Renewable surface electrodes based on dopamine functionalizedexfoliated graphite:

NADH oxidation and ethanol biosensing

P. Ramesh, P Sivakumar, S. Sampath *

Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India

Received 7 January 2002; received in revised form 25 March 2002; accepted 12 April 2002

Abstract

Exfoliated graphite (EG) was modified by covalently attaching dopamine (DA) (3,4-dihydroxyphenethylamine) through amide

linkages, using �/COOH groups introduced on the EG surface. The modified material was characterized by FT-IR spectroscopy, X-

ray photoelectron spectroscopy and electrochemical techniques. Composites of DA modified EG dispersed in organically modified

silicates were prepared by a sol�/gel process. Electrodes were fabricated by casting the composites in glass tubes. The sol�/gel based

electrodes were found to be active for the electrocatalytic oxidation of NADH and biosensing of ethanol in presence of NAD� and

alcohol dehydrogenase enzyme. The modified composite electrodes were found to be stable for several months. The surface of the

electrode could be renewed just by mechanically polishing the electrode using emery sheets. The modified EG was also pressed and

restacked in the form of a pellet and the use of this material as a binderless bulk-modified electrode was also demonstrated. The

performance of sol�/gel derived composite EG electrodes with binderless bulk-modified EG electrodes was compared. # 2002

Elsevier Science B.V. All rights reserved.

Keywords: Exfoliated graphite; Chemically-modified electrode; NADH oxidation; Biosensors; Sol�/gel process; Ethanol

1. Introduction

Amperometric determination of NADH is of great

interest since a vast number of dehyrogenase enzymes

that catalyze oxidation or reduction of various sub-

strates depends on the NAD�/NADH couple for the

enzyme regeneration [1�/5]. Construction of bio-cataly-

tic systems based on dehydrogenase enzymes requires

the regeneration of NAD� for the enzymatic reaction.

However, the regeneration of NAD� by electrochemical

oxidation of NADH on bare carbon and metal electro-

des suffers from high over-potential (h ) (0.6�/0.9 V vs.

SHE) requirements [6]. This makes bio-sensing using

dehydrogenase enzymes prone to interference. Addi-

tionally, cation radicals produced by the one-electron

oxidation of NADH adsorb on the electrode surface

thus leading to electrode fouling. This hinders the use of

these electrodes for accurate detection of NADH.

Attempts have been made to overcome these difficulties

by using mediators to reduce the over-potential required

for the regeneration of NAD�. Several mediators such

as ortho - and para -quinones [7�/29], ferrocene [30], Ni�/

hexacyanoferrate [31], phenoxazine, phenathiazines and

diimines [32�/35] have been reported to reduce the over-

potential requirements. The NAD�/NADH redox reac-

tion involves two electrons and hence a mediator

capable of transferring two electrons is expected to be

efficient. This may also reduce the formation of the one

electron product (i.e) cation radical, thereby reducing

the poisoning of the electrode surface [6]. The redox

mediators should also act as a site for hydride transfer

and delocalise the charge within the mediator [36].Quinones and several substituted quinones belong to

this category and have been employed for this purpose

[7�/29]. Tse and Kuwana have reported the use of several

quinones as freely diffusing mediators for the electro-

catalysis of NADH oxidation in the solution phase [9].

It was found that ortho -quinones are good mediators for

* Corresponding author. Tel.: �/91-80-3092-825; fax: �/91-80-3600-

683

E-mail address: sampath@ipc.iisc.ernet.in (S. Sampath).

Journal of Electroanalytical Chemistry 528 (2002) 82�/92

www.elsevier.com/locate/jelechem

0022-0728/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 0 2 2 - 0 7 2 8 ( 0 2 ) 0 0 8 8 8 - 4

this purpose. Carlson and Miller also demonstrated that

ortho -quinone based mediators catalyze the reaction a

100 times faster than para -quinones and 106 times faster

than the one electron mediators [28]. As for themodification of the electrode surface, several techniques

such as physical adsorption [7,8], electrodeposition [11�/

18] and covalent attachment of ortho -quinone [9,10] and

other mediators with related structures have been

reported. Electrodeposited films of dihydroxybenzalde-

hyde (DHB) on glassy carbon (GC) have been studied as

well [11�/13]. The o -isomers of 3,4-DHB and 2,3-DHB

show very strong catalytic activity while the p-isomer(2,5-DHB) showed a negligible electrocatalytic effect

[13]. Dopamine (DA) can give rise to an ortho -quinone

structure on oxidation and hence can act as a good

mediator. However, the use of DA as a freely diffusing

mediator in the solution phase leads to a cyclization

product formed through the free amine end [9]. This

affects the reversibility of the mediator and in turn the

NADH sensing, thus limiting the use of DA as amediator in the solution phase. This difficulty may be

overcome by covalently attaching DA to an electrode

surface through the free amine end. This will avoid both

cyclization and also the leaching of the mediator from

the modified electrode. Coatings based on DA-incorpo-

rated polymers on carbon electrodes have been em-

ployed for NADH oxidation [27]. Slow diffusion of the

analyte into the polymer film resulted in a non-linearcurrent response with time. Additionally, swelling of the

polymer film reduced the long-term operation of the

sensor in a liquid environment. Electrochemical attach-

ment of DA on to GC was also demonstrated for

NADH catalysis [29]. However, electrochemically as-

sisted attachment is controlled by the adsorption of

catechol and hence, the coverage is dependent on the

oxygen functional groups available on the surface.Surface modified electrodes, however, suffer from

fouling due to adsorption of radical intermediates.

Hence, these electrodes cannot be used for successive

measurements. A surface renewable, bulk-modified

electrode can overcome surface fouling by providing a

fresh surface after every measurement. Towards this

goal, the use of bulk-modified electrodes based on

carbon paste has been reported [37]. These electrodessuffer from instability arising from the presence of

binder. Secondly, leaching of the modifier during

operation poses difficulties in the use of these electrodes

for long-term operations [38,39]. Another class of bulk-

modified electrodes based on graphite�/silicate compo-

sites (carbon�/ceramic electrodes (CCE)) have been

reported recently [40,41]. Meldola’s blue has been used

as a mediator that was physically incorporated into thegraphite�/silicate matrix [42]. Leaching of the modifier

occurred during repeated cycling. Hence, covalent

attachment of the mediator and also avoidance of

binder material is expected to offer better stability for

NADH oxidation than the systems reported so far and

provide an efficient way of developing enzyme-based

sensors.

Exfoliated or expanded graphite (EG) is a low densitymaterial with high temperature resistance [43]. A large

increase in the c lattice upon thermal decomposition of

graphite intercalation compounds results in a puffed-up

material having a high degree of flexibility [44]. EG has

better homogeneity than any other graphite substrate

[45]. This can be compressed or restacked without a

binder. The restacking mechanism is reported to involve

the interlocking of the layers during compression [46]. Itis used for seals, catalyst supports and gaskets. The use

of EG in electrochemistry has not been explored in

detail. There are only a few reports on the electrochem-

istry of EG [47�/49]. Frysz and Chung have reported the

electrochemical properties of EG based electrode mate-

rial in aqueous electrolytes [47]. We have recently

demonstrated the use of EG as binderless, bulk-mod-

ified electrodes for electrocatalytic applications [50,51].In this paper, we report on the covalent functionaliza-

tion of EG with DA and its characterization by

spectroscopic and electrochemical methods. Preparation

of CCEs using the modified EG and its application in

the electrocatalysis of NADH and biosensing of ethanol

using a dehydrogenase enzyme are reported. The use of

recompressed DA modified EG electrodes without any

binder is also explored for sensing applications. Theperformance of sol�/gel derived CCEs and binderless

recompressed EG (RE) electrodes are compared.

2. Experimental

2.1. Chemicals

All the chemicals used were of AR grade. Methyl-trimethoxysilane (MTMOS) was a product of Aldrich,

USA. DA, yeast alcohol dehydrogenase (E. C. 1.1.1.1.

440 units mg�1, obtained as 90% pure lyophilized

powder), NADH (98%), NAD� (99%) were products

of Sigma, USA. Dicylohexylcarbodiimide (DCC) was

obtained from Fluka, Switzerland.

2.2. Functionalization of EG

Natural graphite particles (Stratmin, NJ, 300�/400

mm) were intercalated using HNO3�/H2SO4 (1:3) mix-

ture for 24 h and washed well with water. The resulting

intercalated graphite was then subjected to a thermal

shock at 800 8C for 1 min to obtain EG. EG is a flaky

material with a low density. The EG was then treated

oxidatively to introduce oxygen containing functionalgroups on the surface. EG (1 g) was heated, with

stirring, at 100 8C for about 4 h in HNO3�/H2SO4

(1:3) mixture to introduce carboxyl functional groups on

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/92 83

the EG. Excess acid present was filtered and the solid

was washed with distilled water and subsequently

treated with 5% NaOH. Finally dilute HCl was used

to neutralize the excess base and the resulting oxidized

EG was washed with water and dried in a desiccator

over CaCl2.

Covalent attachment of redox molecules requires

oxygen containing functional groups on the graphite

surface. Carboxyl functional groups that are introduced

during oxidative pre-treatment were used to attach the

primary amine of DA to form an amide bond as given in

Scheme 1. Covalent modification was carried out using

a reported procedure [10]: 250 mg of oxidized EG was

treated with 60 mg of DCC for about 5 min in 20 ml of

CHCl3 to activate the carboxyl groups present on the

EG surface. DA (50 mg) was then added and the stirring

continued for 4 days. After the reaction is completed the

modified material was soxhlet extracted with MeOH for

a further 4 days.

2.3. Preparation of modified electrodes

Two types of electrode were prepared for the electro-

chemical studies. A CCE was prepared by a sol�/gel

process, as follows: 250 ml of MTMOS, 100 ml of water

and 50 ml of 1 M HCl were taken in a glass vial and

mixed thoroughly. DA modified EG (20 mg) was then

added and the resulting slurry was molded in glass

tubes. These were left to dry at room temperature for 4

days and then in an oven at 60 8C for 4 days to

complete the cross-linking. All electrodes were polished

using 600-grit emery paper followed by a 1200-grit

emery paper before use. Copper wire served as the

electrical contact for the CCEs. The surface of the CCE

was cleaned well with twice distilled water after polish-

ing and used for electrochemical experiments. DA

physisorbed on EG, used for comparative studies was

prepared as follows: 2 mg of DA was dissolved in 25 ml

of distilled MeOH and then 200 mg of EG was added to

it. This was stirred well and the solvent was slowly

evaporated at r.t. Unmodified EG�/silicate and DA

physisorbed EG�/silicate composite CCEs were pre-

pared using the similar protocol mentioned above.

Another set of electrodes was prepared without the

silicate binder (RE). DA modified EG was pressed at apressure of 6 tons cm�2 for about 5 h to obtain a

compact pellet. This pellet was found to be strong and

highly conductive. The resistance between two points on

either side of the pellet was found to be 1�/2 V. This

pellet was cut into small pieces, which were mounted in

glass tubes and made into electrodes using silver epoxy

as the contact between the pellet and the copper wire.

The surfaces of the RE pellets were polished with 600-and 1500-grit emery sheets followed by 4/0, 5/0 and 6/0

emery polishing papers to obtain a polished surface.

Roughness on these electrode surfaces was created by

scratching the electrode surface against emery sheets in

the same direction.

2.4. Techniques

FT-IR spectra of EG samples were obtained using aBruker Equinox 55, IR spectrophotometer (Karlsruhe,

Germany). A very small amount of graphite sample was

mixed uniformly with KBr and pressed into pellets.

Transmittance spectra were recorded from 400 to 4000

cm�1 at a resolution of 4 cm�1. A VG Scientific II

ESCA-3 (UK) with Al�/Ka radiation (1486.6 eV) was

used to obtain XPS spectra. XPS (X-ray photoelectron

spectroscopy) measurements were carried out withpressed pellets. A JEOL (model JSM 840A, Japan)

scanning electron microscope operating at 20 kV was

used to obtain micrographs. SEM experiments were

carried out using unmodified and modified EG powder

as well as pellets.

All electrochemical experiments were carried out in a

single compartment cell with a Pt foil and saturated

calomel electrode (SCE) as counter and referenceelectrodes, respectively. The polished EG working

electrode surface was thoroughly washed with distilled

water and immediately used in the electrochemistry

experiments. The electrolyte solutions were purged

with purified nitrogen for 20 min prior to the start of

the experiments and a nitrogen atmosphere was main-

tained above the solution level during the experiments.

The studies were carried out using either a CHI 660Aelectrochemical analyzer from CH Instruments (TX,

USA) or a Versastat IITM from EG&G PARC (NJ,

USA).

3. Results and discussion

3.1. Characterization of the DA modified EG

EG has a very low density of 3.3 g l�1. A detailed

characterization of EG and the oxidatively pretreatedScheme 1.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/9284

EG using SEM, XRD, IR, elemental analysis and

titration methods has been reported earlier [51]. Fig. 1

shows the SEM picture of EG and DA modified EG.

EG has a worm like structure and the expanded layer of

graphite is clearly seen (Fig. 1A). After the oxidative

pre-treatment a change in surface morphology of EG is

observed. Oxidized EG has a particulate structure rather

than a layer structure. The morphology of DA modified

EG resembles oxidized EG with a particle size of 500 mm

diameter (Fig. 1B).

XPS is employed to characterize the functional groups

and the elements present on the DA modified EG. XPS

of unmodified EG and DA modified EG has been

carried out to confirm the covalent modification. High-

resolution XPS spectrum of the C1s region of unmodi-

fied EG shows graphitic, phenolic and carboxyl func-

tional groups in the ratio of 77:4.4:18.7 [52]. Covalent

modification of DA on the EG surface will result in

amide bond formation and an increase in hydroxyl

functional groups. Hence, DA modified EG is expected

to have C�/H (283.4 eV), graphitic (284.3 eV), phenolic

(286.1 eV) and amide (289.1 eV) functional groups.

Indeed the high-resolution spectrum of the C1s region

reveals different types of carbon present on the DA

modified EG matrix (Fig. 2). Non-linear curve fitting

has been employed to deconvolute the spectrum and a

multiple Gaussian function is used to fit the data. It is

found that DA modified EG consists of

57.1:14.05:13.87:13.56 of graphitic/phenolic/amide/C�/

H groups. Comparison of the amount of functional

groups present on the un- and DA modified EG, reveals

a relative increase in the phenolic functional group on

the modified material, thus confirming the covalent

modification. Relative ratios of different elements pre-

sent on DA modified EG are 15, 27, and 1.8 for C/O, C/

N and O/N, respectively. The ratio of N/C is observed to

be 3.7 on the DA functionalized EG as opposed to the

ratio of 1.3 obtained using an electrochemical procedure

to attach DA on GC [29]. This may be due to the result

of a higher loading of DA on EG than on GC.

FT-IR is used to identify the functional groups

present on DA modified EG. FT-IR spectra of un-

modified EG, oxidized EG, DA modified EG, DA

adsorbed EG, and neat DA are obtained and compared

to confirm the covalent modification. Unmodified EG

shows phenolic, alcoholic and carboxyl functional

groups at 1059, 1124 and 1650 cm�1, respectively.

Oxidative pre-treatment increases the carboxyl func-

Fig. 1. Scanning electron micrographs of: (A) EG; and (B) DA

modified EG.

Fig. 2. Deconvoluted C1s region of the XPS of DA modified EG.

Circles represent the experimental data and the solid line represents the

non-linear fit.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/92 85

tional groups on the EG surface as is evident from the

increase in the intensity of the 1650 cm�1 band [51]. The

carboxyl functional groups were used to attach DA on

EG through the amide bond as shown in Scheme 1. The

amide bonding shows characteristic bands, amides I and

II, that correspond to carbonyl and N�/H bending and

they generally appear at 1650 and 1655�/1620 cm�1. In

the case of DA modified EG (Fig. 3A) both bands

overlap to give a broad band at 1630 cm�1. The amide

II band occurs near 1655�/1620 cm�1 and is generally

observed under the envelope of the amide I band [53]. In

the present study, the N�/H and O�/H stretching modes

overlap to give a broad band at 3427 cm�1. Pure DA,

which is a hydrochloride salt, shows a primary amine

stretch and O�/H stretching between 3000 and 3300

cm�1. After modification primary amine bands disap-

pear showing that the modification, indeed, has taken

place. However, in the case of DA physisorbed EG (Fig.

3B), a band corresponding to the primary amine is

present, showing the difference between physical ad-

sorption and covalent modification. Peaks correspond-

ing to C�/O stretching of phenolic groups at around

1100 and 1250 cm�1 are present on DA modified EG.

CH2 stretching of DA at 2850 and 2920 cm�1 are also

observed in DA modified EG.3.2. Electrochemistry of DA modified EG electrodes

Fig. 4 shows the cyclic voltammogram (CV) of a DA

modified EG�/CCE in a phosphate buffer of pH 7.2. The

CV shows quasi-reversible oxidation/reduction behavior

of the attached DA. The formal potential is observed to

be 0.12 V at a scan rate of 50 mV s�1 and it remains the

same at all scan rates. The peak separation, DEp, is 0.06

V at a scan rate of 50 mV s�1 and this is almost constant

for the scan rate range from 20 to 150 mV s�1. Peak

currents are found to increase linearly with scan rate up

to 120 mV s�1 which is characteristic of an electrode

confined redox species (Fig. 4). Beyond 120 mV s�1, a

deviation from linearity is observed. This may be due to

the proton transfer limitation as was reported earlier for

the anthraquinone modified EG [50,51] electrodes. The

apparent surface coverage of DA modified on EG�/CCE

based on the CV is 3.7�/10�11 mol cm�2. The formal

potential of DA observed in the present study is

comparable to that of 3,4-DHB reported by Abruna

and coworkers [11,13]. 3,4-DHB showed a very small

DEp and the peak current was observed to be linear up

to 500 mV s�1 [13]. Electrochemically attached DA on a

GC surface showed a DEp of 0.01 V and the peak

currents were linear up to 400 mV s�1 [29].

The physisorbed DA shows different electrochemical

characteristics as compared to the covalently functiona-

lized material. The redox currents decrease continuously

with cycling, due to leaching of the mediator. The

reduction is approximately 45% for 10 cycles. TheFig. 3. FT-IR of: (A) DA modified EG; and (B) DA physisorbed EG.

The marked vibrational peaks are explained in the text.

Fig. 4. Plot of anodic peak current vs. scan rate of DA modified EG�/

CCE. Inset shows a representative CV of the same electrode in a

phosphate buffer of pH 7.2 at a scan rate of 50 mV s�1.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/9286

formal potential of physisorbed DA is observed to be

0.16 V and is different from the covalently modified

material. A peak potential separation of 0.1 V is

observed at a scan rate of 50 mV s�1 and it is found

to increase with scan rate (i.e. 0.09�/0.23 V for a change

of scan rate from 20 to 150 mV s�1). The peak potential

difference, DEp, observed for the physisorbed DA (0.1

V) compared with that observed for covalently modified

DA (0.06 V) is an indication of the sluggish kinetics of

physisorbed DA.

The CV obtained using the polished surface of the DA

modified EG�/RE electrodes without any binder shows a

low electrochemical activity (not shown) compared with

roughened electrodes (Fig. 5). Oxidative pre-treatment

introduces carboxyl functional groups on the edge

planes and subsequently DA is covalently attached

only on the edge planes of EG. Compression and

polishing of the electrode surface leads to the exposure

of a basal plane oriented surface with low electroche-

mical activity. Roughening the electrode surface exposes

more edge planes containing the attached DA and

consequently better electrochemical activity is observed

with rough surfaces. Hence, further experiments are

carried out using (400-grit) roughened electrode sur-

faces. The observed peak currents for DA are found to

increase linearly with scan rate and above 120 mV s�1,

the peak currents tend to deviate from linearity as

observed with the EG�/CCE. The formal potential is the

same as that observed in the case of DA modified EG�/

CCEs. The DA modified EG�/RE electrode is found to

be very stable during continuous potential cycling and

no leaching of DA is observed. This is confirmed bychecking the buffer solution for any dissolved electro-

active species, after repeated cycling.

The electroactivity of DA modified electrodes with

and without binder is observed to be similar in terms of

formal redox potentials and variations of peak currents

and potentials with scan rate. The silicate binder in the

case of CCE does not change the electrochemical

behavior of DA. However, the difference in currentvalues shown in the CVs (Figs. 4 and 5) is due to the

difference in the area of the electrodes. The cyclization

of DA is not observed in the modified electrodes, as

expected. This is confirmed by the absence of any

additional redox active peaks around �/0.25 V that

have been reported to be due to the cyclized product in

the solution phase [9]. The redox potential of DA is

dependent on the electrolyte pH since it involves aproton coupled electron transfer. Differential pulse

voltammetry has been employed to find out the exact

peak potentials at different pH values. A plot of formal

potential versus solution pH gives a slope of 60 mV per

pH up to a pH of 10 (figure not shown). This

observation is in correlation with the mechanism

reported for DA oxidation that involves two electrons

and two protons up to a pH of 10. The pKa of the DA is8.87 and the mechanism of oxidation beyond pH 10 is

reported to involve two electrons and one proton [27].

This should result in a slope of 30 mV per pH. However,

a slope of 45 mV per pH is observed beyond pH 10. The

exact reason for this observation is presently unknown.

The CV shows a broad peak for the oxidation of DA at

high pH values. This behavior under high alkaline

conditions may be due to the instability of the attachedquinone [10] and this may result in the deviation of the

slope from the expected trend. It should also be pointed

out that the polymer bound DA and the DHB modified

electrodes yielded slopes of 40 and 38 mV per pH,

respectively, in the pH range beyond the pKa [13,27] of

hydroquinone.

3.3. Electrocatalysis of NADH oxidation

3.3.1. Carbon�/ceramic, DA modified EG electrodes

A sol�/gel derived, unmodified EG�/CCE shows stable

background current densities of the order of (3.049/

0.01)�/10�7 A cm�2 in the potential region �/1�/1.2

V, in a phosphate buffer of pH 7.2. The geometric area

is used to determine the current density since the active

area is unknown in the case of silicate based sol�/gel

modified electrodes. XPS data on unmodified EGparticles show a lack of carbonyl/quininoid functional

groups on EG. This may result in a very low residual

electrochemical activity towards the electrocatalysis of

Fig. 5. Plot of anodic peak current vs. scan rate of DA modified EG�/

RE. Inset shows a representative CV of the same electrode in a

phosphate buffer of pH 7.2 at a scan rate of 50 mV s�1.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/92 87

quinone type compounds. The presence of phenolic

functionalities on the EG surface probably results in the

capacitive currents observed. Fig. 6 shows the oxidation

of 1 mM NADH at a scan rate of 50 mV s�1 on an

unmodified EG�/CCE. The oxidation is found to occur

at around 1.2 V. Adsorption of oxidation products is

also observed in the potential region of 0.9 V. A plot of

adsorption current at 0.9 V versus scan rate shows a

linear increase confirming the adsorption of intermedi-

ates (not shown). The oxidation potential of NADH is

reported to be at around 0.6�/0.7 V versus SCE on GC

surfaces [42]. ortho -Quinone type functional groups

present on the GC surface are known to catalyze

NADH oxidation. It is clear from the voltammogram

shown in Fig. 6 that the oxidation potential for NADH

is more positive on unmodified EG electrodes than that

observed on GC and other composite electrodes [42].

The high over-potential requirement for oxidation of

NADH on unmodified EG may be due to the lack of

quinone-type functional groups on the unmodified EG

surfaces as pointed out earlier.

Fig. 7A shows the CVs of DA modified CCE in the

absence and in the presence of NADH. The voltammo-

grams are carried out at a scan rate of 5 mV s�1. The

catalytic currents are observed to start at around 0.05 V

and become saturated at 0.25 V (Fig. 7A). A plot of

catalytic current versus concentration at 0.15 V shows a

linear response up to 5 mM (Fig. 7B). Steady state

measurements carried out at 0.15 V show a linear

response up to 0.3 mM and a dynamic response up to

1.2 mM (Fig. 8A). The response time is rather fast and is

of the order of 4�/5 s. NADH oxidation using physi-

sorbed DA is carried out for comparison purposes.

Electrocatalytic oxidation of NADH using cyclic vol-

tammetry at a scan rate of 5 mV s�1 shows that the

catalytic currents start at around 0.05 V and become

saturated at 0.25 V. A plot of catalytic current versus

Fig. 6. CV of 1 mM NADH on an unmodified EG�/CCE in a

phosphate buffer of pH 7.2. The scan rate used is 50 mV s�1.

Fig. 7. (A) Electrocatalytic oxidation of NADH on DA modified EG�/

CCE in a phosphate buffer of pH 7.2. The scan rate used is 5 mV s�1:

(1) bare EG�/CCE; (2) with 1.21 mM; and (3) with 3.11 mM NADH

additions. (B) Calibration plot of catalytic current vs. concentration at

0.15 V based on the voltammograms.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/9288

concentration at 0.15 V show a linear response up to 5

mM. Steady state experiments at 0.15 V show a linear

response up to 0.4 mM and a dynamic response up to

1.4 mM. The response time is slow and is of the order of

70 s. (Fig. 8B). Adsorbed quinones are known to lie

parallel to the basal plane of the graphite, which restricts

its orientation [7,8]. The slow response observed in the

present studies may due to the unfavorable orientation

of the adsorbed DA on the graphite surface.

3.3.2. Binderless, DA modified EG electrodes

Cyclic voltammetry experiments have been carried

out in a phosphate buffer of pH 7.2 using an electrode

roughened with a 400-grit emery sheet. Catalytic cur-

rents are observed to start at 0.05 V and become

saturated at around 0.2 V (not shown). Catalytic

currents at 0.15 V, as a function of the concentration

of NADH are observed to be linear up to approximately

5 mM. Steady state experiments at 0.15 V show a

response time of 4�/5 s and a linear range up to 1 mM

(Fig. 9).

The linear range observed with the DA modified EG�/

RE electrodes (0.02�/1 mM) electrodes is comparable

with DHB modified electrodes (0.01�/1.2 mM) which

covers the values of a great relevance in biosensor design

and applications [13]. The detection limit of DA

modified EG�/CCE is 20 mM whereas for the DHB

modified electrodes it is 10 mM. The dynamic range for

the DA modified EG�/RE (up to 5 mM) electrodes is

comparable with that of DHB modified electrodes [13].

The dynamic ranges reported for the Meldola’s blue

modified CCE (up to 50 mM) and the toluidine blue

modified restacked EG (up to 30 mM) are higher than

the ortho -quinone modified electrodes [42,50].

Comparison of the data on the DA modified EG�/

CCE and EG�/RE reveals small differences that may

arise due to different diffusion characteristics on the two

surfaces. Sol�/gel derived electrodes are known to be

porous and the restacked electrode may be approxi-

mated to a planar surface though it is roughened before

the experiments. The comparison in the steady state

Fig. 8. Plot of catalytic current vs. concentration of NADH at 0.15 V

in the steady state mode. (A) DA modified EG�/CCE (inset) I �/t

response for the additions of 0.185, 0.181 and 0.178 mM (1, 2, 3,

respectively) of NADH, and 4 corresponds to dilution with buffer. (B)

DA physisorbed EG�/CCE (inset) I �/t response for the additions of

0.039, 0.039 and 0.097 mM (1, 2, 3, respectively) of NADH.

Fig. 9. Plot of log (catalytic current) vs. log [NADH] for DA modified

EG recompressed electrode (400-grit roughness) in a phosphate buffer

of pH 7.2. Inset: plot of catalytic current vs. concentration of NADH

of the same.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/92 89

conditions also reveals that the linear and dynamic

ranges for the silicate based EG electrodes are lower

than the restacked EG electrodes without any binder.

This may be due to the adsorption properties of thesilicate binder that may poison the electrode surface

leading to lower linear and dynamic ranges. Indeed, it is

found that the unmodified EG based CCE leads to

higher adsorption currents for NAD� than the re-

stacked EG based electrodes (not shown).

3.4. Ethanol biosensing

Alcohol sensing is carried out in a phosphate buffer of

pH 7.2 with a DA modified EG�/CCE using alcohol

dehydrogenase enzyme and NAD� in the solution

phase. Fig. 10A shows the linear response of the

electrode to the alcohol concentration in the bulk of

the solution. The linear range is observed to be from 1 to

4 mM and the dynamic response extends up to 50 mM inthe steady state mode at 0.15 V. The inset of Fig. 10A

shows the steady state response of the DA modified

EG�/CCE for various additions of ethanol. The re-

sponse time is of the order of 6�/7 s in the linear range.

The DA modified EG�/RE electrodes show a linear

response up to 40 mM (Fig. 10B) and the response timeis of the order of a few seconds. The difference in the

linear ranges between the two configurations is large

though the active material is the same DA functiona-

lized EG. This may be due to the difference in diffusion

characteristics and is being probed further. Blood

alcohol concentrations can vary from 10 to 50 mM

depending on the degree of intoxication from haziness

to complete drunkenness [60]. Similar intoxication canlead to an alcohol content variation in urine samples

from 10 to 100 mM [31]. Alcoholic beverages have very

high alcohol contents (5�/45% v/v). The samples can be

diluted for analysis with the EG electrodes.

A comparison of the performance of DA modified

EG electrodes with other systems shows that the linear

range of the modified EG electrode overlaps well with

many electrodes reported in the literature. Metal�/1,10-phenanthroline-5,6-dione complex modified electrodes

show similar linear and dynamic ranges when compared

with DA modified EG�/RE electrodes [14]. Methylene

green, toluidine blue and yeast modified carbon based

electrodes showed linear ranges of 0.04�/6; 1�/6 and

0.002�/0.03 mM for ethanol, respectively [54,50,55]. An

electrode modified with a polymer of toluidine blue

showed a linear range of 0.05�/1 mM [56]. Rutheniumloaded activated carbon and carbon paste electrodes

without any mediator showed a rather large linear range

of 1.7�/17 and 10�/150 mM, respectively. However, these

electrodes operate at high over-potentials (0.6�/0.7 V)

and are prone to various interferences [57,58]. A Ni�/

hexacyanoferrate modified electrode showed a linear

range up to 10 mM [31].

As for the response times, the EG electrodes showbetter characteristics than the other systems. The

toluidine blue modified EG�/RE showed a response

time of 15�/20 s. The alcohol sensors based on methylene

green modified carbon paste, Ni�/hexacyanoferrate and

metal�/1,10-phenanthroline-5,6-dione complex modified

electrodes show high response times, of the order of 50�/

60 s. The present sensor is fast and the response time is

6�/7 s.

3.5. Interferences

Interference in the NADH oxidation has been tested

on the DA modified EG�/CCE electrodes. Acetamino-

phen undergoes oxidation at 0.25 V whereas ascorbic

acid oxidation is observed to start at 0 V on DA

modified EG�/CCE electrodes while the attached DA

starts to undergo oxidation at 0.05 V. Steady stateexperiments reveal that even a high concentration (0.8

mM) of acetaminophen does not interfere with the

signal of (0.2 mM) NADH oxidation at 0.15 V while

Fig. 10. Plot of catalytic current vs. concentration of ethanol at 0.15 V

in the steady state mode. (A) DA modified EG�/CCE (inset) I �/t

response for the additions of 3.67, 5.22, 8 mM (1, 2, 3, respectively) of

alcohol. (B) DA modified EG recompressed electrode.

P. Ramesh et al. / Journal of Electroanalytical Chemistry 528 (2002) 82�/9290

the interference from ascorbic acid could not be

eliminated. It is known in the literature that ascorbic

acid is an interferent for NADH oxidation [9,10].

Acetone and hexane are other compounds that are ofinterest in this context. The current response for 0.02 M

ethanol is not altered by the additions of 0.015 M

acetone and 0.008 M hexane while acetone and hexane

are found to interfere with the alcohol signal in the case

of a semiconductor based gas sensor [60].

3.6. Stability and surface renewability

The stability of the DA modified EG electrode is

found to be very good. The operational stability of the

electrodes in the steady state mode at 0.15 V shows thatthe signal remains constant for about 30 min. The

storage stability of the modified EG recompressed

electrodes is comparable with the sol�/gel composites

and is of the order of 4 months without any loss of

electroactivity. This may be compared with the stability

of the electrodes reported earlier for NADH oxidation.

The ortho -quinone modified GC electrodes have been

reported to be stable only for a few cycles in the presenceof NADH [9,10]. The storage stability of the bulk-

modified electrodes for the NADH oxidation based on

carbon paste and graphite epoxy electrodes are also

reported to be about 2 weeks [54,59].

Surface modified electrodes are prone to poisoning,

thus leading to loss of activity. The adsorption of

intermediates and NAD� may be responsible for the

loss of activity. Hence, it is desirable to have bulk-modified electrodes for the NADH and alcohol detec-

tion in the solution phase. The surface of the DA

modified EG electrodes can be renewed easily by

polishing using a SiC emery paper. This exposes a fresh

surface that is modified in an identical fashion to that of

the previous surface. Repeated polishings of the mod-

ified electrode yield currents with a standard deviation

of 5%.

4. Conclusions

Electrocatalytic oxidation of NADH and biosensing

of ethanol on sol�/gel composites and binderless recom-

pressed DA modified EG electrodes have been demon-

strated. The sol�/gel composite offers a porous,

hydrophobic surface for operation, leading to a good

response. The porous nature of the sol�/gel electrode canbe used for gas phase sensing of ethanol and is being

pursued. Sol�/gel electrodes and binderless electrodes

can offer better operational and storage stability over

carbon paste electrodes.

Acknowledgements

The authors wish to acknowledge DST and CSIR,

New Delhi, India for financial support. P. Bera isthanked for the help in recording XPS spectra.

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