Accepted Manuscript

Title: General approach for electrochemical functionalizationof glassy carbon surface by in situ generation of diazonium ionunder acidic and non-acidic condition with a cascade protocol

Author: Hamid Salehzadeh Davood Nematollahi VahidKhakyzadeh Banafsheh Mokhtari Luke C. Henderson

PII: S0013-4686(14)01350-4DOI: http://dx.doi.org/doi:10.1016/j.electacta.2014.06.134Reference: EA 22996

To appear in: Electrochimica Acta

Received date: 28-4-2014Revised date: 12-6-2014Accepted date: 13-6-2014

Please cite this article as: H. Salehzadeh, D. Nematollahi, V. Khakyzadeh, B.Mokhtari, L.C. Henderson, General approach for electrochemical functionalizationof glassy carbon surface by in situ generation of diazonium ion under acidicand non-acidic condition with a cascade protocol, Electrochimica Acta (2014),http://dx.doi.org/10.1016/j.electacta.2014.06.134

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General approach for electrochemical functionalization of glassy carbon

surface by in situ generation of diazonium ion under acidic and non-acidic

condition with a cascade protocol

Hamid Salehzadeh,a Davood Nematollahi,*a Vahid Khakyzadeh,a Banafsheh Mokhtari,a Luke

C. Hendersonb,c

Faculty of Chemistry, Bu-Ali-Sina University, P. O. Box 65174, Hamedan, Iran.

Tel.: +98 811 8282807; Fax: +98 811 8257407. E-mail: nemat@basu.ac.ir.

bStrategic Research Centre for Chemistry and Biotechnology, Deakin University, Geelong,

Victoria, Australia , 3216

cInstitute for Frontier Materials, Deakin University, Geelong, Victoria, Australia , 3216

Abstract

Immobilization of catechol derivatives on GC electrode surfaces can be performed by

in situ generation and reduction of nitrocatechol. We present the oxidative nitration of

catechol in the presence of nitrous acid followed by electrochemically reduction of

the generated nitro aromatic group to the corresponding amine group and its

conversion to diazonium cation at the electrode surface to yield a surface covalently

modified with catechol. In this manner, some derivatives of catechol can be

immobilized on the electrode surface. Whole of the process is carried out in

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Triethylammonium acetate ionic liquid as an inert and neutral medium (pH~7.0).

Surface coverage can be easily controlled by the applied potential, time and

concentration of catechol. After modification, the electrochemical features of

modified surface have been studied. Also modified GC electrode exhibited

remarkable catalytic activity in the oxidation of NADH. The catalytic currents were

proportional to the concentration of NADH over the range 0.01-0.80 mM. This

condition can be used for modification of GC surfaces by various aromatic molecules

for different application such as design of sensors and biosensors.

Keywords: Electrochemical derivatization, Nitrocatechol, Diazonium ion, Triethylammonium

acetate.

1. Introduction

In recent year’s modification of surface with organic films have performed by several

methodologies, including chemisorption and physical adsorption [1]. Between them, one of

the most dominant procedures is the covalent attachment of aryl groups by electroreduction

of diazonium salts [1-4]. The functionalization of surfaces including carbon materials, metals,

electrodes and powders during the past decade has been widely performed by this procedure

[1-7]. Various substituted aryl groups can be immobilized onto a variety of surfaces in order

to change their properties for different applications [8-10], including bioelectrochemistry

[11], molecular electronics [12], immobilization of organometallics [13] and corrosion

protection [14], design of novel high-performance redox surfaces [10,15,16], (bio)sensors

[17,18], biocatalysts [19], and hybrid molecule-on-semiconductor nanoelectronic devices

[20].

Functionalization of carbon substrates by electroreduction of aryl diazonium salts is

affected, by multitude of factors such as: (i) the applied potential, (ii) the electrolysis time

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[21-23], (iii) concentration and electronic nature of the diazonium salt [24,25] (iv) the type of

carbon material used, and (v) the type of solvent [26,27]. Nonetheless, modification of

surfaces by this method suffers from high reactivity of aryl diazonium salts and so controlling

the extent of reaction can be challenging. It has been observed that surface grafted aryl

groups are attacked by aryl radicals which generally results in multiple attachments and

multilayer formation [28,29]. To resolve this problem, introduction of bulky substituents on

the ArN2+ moieties was proposed to limit the secondary radical reactions. Using this strategy,

ultrathin organic layers were obtained [25,30]. In other work, 2,2-diphenyl-1-picrylhydrazyl

was used as a radical scavenger to control, or prevent, the polymerization of the

electrogenerated aryl radicals on a carbon electrode [31]. In other strategies, to overcome the

high reactivity of diazonium ions, some precursors were used for in situ generation of these

ions. Recently, Tour's research group developed a convenient procedure for in situ

functionalizing of Si(100) and single-wall carbon nanotubes (SWNTs) in acidic aqueous

media using organic triazenes [32,33]. Also, a two-phase laminar flow procedure was applied

by Downard's group to form an aryldiazonium ion from an aryltriazene precursor at the

interface between the two streams including aryltriazene and acid [34]. In other work,

Cougnon et al. reported an organo-layer modification procedure based on stepwise reduction

of nitro precursors for in situ generation of diazonium ion [35-37]. In these works, the

modification could be carried out at room temperature and the problem of high reactivity of

aryldiazonium ions was reduced.

Nevertheless generation of diazonium ion in surface modification by precursors

(compounds containing amine, nitro or aryltriazene) requires the use of acidic environment

[32-37]. This condition may be a major limitation for the surface grafting of compounds

possessing acid-sensitive functional groups [38]. Thus there is a need for inert conditions to

overcome this limitation to expand the chemical space available by the methodology.

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In 2011, Daasbjerg and co-workers immobilized organic layers onto surface of a GCE by

electrochemical reduction of in situ generated diazonium ion from phenyltriazene

functionality in acetonitrile and non-acidic media by applying double-pulse potentials [39]. In

this work, the required protons for the conversion of phenyltriazene to the corresponding

diazonium ion is prepared from oxidation of N,N'-diphenylhydrazine by an oxidative pulse. A

reductive pulse converts diazonium ion to its reduced form.

In other work, reported by Cougnon et al., activated carbon was functionalized via the

spontaneous reduction of sulfonic acid functionalized phenyldiazonium salts generated in situ

in neutral water. In this elegant approach the proton source originate from sulfonic acid

substituent, thus requiring no additional acidic additive to promote reaction progression [40].

Spontaneous grafting of diazonium salts in a neutral aqueous medium was also reported [41].

In a different work, spontaneously reaction of diazoates, produced from reaction of hydroxyl

ions with diazonium salts in a basic condition, with Fe and Au surfaces was reported to

modification with aryl groups [42].

Function of more than 300 dehydrogenase based enzymes in living system is dependent

to the enzyme cofactor nicotinamide adenine dinucleotide (NAD+) [43]. NAD+ dependent

dehydrogenase biosensing device acts based on the quantification of enzymatically generated

NADH. The concentration of NADH which produced is enzymatically proportional to the

amount of available substrate in the sample. Though the redox potential of NAD+/NADH

couple is -0.56 V vs SCE at pH 7.0 [44], but due to slow electron transfer and dimerization of

intermediates, high positive potential, as large as 0.8-1 V, is required for the oxidation of

NADH on common electrodes [45]. Various methodologies have been used to improvement

of the electron transfer reaction and to decrease the overpotential of NADH oxidation [43].

The oxidation of NADH has been traditionally facilitated by the redox mediator based

electrodes [46-48].

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In the current work, we present a new general approach to covalently immobilize

catechol derivatives onto the GCE surface. We describe, in situ chemically generated

nitrocatechol, in the bulk solution, which is electrochemically reduced to aminocatechol

followed by immediate conversion to the corresponding diazonium ion that is

electrochemically reduced and immobilized onto the surface of GCE. The entire process is

done in aqueous triethylammonium acetate (TEAA) solution which is a neutral buffered

solution (pH ~ 7.0) and also is a source of protons for various steps of modification, e.g.

(generation of nitrous acid required for chemical oxidative nitration of catechol, etc). This

procedure can be applied to electro-grafting of other amino and nitro aryl groups to

appropriate applications. Finally, we will show that glassy carbon electrode modified with

catechol exhibit a good catalytic activity in the electrooxidation of NADH.

2. Experimental

2.1. Reagents and Apparatus

4-Nitrocatechol, catechol, 3-methoxycatechol, 3-methylcatechol, triethylammonium

acetate (TEAA) and sodium nitrite were obtained from Sigma-Aldrich. All other chemicals

used in this investigation were of analytical grade. Cyclic voltammetry, controlled-potential

coulometry and preparative electrolysis were performed using an Autolab model PGSTAT

302N potentiostat/galvanostat. The working electrode used in the voltammetry experiment

was a glassy carbon disc (1.8 mm diameter) and platinum wire was used as counter electrode.

The working electrode used in controlled-potential coulometry was an assembly of four

carbon rods (6 mm diameter and 4 cm length) and a large platinum gauze constitute the

counter electrode. The working electrode potentials were measured versus Ag/AgCl electrode

(saturated KCl) (all electrodes from AZAR electrode). The glassy carbon electrode was

polished using alumina slurry (from Iran Alumina Co.).

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2.2. Controlled-potential coulometry

70 ml of aqueous phosphate buffer solution (c = 0.1 M, pH = 7.0) containing catechol

(0.25 mmol) and sodium nitrite (0.25 mmol) was subjected to electrolysis at 0.22 V versus

Ag/AgCl in a divided cell. The electrolysis was terminated when the decay of current became

more than 95%. At the end of electrolysis the yellow product was extracted in ethylacetate.

After drying of ethylacetate, the product was characterized by IR and cyclic voltammetry.

2.3. Chemical nitration of catechol

To a solution of catechol (0.5 mmol) in 30 ml of aqueous phosphate buffer (pH 3.0, c =

0.2 M), sodium nitrite (1.5 mmol) was added. The solution was stirred for approximately 45

min. Then, the reaction mixture was neutralized to pH = 7.0 by addition of sodium carbonate

and then product was extracted into ethylacetate. After drying, the product was characterized

by IR, 1HNMR and cyclic voltammetry.

2.3. Electrografting of Electrode Surface

Glassy carbon electrode was polished with alumina powder (0.05 μm) on a polishing

cloth and rinsed thoroughly with water. The polished electrode was then modified in situ

under potentiostatic condition at −0.60 V for catechol or by potential cycling over the range

of 0.0 to -0.9 V vs. Ag/AgCl in aqueous solution containing HCl (pH = 1.0) or TEAA (1/1

v:v water/ TEAA, pH ~ 7.0) as a proton source, catechol and NaNO2. Finally, the as prepared

catechol modified GCE was thoroughly rinsed with distilled water. In all cases, the electrode

modification was carried out at room temperature in an electrolytic mixture. The redox

activity of the catechol modified GCE was quantified by cyclic voltammetry in aqueous

buffered solutions.

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3. Results and discussion

Cyclic voltammogram of catechol (Figure 1, curve a) shows one anodic peak (A1) and its

cathodic counterpart (peak C1) which correspond to the transformation of catechol to o-

benenzoquinone and vice-versa via a quasi-reversible two-electron process. In this figure,

cyclic voltammogram of nitrite ion (curve b) shows an irreversible oxidation peak situated at

0.85 V vs. Ag/AgCl which is attributed to the oxidation of nitrite to nitrate ion [49-51].

Figure 1

Reaction of nitrite ion with electrochemically generated o-benzoquinone was studied by

cyclic voltammetry and controlled potential coulometry. In the presence of nitrite ion, the

voltammogram of catechol shows a decrease in the cathodic peak C1 and the appearance of a

new anodic peak (A3) in the more positive potentials which is related to electrochemical

oxidation of produced 4-nitrocatechol [52]. More voltammetric studies were performed by

increasing of the potential scan rate. Our data shows that at high potential sweep rates, the

peak current ratio (IpC1/IpA1) is about one, while, the current of peak A3 (IpA3) is nearly zero.

These data confirm the reactivity of electrochemically generated o-benzoquinone toward

nitrite ion.

Controlled-potential coulometry of aqueous phosphate buffer solution (c = 0.2 M, pH =

7.0) containing catechol (0.25 mmol) and sodium nitrite (0.25 mmol) at 0.22 V versus

Ag/AgCl was also performed. Cyclic voltammetric analysis, carried out during the

electrolysis, shows the progressive formation of anodic peak A3, parallel to the disappearance

of the peak A1.

The plot of IpA1 vs. Q shows that IpA1 disappears when the charge consumption becomes

about 2e- per molecule of catechol. All of these analysis confirm the reaction of nitrite ion

with electrochemically produced o-benzoquinone and generation of 4-nitrocatechol (4-NC).

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As previously reported, nitrous acid (pKa = 3.27) [49,50] can act as an oxidizing agent

for compounds such as catechols and converts them to the corresponding o-benzoquinone via

a two-electron process [49,50]. The reaction pathway is shown in Scheme 1. The oxidation of

4-NC is more difficult than the oxidation of starting molecule by virtue of the presence of

electron-withdrawing nitro group on the catechol ring and therefore, its oxidation is

circumvented [52].

The progress of the chemical nitration of catechol with NO2- in acidic media was tracked

by cyclic voltammetry (Figure 2, Part I). As shown during the oxidation of catechol by

nitrous acid, anodic peak A1 and its cathodic counterpart (peak C1) decreases and disappears

after 40 minutes. In addition, anodic peak A2 (related to the oxidation of NO2-) decreases. On

the other hand, a new anodic peak (A3) which corresponds to the oxidation of chemically

produced 4-nitrocatechol appears at more positive potentials relative to the potential of peak

A1 [49-52]. Similar data was obtained for chemical nitration of catechol by NO2- in aqueous

solution of TEAA ionic liquid.

In addition, formation of 4-nitrocatechol during reaction of catechol by nitrous acid was

investigated by tracking of cathodic peak C2 The cyclic voltammetric curves show the

progressive increase of the peak C2 which is related to the reduction of chemically produced

4-nitrocatechol to the corresponding amine group (Scheme 1) (Figure 2, Part II).

Figure 2

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Working electrode

Reference electrodeCounter electrode TEAA

Catechol

H2O

NaNO2

NaNO2HOOH

NO2

HOOH

NH2

Step One: Catalytic Oxidation of Catechol

Step Two: Attacking of Nitro group and Reduction of it

+ 6 e

+ 6 H

NaNO2 TEAA

HOOH

N2

- N2

+1 e

HOOH Surface Functionalization

of the Working Electrode

Step Three: In situ generation of diazonium ion

Step Four: Surface functionalization of the working electrodeonto surface of the working electode

In solution

Nitrogen Purging

(II)

(III)

(IV)

(V)

OHOH

OHOHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OH

Wor

king

Elec

trod

e

Working Electrode

2 NaNO2

NOHO N

HO ONO O H2O

O

O

(I)

N O

HOO

H

N O

OO

NO

H

NO H

[TEAA] -H

TEAA

Scheme 1. Modification of GCE by electroreduction of in situ generated 4-nitrocatechol

For more confirmation, chemically formation of 4-nitrocatechol in acidic condition was

also tracked by UV–vis absorption spectroscopy. Our data show that, catechol has an

absorption peak (P1) at 273 nm while 4-nitrocatechl has two absorption peaks (P0 and P2) at

233 and 333 nm, respectively. During the reaction of catechol with nitrite ion at pH 3.0, peak

P1 is decreased and peaks P0 and P2 are increased, which shows the formation of 4-

nitrocatechol during the chemical reaction.

Possible derivatization of GCE surface by reduction of diazonium salt has been shown

by Cougnon et al in which glassy carbon electrode was modified by a protected catechol

structure (1,2-bis-(tertbutyldimethylsilanoxy)-4-(4-nitrophenylethynyl)-benzene) [36,37].

With respect to these data, we coupled in situ nitration of catechol with electrochemical

reduction of produced 4-nitrocatechol for one-pot functionalization of GCE. All of stages

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were carried out initially in acidic aqueous solution (HCl, pH = 1.0) and then independently

in buffered aqueous solution by TEAA ionic liquid with pH ~ 7.0 and results of two

conditions were compared. Figure 3 parts I and II shows the successive cyclic

voltammograms of 4-nitrocatechol and catechol respectively obtained at GCE in aqueous

solution containing potassium nitrite and HCl (pH = 1.0). In both cases, upon the first scan of

the GCE to negative potentials, voltammogram shows a cathodic peak (C2) at −0.52 V versus

Ag/AgCl. As shown, following nine cycles over the -0.2V to -0.65 V range vs. Ag/AgCl at a

scan rate of 100 mV s-1, potential of this peak (C2) shifts negatively and its current totally

disappears. After thorough rinsing with acetonitrile and distilled water, the blocking effect of

the electrode response persists. Fig 3, part II shows that similar to 4-nitrocatechol, in situ

chemically generated 4-nitrocatechol is electrochemically reduced to the corresponding 4-

aminicatechol [35]. In a similar way, cougnone et.al are shown that multicyclic

voltammograms of 1,2-bis-(tertbutyldimethylsilanoxy)-4-(4-nitrophenylethynyl)-benzene

results to its grafting on to the GCE surface [37].

Figure 3

In the next step, we investigated the possibility of GCE modification at neutral pH. But

in these conditions, the dissociation equilibrium of nitrous acid lies predominantly to the right

and thus is mainly in its nitrite ion form. Thus the nitrous acid concentration, as an oxidizing

agent, is not sufficient for the oxidation of catechol. So, in order to perform GCE surface

modification in neutral conditions, TEEA (an ionic liquid with pH ~ 7.0) was used.

Repetitive cyclic voltammograms of 4-nitrocatechol (Figure 4, part I) and catechol (Figure 4,

part II) in TEEA aqueous solution (pH ~ 7.0) containing sodium nitrite are shown. As seen,

similar to HCl solution, a cathodic peak C2 related to the reduction of nitro aromatic group

appears and its potential shifts to the negative right. In addition, its current decreases under

subsequent cycles and finally disappears following the twentieth cycle. The results, obtained

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in both acidic and neutral conditions, indicate that similar to 4-nitrocatechol, in situ produced

4-nitrocatechol (Scheme 1), is reduced to the corresponding 4-aminocatechol during the

cathodic peak C2 (Scheme 1, step III). In the presence of nitrous acid, cathodically generated

4-aminocatechol rapidly reacts with HNO2 to produce corresponding diazonium ion (Scheme

1, step IV). After this, the generated diazonium ion is further reduced on the surface of glassy

carbon electrode, resulting to the formation of aryl radicals. Reaction of these radicals, with

unsaturated carbon atoms of the GCE, yields catechol layer covalently attached to the GCE

surface (Scheme 1, step V). The formation of this layer prevents subsequent reduction, and

consequently cathodic peak shifts to the negative potentials and its current decreases and

finally disappears.

Figure 4

3.1. Modified Electrode

In Figure 5, CVs related to the catechol electrografted glassy carbon electrode using

discussed procedures are presented. In all of them, the modified electrode shows a reversible

peak at 0.19 V in which anodic peak A1 is related to the oxidation of catechol to the

corresponding o-benzoquinone and cathodic peak C1 corresponds to the revers reaction. In

Figure 6, cyclic voltammograms correspond to the modification of GC electrode in the

presence and absence of NaNO2 or proton source are presented. Curve a is related to the

modification in solution containing catechol, NaNO2 and HCl. Modification condition of

cyclic voltammogram b is as same as a but in the absence of NaNO2 and modification

condition of cylic voltammogram c is as same as a but in the absence of HCl. As seen, only in

the presence of all reagents required for modification (catechol, NaNO2 and proton source)

catechol can be immobilized on to the GCE (Figure 6, curve a). CVs corresponding to the

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electrografted glassy carbon electrode in these conditions are also shown in Figure 6, curves

d-f. These data also confirm the results obtained from the comparison of curves a-c.

Figure 5

Figure 6

Figure 7 (part I) represents the normalized cyclic voltammograms of immobilized

catechol at different scan rates (these voltammograms were obtained by dividing the current

by the scan rate, I/ν). As shown, IpA1/ν and IPC1/ν remain nearly constant, upon increasing the

potential sweep rate, implying that electron transfer process is related to the electrografted

species. In addition, the cathodic to anodic peak current ratio (IpC/IpA) is about one at given

scan rates, indicating a reversible electron transfer process. A fast electron transfer at the

electrode surface is indicated by small peak potential separation (although not zero) and

independence of peak shape from scan rate. The formal potential of voltammograms is also,

independent of the potential scan rate so that the transfer coefficient (α) was anticipated to be

equal to 0.5 [48]. By plotting of log IpA1 vs. log v, a linear relationship with equation log IpA1

= 1.06 log v -2.34 (R2 = 0.9993) was obtained. It was reported that when the slope of log Ip

against log v is 0.5, the electrochemical reaction is a diffusion controlled process, while when

the slope equals to 1.0, the electrochemical reaction occurs via an adsorption-controlled

process [53,54].

Contrary to the low potential sweep rates (Figure 7, part I), at high potential sweep rates,

∆Ep values increased significantly with increasing sweep rate, suggesting a limitation in the

kinetics of charge transfer (Figure 7, part II). When ∆Ep > 200/n (at high potential scan rates),

the heterogeneous electron transfer rate constant (ks), (assuming that a monolayer of catechol

is adsorbed onto the surface of electrode) can be calculated by plotting Ep vs. log ν (see Table

1) [55]. Heterogeneous electron transfer rate constant of GC and Au electrodes modified by

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catechol derivatives was also calculated previously [56,57]. Our results are very close to the

values reported in Ref. 56 and are higher than Ref. 57.

Figure 7

Modification of the electrode surface has been influenced by the electrolysis potential,

time and concentration of catechol. Maximum surface coverage (Γ) was found to be at the

potential of -0.60 V, time of 250 s and in 1.0 mM of catechol or 4-nirocatechol. The GCE

was also modified with 3-methylcatechol and 3-methoxycatechol by the same method

described for catechol (time 250 s and c = 1.0 mM). The optimum potential of modification

of 3-methylcatechol and 3-methoxycatechol are -0.64 and -0.70 V vs. Ag/AgCl, respectively.

Parameters related to the catechol derivatives modified electrodes including surface coverage

(Γ), anodic and cathodic electron transfer rate constants ( sA and ks

C) and E1/2 are given in

Table 1. As shown, with respect to substituent of 3-methylcatechol and 3-methoxycatechol,

surface coverage for these derivatives is lower than that for catechol.

Table 1

As shown in Table 1, the calculated surface coverage (Γ) was found to vary in the order

catechol > 3-methylcatechol > 3-methoxycatechol. The observed trend is expected, since the

amount of surface coverage (Γ) is dependent on the size of the substituted group on the

catechol ring. The presence of methyl or methoxy groups on catechol ring causes a decrease

in Γ. Furthermore, the heterogeneous rate conxstant (ks) of the immobilized catechol derivatives

for both anodic and cathodic peaks, varies in the order catechol > 3-methoxycatechol > 3-

methylcatechol. The observed trend, confirms that catechol itself is more suitable mediator

than 3-methylcatechol and 3-methoxycatechol for the catalytic oxidation of NADH.

Because of the participation of proton(s) in the oxidation of catechols, the response of

catechol modified electrode would be pH dependent. The voltammetric response of the

catechol-bonded electrode was studied in buffer solutions with pH values from 1.0 to 8.0. It

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was found that, both reduction and oxidation peak potentials shifted to the negative potentials

by increasing pH and half-wave potential showed a linear dependence with pH, with a slope

very close to the 59 mV/pH unit which is expected for a two-proton, two-electron process

according to Nernstian behavior. The electrochemical response and surface coverage of the

modified electrode remained nearly constant in all pHs, showing the stability of the catechol

bonded electrode over the pH range 1.0-8.0 [48].

3.2. Electrocatalysis

One of the aims of the present work is to examine the electrocatalytic oxidation of

NADH by the surface modified catechol. It is previously reported that quinones (specially, o-

quinones compared to the p-quinones) are an excellent mediator for the oxidation of NADH

[41]. Cyclic voltammetric response of the catechol bonded electrode was obtained in pH =

7.0 in the absence and in the presence of NADH (1.0 mM) in order to the study of its

electrocatalytic activity toward NADH oxidation (Figure 8). In the absence of NADH, a well-

defined voltammogram for the catechol on the electrode could be obtained at the formal

potential of 0.19 V vs. Ag/AgCl. The occurrence of a catalytic reaction in the presence of

NADH is supported by the increase of IpA1 and decrease of IpC1, which could indicate that

electro-catalytically active o-quinone formed at the surface of the electrode reacted with

NADH to form NAD+ and catechol. The catechol, which is regenerated at the electrode

surface subsequently reoxidized and again is accessible for another catalytic cycle (Scheme

2).

Figure 8

Scan rate-dependence studies further support the creation of catalytic current in the

oxidation of NADH by modified electrode. Normalized voltammograms (I/ν1/2) of the

modified electrode in the presence of NADH at different scan rates are shown in Figure 8,

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part II. By decreasing the potential scan rate (longer time scale), the reaction between the

electro-generated o-quinone on the surface of electrode and NADH via the EC' mechanism

has sufficient time to occur (Scheme 2). At shorter time scale (higher scan rates), the anodic

response for the mediated oxidation of NADH by catechol is not readily apparent. These

observations indicate that in longer time scale the catalytic reaction can be carried further and

thus lead to the higher catalytic signal.

 

OHHO

OO

2NADH 2NAD+

-2e- -2H+

Scheme 2. Catalytic oxidation of NADH by surface confined catechol.

 

It was also found that the catalytic current of surface modified electrode in the presence

of NADH depends on the solution’s pH. The anodic peak current (IpA1) increases firstly with

increasing pH, reaches to a maximum value at pH 7.0, and then decreases. Thus we choose

pH = 7.0 as an optimum pH for catalytic oxidation of NADH.

Figure 9 shows the electrocatalytic activity of surface bounded catechol toward the

oxidation of different NADH concentrations. The current of the GC-modified electrode

linearly responds to the NADH concentration over the two linear ranges consisting of 0.01-

0.15 and 0.15-0.80 mM, which covers values of a great relevance in biosensor design and

applications. In addition, for the modified electrode, detection limit of 6.0 μM was estimated.

As anticipated for a Michaelis- Menten-type reaction, the catalytic signal was leveled off at a

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high concentration (>0.8 mM) which was ascribed to kinetic limitations. The linear range of

this method is consistent with that of method reported previously [56], while, the detection

limit of this method is significantly improved.

Figure 9

We have also investigated the reaction mechanism at pH = 7.0. By considering the

catalytic reaction as Eqs. (2) and (3), the reaction current, Ip, can be obtained using Eq. (4):

Mred Mox + 2e- + 2H+ (2)

NADH + Mox NAD+ + Mredk1 (3)

Ip = nFAΓk1C* (4)

Here Γ represents the surface coverage of the mediator and C* is the bulk concentration

of NADH. The catalytic reaction proceeds via an intermediate complex (Eq. 5) [57].

NADH + Mox NAD+ + Mredk+1

k-1[NADH-M]

k+2 (5)

According to the Michaelis–Menten type reaction model, and using the Lineweaver–

Burk type equation for the reaction dynamics:

k1 =k+2

Km + C*(6)

where Km is Michaelis parameter. The plots of k1 and k -11 vs. NADH concentration (C*) are

shown in Figure 10. Plot of k -11 vs. NADH concentration gives a straight line indicating that

in the catalytic oxidation of NADH by modified electrode an intermediate complex is

involved. The reaction parameters including, Km and k+2 can also be determined from the

slope and the intercept of the plots; they were 0.26 mM and 5.02 s−1, respectively. These

values are consistent with that of values reported previously [57-59].

Figure 10

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4. Conclusions

In summary, we have demonstrated that catechols can be successfully electrografted onto

GC surfaces via their chemical nitration and then electrochemical reduction. This method

involves the in situ chemical generation of 4-nitrocatechol from catechol and NO2- followed

by electrografting of diazonium ion produced from 4-aminocatechol which itself generated

from electrochemical reduction of created 4-nitrocatechol. Required protons (for conversion

of nitrite ion to nitrous acid and also for diazotization reaction) can be originated from HCl

(pH = 1.0) or TEAA (pH ~ 7.0) aqueous solutions. By electrochemical reduction of nitro

aromatic to amino aromatic group, diazonium salt is generated exactly in the adjustment of

electrode surface to be modified and also diazotization reaction of produced amine occurs in

non-acidic media resulted from TEAA aqueous solution. As a result, directly anchoring of

molecules containing functionalities that may be capable of reacting with the diazonium

group is possible. Finally, as an application modified electrode surface was used for the

catalytic oxidation of NADH at less positive potential. The catalytic currents are proportional

to the concentration of NADH in solution. Obtained results were very promising and could be

effectively applied in the field of electrocatalysis and electrochemical biosensors.

Acknowledgments

We acknowledge the Bu-Ali Sina University Research Council and the Center of

Excellence in Development of Environmentally Friendly Methods for Chemical Synthesis

(CEDEFMCS) for their support of this work. Also, the institute for frontier materials and the

strategic research center for biotechnology and chemistry are thanked for their continual

support of LH.

 

 

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Supplementary materials

Supplementary materials associated with this article can be found, in the online version, at

http://dx.doi.org...

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Figure Captions

Fig. 1. Cyclic voltammograms of (a) catechol (1.0 mM) and (b) sodium nitrite (3.0 mM) at

glassy carbon electrode, in phosphate buffer solution (pH 3.0, c = 0.2 M). Scan rate: 500

mV s-1. Temp. = 25 ± 1 oC.

Fig. 2. Cyclic voltammograms of catechol (1.0 mM) in the presence of sodium nitrite (3.0 mM)

during chemical reaction in aqueous phosphate buffer (pH = 3.0) at various times; reaction

times are 1, 5, 10, 15, 20, and 40 min from (a) to (f), respectively. Part I: Es = -0.1 and Ef =

1.0 V vs. Ag/AgCl. Part II: Es = 0.0 and Ef = -0.9 V vs. Ag/AgCl. Scan rate 500 mV s-1.

Temp. = 25±1 ◦C.

Fig. 3. a) First, b) second, c) third and d) ninth cycles of cyclic voltammograms of (I) 4-

nitrocatechol (1.0 mM) and (II) catechol (1.0 mM) at a bare glassy carbon electrode, in

water solution containing sodium nitrite (5 mM) and HCl (pH = 1.0). Scan rate: 100 mV

s−1. Temp. = 25±1 oC.

Fig. 4. a) First, b) Second, c) fifth, d) tenth and e) twentieth cycles of cyclic voltammograms

of 4-nitrocatechol (1.0 mM) (left) and catechol (1.0 mM) (right) at a bare glassy carbon

electrode, in aqueous/TEAA (1:1) solution containing sodium nitrite (5 mM) (pH = 7.0).

Scan rate: 100 mV s−1. Temp. = 25±1 oC.

Fig. 5. Cyclic voltammograms of electrografted catechol in various conditions (I) from 4-

nitrocatechol precursor in the presence of HCl (pH = 1.0) (II) from catechol precursor in

the presence of HCl (pH = 1.0) (III) from 4-nitrocatechol precursor in the presence of

TEAA (pH = 7.0) (IV) from catechol precursor in the presence of TEAA (pH = 7.0).

Concentration of NaNO2 = 5 mM, TEAA/water ratio is 1/1 v:v. Scan rate: 1000 mV s−1.

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Fig. 6. Cyclic voltammograms for modification of GCE (a) catechol (1.0 mM) in the

presence of sodium nitrite (5 mM) and HCl (pH = 1.0), (b) catechol (1.0 mM) in the

presence of HCl (pH = 1.0) and (c) catechol (1.0 mM) in the presence of sodium nitrite (5

mM). Corresponding cyclic voltammograms for modified electrode in aqueous solution

containing phosphate buffer (pH = 7.0). d, e and f are related to a, b and c respectively.

Scan rate 1000 mV s-1.

 

Fig. 7. Part I: Normalized cyclic voltammograms of the surface-confined catechol in

phosphate buffer solution (pH = 7.0, c = 0.10 M) at (low) potential scan rates: 0.05, 0.10,

0.25, 0.50 and 1.00 V s-1. Part II: Cyclic voltammograms of above electrode at (high)

potential scan rates: (a) 5, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50 and (g) 60 V s-1. Part III:

Experimental variation of Ep vs. the log v for part II. Part IV: Magnification of the same

plot for high sweep rates.

 

Fig. 8. Part I: Cyclic voltammograms obtained for the surface-modified catechol in phosphate

buffer solution (c = 0.2M, pH = 7.0). (a) In the absence and (b) in the presence of NADH

(1 mM). Scan rate: 20 mV s-1. Part II: Normalized cyclic voltammograms (I/ν1/2) in the

presence of NADH (0.5 mM) at different potential sweep rates: (a) 10, (b) 25, (c) 50 and

(d) 75 mV s-1.

Fig. 9. Part I: Cyclic voltammograms of the GC-modified catechol electrode in the presence

of different concentration of NADH in phasphate buffer (pH 7.0, c = 0.20 M). [NADH]:

(a) 0.000 (b) 0.010, (c) 0.050, (d) 0.075, (e) 0.100, (f) 0.150, (g) 0.200, (h) 0.400, (i)

0.600, and (j) 0.800 mM. Scan rate: 20 mV s-1. Parts II, III and IV show the corresponding

calibration plots.

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Fig. 10. Plots of k1 and k -11 vs. NADH concentration for the CV responses of the modified

electrode. Experimental conditions are as same as Fig. 9.

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Table 1. Surface coverage (Γ), heterogeneous rate constants and E1/2 of derivatized GCE prepared by

reduction of diazonium cations in situ produced from nitrocatechol derivatives.

Redox mediator

OHHO

OHHO

OHHO O

Γ[a] 8.0 5.8 5.1

E1/2 0.214 0.138 0.130

ks (anodic peak)[b] 67.9 24.8 42.3

ks(cathodic peak)[b] 54.6 20.4 33.6

[a] Anodic surface coverage (in mol cm-2 × 1011) [b] Heterogeneous electron transfer rate constant (s−1)

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GRAPHICAL ABSTRACT

General approach for electrochemical functionalization of glassy carbon

surface by in situ generation of diazonium ion under acidic and non-acidic

condition with a cascade protocol

Hamid Salehzadeh, Vahid Khakyzadeh, Davood Nematollahi* Banafsheh Mokhtari and Luke

C. Henderson

Graphical Abstract (for review)

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Highlighted

Selective electrochemical determination of NADH.

Electrochemical functionalization of glassy carbon surface by in situ

generation of diazonium ion.

Oxidative nitration of catechols.

Electrografting of catechols onto the GC surface.

Research Highlights

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-35

-15

5

25

45

65

85

105

-0.15 0.05 0.25 0.45 0.65 0.85 1.05

I/m

A

E vs. (Ag/AgCl)/V

A1

C1

A2

a

b

A1

C1

A2

a

b

Figure 1

Figure 1

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-14

6

26

46

66

86

106

-0.15 0.1 0.35 0.6 0.85 1.1

I/µ

A

E vs. (Ag/AgCl)/V

A1

C1

A3

A2

a

f a

f

f

a

Part I

-65

-55

-45

-35

-25

-15

-5

5

-1 -0.75 -0.5 -0.25 0

I/m

A

E vs. (Ag/AgCl)/V

C2

a

f

Part II

Figure 2

Figure 2

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-180

-160

-140

-120

-100

-80

-60

-40

-20

0

-0.75 -0.55 -0.35 -0.15

I/µ

A

E vs. (Ag/AgCl)/V

a

C2

Part I

d

-160

-140

-120

-100

-80

-60

-40

-20

0

-0.75 -0.55 -0.35 -0.15

I/µ

A

E vs. (Ag/AgCl)/V

a

d

C2

Part II

Figure 3

Figure 3

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-59

-49

-39

-29

-19

-9

1

-1 -0.8 -0.6 -0.4 -0.2

I/µ

A

E vs. (Ag/AgCl)/V

C2

a

e

Part II

-62

-52

-42

-32

-22

-12

-2

-1 -0.8 -0.6 -0.4 -0.2

I/µ

A

E vs. (Ag/AgCl)/V

a

e

C2 I

Part I

Figure 4

Figure 4

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-11

-7

-3

1

5

9

13

17

-0.05 0.05 0.15 0.25 0.35

I/µ

A

E vs. (Ag/AgCl)/V

A1

C1

I

-14

-10

-6

-2

2

6

10

14

-0.05 0.05 0.15 0.25 0.35

I/µ

A

E vs. (Ag/AgCl)/V

A1

C1

II

-15

-11

-7

-3

1

5

9

13

17

-0.05 0.05 0.15 0.25 0.35

I/µ

A

E vs. (Ag/AgCl)/V

C1

III

A1

-15

-11

-7

-3

1

5

9

13

17

-0.05 0.05 0.15 0.25 0.35

I/µ

A

E vs. (Ag/AgCl)/V

C1

A1 IV

Figure 5

Figure 5

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-200

-170

-140

-110

-80

-50

-20

10

-1 -0.8 -0.6 -0.4 -0.2 0

I/µ

A

E vs. (Ag/AgCl)/V

C2

d

e

f

-13

-9

-5

-1

3

7

11

15

0 0.1 0.2 0.3 0.4

I/µ

A

E vs. (Ag/AgCl)/V

A1

C1

a

b

c

Figure 6

Figure 6

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-350

-250

-150

-50

50

150

250

350

-0.1 0 0.1 0.2 0.3 0.4 0.5

I/µ

A

E vs. (Ag/AgCl)/V

a

g A1

C1

Part II

0.05

0.13

0.21

0.29

0.37

-2.2 -1.2 -0.2 0.8 1.8

E p/V

log (ν/V s-1)

Part III Ep = 0.17 log v + 0.036 R² = 0.9879

Ep = -0.15 log v + 0.367 R² = 0.9921

0

0.1

0.2

0.3

0.4

0.5

1 1.2 1.4 1.6 1.8 2

E p/V

log (ν/V s-1)

Part IV

Figure 7

-0.015

-0.011

-0.007

-0.003

0.001

0.005

0.009

0.013

0.017

-0.1 0.05 0.2 0.35

(I/v)/m

A. s

. mV

-1

E vs. (Ag/AgCl)/V

A1

C1

Part I

Figure 7

Page 38 of 40

Accep

ted

Man

uscr

ipt

-0.2

0

0.2

0.4

0.6

0.8

-0.2 -0.1 0 0.1 0.2 0.3 0.4

I/µ

A

E vs. (Ag/AgCl)/V

A1

C1

a

b

Part I

-0.1

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

-0.1 0 0.1 0.2 0.3 0.4 0.5

(I/ν

1/2

)/m

A s

1/2

mV

-1/2

E vs. (Ag/AgCl)/V

A1

a

C1

d

Part II

Figure 8

Figure 8

Page 39 of 40

Accep

ted

Man

uscr

ipt

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

-0.1 0 0.1 0.2 0.3 0.4 0.5

I/µ

A

E vs. (Ag/AgCl)/V

A1

a

j

Part I

0

0.15

0.3

0.45

0.6

0.75

0.9

0 0.2 0.4 0.6 0.8

[NADH]/mmol dm-3

Catalytic Current/mA

Part II

Ip = 2.24C + 0.06 R² = 0.9989

0.05

0.15

0.25

0.35

0.45

0 0.04 0.08 0.12 0.16 [NADH]/mmol dm -3

Catalytic Curren/mA

Part III

Ip = 0.84C + 0.27 R² = 0.9923

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.1 0.3 0.5 0.7 [NADH]/mmol dm-3

Catalytic Current/mA

Part IV

Figure 9

Figure 9

Page 40 of 40

Accep

ted

Man

uscr

ipt

4

6

8

10

12

14

16

0 0.2 0.4 0.6 0.8

(k1/1

0-3

)/d

m3 m

ol-1

s-1

[NADH]/mmol dm-3

Part I

y = 0.20C + 0.054 R² = 0.9976

0.05

0.08

0.11

0.14

0.17

0.2

0 0.2 0.4 0.6 0.8 (1

0-3

/k1)/

mo

l s d

m-3

[NADH]/mmol dm-3

Part II

Figure 10

Figure 10