight to equilibrate.

The mediator was incorporated into the enzyme layer by

cycling the modified electrode in a 4 mM solution of the

[M(bpy)2(pic)]Cl salt for 200 cycles at 0.1 V s�1 between

the limits of 0.4 to +1.0 V vs. Ag/AgCl|KCl (3 M) for the

ruthenium complex and 0.0 to +0.6 V vs. Ag/AgCl|KCl (3

M) for the osmium complex. The electrolyte employed was

0.5 M KCl in 0.25 M sodium dihydrogen phosphate buffer

adjusted to pH 7.2 with 4 M NaOH. After cycling, the

electrode was rinsed with deionised water and 2�5 Al of4% Nafion applied to the surface, allowing the first layer to

tures of complexes synthesised.


dry before application of the second. When the second

Nafion layer was dry, the modified electrode (M(bpy)2(pic)/

GOx/Naf) was stabilised by cycling through the metal

couple for 200 cycles at 0.1 V s�1 using the same potential

limits as above.

2.3.2. Electrochemical characterisation

The M(bpy)2Cl2 ruthenium and osmium starter com-

pounds and PF6 salts of the [M(bpy)2(pic)]+ complexes

were analysed by cyclic voltammetry using tetrabutylam-

monium perchlorate (TBAP) in dried acetonitrile using a

non-aqueous reference electrode. The reference electrode

employed was a silver wire in contact with an acetonitrile

solution of AgNO3 (0.01 M) and 0.1 M of the same

supporting electrolyte as that employed in the cell and

will be hereafter referred to as Ag/Ag+. o-Picolinic acid

and [Ru(bpy)2(pic)]Cl were analysed in solutions of PBS

vs. Ag/AgCl|KCl (3 M). The [Os(bpy)2(pic)]Cl complex

was studied in a 0.5 M KCl solution made up with 0.25

M sodium dihydrogen phosphate, adjusted to pH 7.2

using 4 M NaOH, with an Ag/AgCl|KCl (3 M) reference.

All solutions were degassed with premium grade argon

for a minimum of 5 min prior to analysis.

The oxidation peak potential for each metal complex

(0.76 V vs. Ag/AgCl|KCl (3 M) for the [Ru(bpy)2(pic)]+

and 0.33 V vs. Ag/AgCl|KCl (3 M) for the [Os(bpy)2(pic)]+) was used for hydrodynamic amperometric experi-

ments in which the modified electrode was immersed in

a continuously stirred solution of PBS. Upon application

of the potential, the background current decreased and

when a steady baseline was achieved, successive addi-

tions of glucose were made to the solution, allowing

steady state currents to be generated, until a total con-

Table 1

Electrochemical data for all starting materials and metal complexes

Ep,a (V) Ep,c (V)

Ru(bpy)2Cl2a �1.829 �2.029

0.074 0.014

1.744 –

Picolinic acidb 1.156 –

�0.699 –

[Ru(bpy)2Pic]PF6a �1.713 �1.771

�1.942 �2.003

0.600 0.547

2.066 –

[Ru(bpy)2Pic]Clb 0.803 0.738

Os(bpy)2Cl2a �1.866 �1.930

�0.307 �.0368

[Os(bpy)2Pic]PF6a �1.988 �1.922

�1.732 �1.653

0.149 0.196

1.566 –

[Os(bpy)2Pic]Clb 0.382 0.315

a At GCE in 0.1 M tetrabutylammonium perchlorate, scan rate=0.1 V s�1 vs. Ab At GCE in PBS, scan rate=0.1 V s�1 vs. Ag/AgCl|KCl (3 M).

centration of 10 mM glucose had been added. This

experiment was repeated in triplicate with the same

modified electrode.

2.4. Apparatus

Cyclic voltammetry and amperometry experiments were

carried out using a CH Instruments CHI 750 potentiostat. A

single-compartment electrochemical cell was used with a

platinum counter electrode and Ag/AgCl|KCl (3 M)

reference for aqueous solutions and Ag/Ag+ for non-

aqueous solutions. NMR studies were carried out on a Joel

300 MHz spectrometer.

3. Results and discussion

3.1. Electrochemical characterisation of [Os(byp)2(pic)]+

and [Ru(byp)2(pic)]+ complexes

Electrochemical data for starting material and com-

plexes is summarised in Table 1. Cyclic voltammograms of

[Ru(bpy)2(pic)]PF6 show a reversible one-electron couple

at 0.575 V vs. Ag/Ag+ representing the Ru(II/III) redox

process. The same process for the aqueous salt [Ru(bpy)2(pic)]Cl was at 0.77 V vs. Ag/AgCl|KCl (3 M) (Fig. 2(a)

and (b)).

½RuIIðbpyÞ2ðpicÞ�þf½RuIIIðbpyÞ2ðpicÞ�

2þ þ e�

The potential of this couple is more positive in this

complex than in Ru(bpy)2Cl2 (E1/2=0.044 V vs. Ag/Ag+),

which indicates that the +2 state in this mixed ligand

complex is more stable. Similarly, the [Os(bpy)2(pic)]PF6

E1/2 (V) DE (V) Process

�1.929 0.200 bpy0Ybpy�1

0.044 0.060 RuIIYRuIII

– – Irr. ox. wave


– – Irr. red. wave

�1.744 0.058 bpy0Ybpy�1

�1.973 0.061 bpy�1Ybpy�2




�1.898 0.064 bpy0Ybpy�1

�0.338 0.061 OsIIYOsIII

�1.955 0.066 bpy�1Ybpy�2

�1.693 0.079 bpy0Ybpy�1

0.173 0.049 OsIIYOsIII


0.3485 0.067 OsIIYOsIII

g/Ag+ non-aqueous reference electrode.

Fig. 2. (a) [Ru(bpy)2(pic)]PF6 at GCE in 0.1 M tetrabutylammonium perchlorate/acetonitrile at 0.100 V s�1. (b) [Ru(bpy)2 (pic)]Cl at GCE in PBS at 0.100

V s�1. (c) [Os(bpy)2(pic)]PF6 at GCE in 0.1M tetrabutylammonium perchlorate/acetonitrile at 0.100 V s�1. (d) [Os(bpy)2 (pic)]Cl at GCE in PBS at 0.100 V s�1.



and [Os(bpy)2(pic)]Cl salts (Fig. 2(c) and (d)) exhibited a

more positive metal (Os(II/III) couple (E1/2=0.173 and

0.348 V vs. Ag/Ag+, respectively)) compared with E1/2=

�0.338 V vs. Ag/Ag+ for Os(bpy)2Cl2.

Ghatak et al. [13] reported the gradual decrease in the

potential of the Ru(III) couple in the series [Ru(bpy)3]2+

1.30 V, [Ru(bpy)2(pic)]+ 0.75 V, [Ru(bpy)(pic)2] 0.44 V and

Ru(pic)3 �0.09 V[13], which reflects the ability of the

anionic picolinate ligand to stabilise Ru(III) better than the

neutral pyridyl ligand.

It is well known that each bipyridine ligand can

successively accept two electrons into one electrochemically

accessible lowest unoccupied molecular orbital [21]. Hence,

four successive reductions may be expected, but only the

first two of these are observed in the case of [Ru(bpy)2(pic)]PF6 (at E1/2=�1.97 and �1.734 V vs. Ag/Ag+) and

[Os(bpy)2(pic)]PF6 (E1/2=�1.95 and �1.69 V vs. Ag/Ag+)

as solvent reduction precedes them. This is consistent with

results from Couchman et al. [22] who demonstrated that

[Ru(bpy)2(pic)]PF6 showed two reversible one-electron

Fig. 3. (a) Growth of [Os(bpy)2(pic)+] at GOx/Nafion enzyme electrode in 4 mM

cycles 200). (b) Stable redox couple of [Os(bpy)2(pic)]/GOx/Nafion enzyme electr

PBS) at 0.005 V s�1.

waves at �1.87 and �2.21 V vs. Fc/Fc+ corresponding to

the ligand centred processes below.

½RuIIðbpyÞ2ðpicÞ�þ þ e�f½RuIIðbpyÞðbpy�ÞðpicÞ�

þ e�f½RuIIðbpy�Þ2ðpicÞ��

An irreversible oxidation wave at 1.156 V was evident in

the case of picolinic acid alone and was present in

[Ru(bpy)2(pic)]PF6 at 2.06 V (Fig. 1(a)) and [Os(bpy)2(pic)]PF6 at 1.566 V vs. Ag/Ag+ (Fig. 1(c)).

3.2. Immobilisation of [M(bpy)2(pic)]+ metal complexes

within Nafion/glucose oxidase layer

As the aim of this study is to achieve a reagentless sensor

for glucose various immobilisation procedures were inves-

tigated in order to form a stable mediator/enzyme layer.

These included electrostatic deposition by soaking at the

GOx/Nafion modified electrode, manual deposition of the

organic [M(bpy)2(pic)]PF6 salts as both inner and outer

layers and finally potential cycling in a solution containing

[Os(bpy)2(pic)]Cl at 0.1 V s�1. Cycles shown are 1, 5, 10, 25 and 50 (total

ode following growth and potential cycling in fresh electrolyte (0.5 M KCl/


the metal complex at the GOx/Nafion electrode. The latter

method proved to be the most successful and was used

throughout this study (as described in Section 2.3.1).

Fig. 3(a) shows deposition of the [Os(bpy)2(pic)]+

complex from the chloride salt (4 mM) at a GOx/Nafion

modified electrode via potential cycling (200 cycles, at 0.1

V/sec) with a current increase for the Os(II/III) couple from

0.4 to 8.1 AA during growth. The electrode was then washed

with deionised water and two additional layers of Nafion

(4%) were deposited on the surface followed by cycling

(200 cycles at 0.1 V s�1) in fresh PBS until currents were

stable (E1/2 for Os(II/III)=0.328 V with DEp=0.074 V at

0.005 V s�1). The film exhibited a 50% decrease in

electroactivity over this period prior to stabilisation. Fig.

3(b) shows a cyclic voltammogram of the stable film.

Films were prepared in a similar manner from [Ru

(bpy)2(pic)]Cl showing current increases from 3.4 to 14.7

AA upon growth and a 32% decrease in electroactivity upon

stabilisation. The current decrease for both electrodes is due

to leaching of mediator, which was non-specifically

adsorbed during the growth stage.

Fig. 4. Scan rate study for (a) [Ru(bpy)2(pic)]/GOx/Naf] and (b) [Os(bpy)2Pic+]/

square root scan rate for both anodic and cathodic currents.

E1/2 values for the metal redox process of the compounds

in solution (see Table 1) were slightly greater than values

obtained when immobilised in the Nafion layer. [Ru(bpy)2(pic)]Cl in solution gave E1/2=0.77 V and immobilised

Ru(bpy)2(pic)]+ resulted in E1/2=0.746 V, while E1/2 of

[Os(bpy)2(pic)]Cl in solution was 0.348 V and immobilised

[Os(bpy)2(pic)]+ was 0.286 V. The mediator immobilised at

the electrode surface should be more readily oxidised and

this is more evident in the case of the Os film. The high

operating potential of the Ru-based device in particular

requires measures to ensure selectivity such as anti-

interference layers.

Integration of the charge passed upon oxidation gave a

surface coverage of 2.76�10�10–1.72�10�9 mol cm�2 for

[Os(bpy)2(pic)]/GOx/Naf] and 7.35�10�10–3.01�10�9 mol

cm�2 for [Ru(bpy)2(pic)]/GOx/Naf] modified electrodes at

scan rate of 0.005 V s�1. A scan rate study was carried out

for films generated from both metal complexes, in the

absence of glucose, and the peak current dependence on the

square root of scan rate proved a semi-infinite diffusion

control process (Fig. 4(a) and (b)). Estimation of Dapp1/2Cm

GOx/Naf] electrodes in 0.5 M KCl/PBS showing linearity with respect to

ðaÞ

ðbÞ


where Cm is the concentration of redox sites and the

apparent diffusion coefficient Dapp cm2 s�1 represents

diffusion of charge in a matrix, may be achieved from the

slope of the Randles–Sevcik plot.

Ip ¼ 0:4463nFA nF=RTð Þ1=2D1=2app v

1=2Cm ð1Þ

where n=number of electrons, F=Faradays constant,

A=electrode area, v=scan rate and other terms have their

usual meaning. The product Dapp1/2Cm was calculated as

1.488�10�10 mol cm�1 s�1 for films generated from the

[Ru(bpy)2(pic)]/GOx/Naf] modified electrode and 7.503�10�11 mol cm�1 s�1 for [Os(bpy)2(pic)]/GOx/Naf] electro-

des. Cm is related to surface coverage/film thickness but as

film thickness was unknown individual Dapp and Cm values

were not determined.

The immobilisation matrix includes Nafion, which is a

stable cation exchange polymer, comprising of hydrophilic

columns composed of anionic sulphonate groups, hydro-

phobic columns composed of main chains and interlayer

regions. When a cationic material is adsorbed from an

aqueous solution, the material is located in the hydrophilic/

interlayer regions [23]. As charge propagation may follow a

diffusion like process such as electron hopping between

adjacent redox sites, or diffusion of counterions, the

mechanism is determined by the interaction of the matrix

with the redox species. When this interaction is weak and

the product can diffuse charge is transported by a diffusion

mechanism, but when interaction is strong, charge is

transported by a hopping mechanism [17]. This is the

subject of further work but preliminary results into the

mechanism of charge transport in a simple mediator/Nafion

layer indicate that the mechanism is that of diffusion over

the mediator concentration range 2�10�6–10�10�6 M in

HClO4 and LiClO4 electrolytes. The charge transport

diffusion coefficient values are independent of mediator

concentration over this range. Upon oxidation of the 2+ to

the 3+ species the electrostatic interaction between the

cationic complex and the anionic polymer becomes stron-

ger, which suppresses diffusion of the molecule while upon

reduction the interaction becomes weaker and diffusion

becomes possible. This appears to correlate with a greater

Dapp1/2Cm (cathodic) of 9.196�10�11 mol cm�1 s�1 relative

to 5.810�10�11 mol cm�1 s�1 (anodic) for the [Os(bpy)2(pic)]/GOx/Naf] modified film.

3.3. Investigation of [M(bpy)2(pic)]/GOx/Naf (M=Ru or Os)

electrode as mediator for glucose

Fig. 5(a) shows a cyclic voltammogram of the [Os(bpy)2(pic)]/GOx/Naf] modified electrode in absence and presence

of glucose (0–4 mM) at 0.005 V s�1. The voltammogram in

the absence of glucose exhibits a diffusion controlled wave

representing the reversible OsII/III system (surface coverage

1.522�10�9 mol cm�2). Under the same conditions, neither

glucose nor glucose oxidase exhibits any observable

electrochemistry. Upon addition of glucose, a large catalytic

current results at oxidising potentials, which is particularly

apparent at slower scan rates and indicates of the regener-

ation of Os(II) from Os(III) by the reduced form of the

enzyme. Fig. 5(b) shows the [Ru(bpy)2(pic)]/GOx/Naf]

modified electrode grown from 12 mM [Ru(bpy)2(pic)]Cl

in solution (surface coverage 1.627�10�9 mol cm�2)

demonstrating catalytic currents after addition of 3 and 5

mM glucose at 0.25 mV s�1.

Therefore, addition of substrate to the solution results in

the catalytic electrooxidation of glucose according to:

2MðIIÞY2MðIIIÞ þ 2e�

where M=Os/Ru, GOx-FAD is the oxidised form of the

flavin adenine dinucleotide bound to the active site of the

enzyme and GOx-FADH2 is the reduced form which is re-

oxidised by two metal (III) centres of the mediator. The

glucose turnover rate constant (kcat) and the apparent

Michaelis–Menton constant for glucose (KmV (glucose)=

(k�1+k1)/k1) may be estimated from the Lineweaver–Burk

plot.

Following the enzymatic reaction (a) above GOx-FADH2

is re-oxidised by the two metal (III) centres of the complex

(rate constant kmed) with the release of two protons.

Electrons originating in the redox site of the glucose

oxidase are transferred through the protein/mediator net-

work to the electrode surface.

If the glucose and enzyme reaction (a) is faster than the

GOx-mediator reaction (b) the entire oxidation current in the

presence of glucose can be attributed to the catalytic

oxidation process involving GOx(FAD) and the catalytic

current represents GOx turnover, which can be expressed

using the well-known expression [24,25].

Icat ¼ nFACm kmedDmCenzð Þ1=2 ð2Þ

where Cm and Cenz are the concentrations of mediator and

enzyme in surface units andDm is the diffusion coefficient of

the redox mediator. The steady-state plateau or limiting

current is independent of further increases in substrate

concentration meaning that control of this current is solely

by the enzyme mediator reaction. Using the expression

above kmed values of 1.55�106 and 4.0�106 mol�1 cm3 s�1

were calculated for [Ru(bpy)2(pic)]/GOx/Naf] and [Os(b-

py)2(pic)]/GOx/Naf] electrodes (films grown from 4 mM

[M(bpy)2(pic)Cl]) using the Dapp1/2Cm values from the

Randles–Sevcik plots (Fig. 4(a) and (b)) and Cenz=

3.87�10�8 mol cm�3 (this is the homogeneous GOx

ðcÞ

Fig. 5. (a) Electrocatalytic response for 0–4 mM glucose additions at [Os(bpy)2(pic)]/GOx/Naf] modified enzyme electrode (grown from 4 mM

Os(bpy)2(pic)+). Measurements taken at 0.005V s�1, in PBS vs. Ag/AgCl|KCl (3 M) showing diffusion controlled process in absence of substrate and

catalytically controlled current upon substrate addition. (b) Electrocatalytic response for 0, 3 and 5 mM glucose additions, at [Ru(bpy)2(pic)]/GOx/Naf]

modified enzyme electrode (grown from 12 mM Ru(bpy)2(pic)+). Measurements were taken at 0.25 mV s�1, in PBS vs. Ag/AgCl|KCl (3 M).


concentration which was assumed to be comparable with that

immobilised on the electrode). As catalytic currents are

generated in the presence of excess glucose, we assume here

that all the redox sites are involved in the process and

therefore Dapp1/2Cm values generated previously from the

Randles–Sevcik plots may be employed. Zakeeruddin et al.

[26] have investigated a range of novel tris(4,4V-substituted-2,2V-bipyridine) complexes of ruthenium and osmium and

kmed values for many of the complexes studied in this report

are three orders of magnitude lower than that reported here.

This reflects rapid electron transfer from the reduced glucose

oxidase in the case of both Os(bpy)2(pic)+ and Ru(bpy)2

(pic)+. However, the high redox potential represents a

limitation in terms of selectivity of the devices relative to,

e.g., the tris-(4,4V-dimethoxy-2,2V-bipyridine) (Eo=0.225 V)

or the tris(4,4V-diethoxy-2,2V-bipyridine) (Eo=0.21 V) com-

plex of osmium [26]. By appropriate ligand selection, the

electrochemical properties may be tuned to suit the intended

sensor application.

The catalytic current was studied by increasing the

concentration of mediator in the solution from which the

film was grown. Eq. (2) predicts a linear relationship and a

plot of Icat vs. the concentration of Os metal complex in

solution from which the film is grown (which is related to

Cm) is linear over the concentration range investigated (4–

12 mM [Os(bpy)2(pic)]Cl) (r2=0.9997) This experiment

was carried out at a scan rate of 2 mV s�1 to confirm Eq. (2)

above and correlates with the results of Sakura and Buck

[27] for ferrocene monocarboxylic acid in solution.

Voltammetric theory for the pseudo first order catalytic

ECV mechanism (electrochemical step followed by catalytic

chemical step) [28], allows E1/2 and number of electrons

transferred for this process to be confirmed by a plot of E

vs. ln(Iinf�I)/I (where I=current and Iinf is the limiting

current in the presence of 50 mM glucose).

E ¼ E1=2 þRT

nFln

Iinf � Ið ÞI

ð3Þ

This resulted in E1/2=0.27 V from the intercept with

n=1 (from slope) in the case of the [Os(bpy)2(pic)]/GOx/

Naf] electrode and E1/2=0.69 V (n=1) for the [Ru(bpy)2(pic)]/GOx/Naf] electrode (films grown from 4 mM


mediator). Analyses of the Tafel regions of the catalytic

cyclic voltammogram was carried out and resulted in an

anodic transfer coefficient (aa) of 0.63 for [Os(bpy)2(pic)/

GOx/Naf] and 0.48 for [Ru(bpy)2(pic)/GOx/Naf] confirm-

ing the reversible nature of the immobilised redox couple.

The scan rate dependence of the catalytic current was

studied and a plot of Icat/v1/2 vs. log v [27] for the

[Os(bpy)2(pic)]/GOx/Naf] electrode (Fig. 6(a)) does not

change for the diffusion controlled couple (in absence of

glucose) as current is controlled by diffusion and I/v1/2 is

independent of scan rate. However, for the catalytic current

(in presence of 3 mM glucose), a scan rate dependence in

I/v1/2 up to 0.1 V s�1 was observed, after which the modified

electrode behaves like the film alone in the absence of

substrate.

Fig. 6. (a) Icat/v�1/2 vs. log v for [Os(bpy)2Pic

+]/GOx/Naf] modified electrode b

catalytic response using cyclic voltammetry for 3 mM glucose addition at [Os(bp

Fig. 6(b) shows the effect of scan rate on the catalytic

response at an [Os(bpy)2(pic)]/GOx/Naf] modified elec-

trode (grown from 4 mM mediator in solution). Upon

addition of 3 mM glucose, a catalytic oxidation wave is

observed and the reduction wave is eliminated (scan rate

0.005 V s�1). The absence of a reduction wave shows that

at this scan rate the film is maintained in the reduced state

by the transfer of electrons from GOx-FADH2 to Os(III),

i.e., the reduction of the oxidised Os(III) by the reduced

enzyme is more efficient than the reduction at the

electrode surface. At faster scan rates (0.3 V s�1) (also

in the presence of 3 mM glucose), a reduction wave

appears, i.e., the film is no longer completely reduced by

the enzyme mediated electron transfer from glucose at this

scan rate [27].

oth in absence and presence of 3 mM glucose. (b) Effect of scan rate on

y)2(pic)]/GOx/Naf] electrode.

Fig. 7. (a) Calibration curve representing amperometric data (n=3) and (b) corresponding Lineweaver–Burk plot for [Os(bpy)2(pic)]/GOx/Naf] modified

electrode over the range 3.0–10.0 mM glucose.


3.4. Amperometric studies on [M(bpy)2(pic)]/GOx/Naf

(M=Ru or Os) modified electrodes

In the presence of excess immobilised mediator (excess

refers to the mediator concentration from which the films

were grown above which there will be no further increase in

current response, i.e., 12 mM), Imax, which is the maximum

current at saturation concentrations of substrate and apparent

Michaelis–Menton constant KmV values, may be evaluated by

Table 2

Kinetic data for [M(bpy)2(pic)]/GOx/Naf (M=Ru or Os) electrodes

KmV (mM) Imax (A) Imax

[Ru(bpy)2(pic)]/GOx/Naf] 5.7 2.9�10�7 5.1�[Os(bpy)2(pic)]/GOx/Naf] 16.11 2.98�10�7 1.8�a Sensitivity of the sensor.b kcatd l obtained from intercept of Lineweaver–Burk plot.

curve fitting of the steady-state amperometric response to the

electrochemical Michaelis–Menton equation.

1=I ¼ KmV= ImaxCgluc

� �þ 1=Imax ð4Þ

Imax ¼ nFAkcatCenzlð Þ=2 ð5Þwhere I is the steady state current after addition of substrate,

l=thickness of the enzyme layer, Cgluc is concentration of

glucose (mM), Cenz is concentration of enzyme (mM), n is

the number of electrons (1), F is Faradays constant and A is

/Kma (A mM�1) kcatd l

b (cm s�1) kmed (mol�1 cm3 s�1

10�8 2�10�3 1.55�106

10�8 2.25�10�3 4.0�106

)

Table 3

Film stability and biosensor response stability over time for [Os(bpy)2(pic)]/

GOx/Naf] electrode

% Decrease in current

for Os(II/III) couple

% Decrease in response

to glucose (5 mM)

1–30 days 35.6% 70.6%

1–60 days 73.7% 87.7%

1–100 days 89.5% 83.9


area of the electrode (cm2). The Imax/KmV ratio is a measure of

the sensitivity of the sensor and the apparent turnover rate

constant kcat for GOx and its catalytic efficiency kcat/KmVmay

be estimated from this method [29].

Fig. 7 shows the calibration curve generated from

amperometric data for [Os(bpy)2(pic)]/GOx/Naf] modified

electrode (grown from 12 mM mediator in solutions) from

which Lineweaver–Burk plots were generated (Fig. 7(b)).

Aliquots of 0.5 mM glucose were added to the cell under

controlled convection and the current stabilised in 25 s, i.e.,

time required to reach uniform glucose concentration in the

cell. The device exhibited linearity up to 10 mM glucose,

y=5.44�10�8 (F3.73�10�9) A mM�1�1.33�10�7

(F1.49�10�8) A (r2=0.999) and resulted in KmV=16.1 mM

and kcatd l=2.25�10�3 cm s�1. In the same manner, the

[Ru(bpy)2(pic)]/GOx/Naf] (grown from 12 mM mediator in

solutions) modified electrode resulted in KmV=5.7 mM with

linearity up to 2 mMglucose, y=3.89�10�8F(2.04�10�8) A

mM�1. The linearity for both electrodes extended beyond the

expected 0.1�KmV, perhaps as a result of the additional Nafionlayers deposited over the immobilised mediator, which allow

for controlled diffusion of the substrate through the film. The

data was also analysed by the Eadie-Hofstee and Hanes plots

but did not fit these models. Table 2 summarises the kinetic

information obtained for both modified electrodes.

3.5. Enzyme and film stability

The use of a biosensor is normally limited to the lifetime of

the immobilised enzyme and therefore electrodes were inves-

tigated under daily electrochemical measurements for a pe-

riod of 100 days. Table 3 summarises the stability data in the

case of the film itself and the enzyme catalytic response over a

period of 100 days for a [Os(bpy)2(pic)/GOx/Naf] electrode.

Cyclic voltammograms were carried out in 0.5 M KCl/PBS

with addition of 5 mM glucose daily. The Os(II/III) couple

exhibited a decrease in current of 35.6% for the first 30 days

while the response to 5 mM glucose decreased by 70% over

the same period. This suggests a leaching of enzyme from the

film/enzyme denaturation, which appeared to happen at a

more rapid rate than mediator leaching/removal over time.

4. Conclusion

In conclusion, this report describes the synthesis of novel

mediators for glucose based on replacement of the chloride

of M(bpy)2Cl complexes with a picolinate ligand. The

compounds were characterised and then tested as mediators

for regeneration of the glucose oxidase prosthetic group

FADH2. Immobilisation was achieved by potential cycling

at an enzyme/Nafion modified electrode and the convenient

method of immobilisation by electrostatic interaction with

an anionic Nafion layer creates a reproducible device with

good sensitivity and linearity for glucose particularly for the

[Os(bpy)2(pic)/GOx/Naf] electrode. The electrocatalytic

ability of the complexes were investigated using slow scan

rate cyclic voltammetry and the relatively low redox

potential (0.34 V) and high kmed values for the [Os(bpy)2(pic)]/Naf/Gox electrode in particular (4�106 mol�1 cm3

s�1) show that such materials have promise in development

of biosensors. Future work will evaluate alternative ligands,

which decrease the operating potential of the metal process

and allow development of a more selective sensor. This will

include interference evaluation, real sample analysis and

incorporation into a flow cell combined with microdialysis

sampling.

Acknowledgement

The authors would like to acknowledge financial support

from the Irish Postgraduate Research and Development

Programme 2002.

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investigation of novel mediators for a glucose biosensor based on metal picolinate complexes

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