Iwamoto M., Kwon Y.-S., Lee T. (Eds.) Nanoscale Interface for Organic Electronics

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CHAPTER 4

ELECTROCHEMICAL PROPERTIES OF SELF-ASSEMBLED

VIOLOGEN DERIVATIVE AND ITS APPLICATION TO

HYDROGEN PEROXIDE DETECTING SENSOR

Dong-Yun Lee, Hyen-Wook Kang, Sang-Hyun Park and Young-Soo Kwon

Department of Electrical Engineering and NTRC, Dong-A University,

Busan 604-714, Korea

E-mail: yskwon@dau.ac.kr

Viologen derivatives, which have been widely investigated their well-

behaved electrochemistry including electron transfer mediation, are

attractive materials because of their chemical stability, their relatively

simple behavior of redox reaction and their possible practical

applications due to their electrochemical properties. The self-assembled

monolayers (SAMs) were prepared surfaces by use of a viologen. The

electrochemical property of the self-assembled viologen derivatives has

been investigated with a quartz crystal microbalance (QCM), which

has been known as a nanogram order mass detector. After then we

attempted to apply this viologen derivative for biosensor design. A gold

electrode modified by viologen-thiol modification is used to design a

biosensor in corporate with hemoglobin (Hb). By incorporating with

SAMs viologen and Hb, viologen can act as an electron transfer

mediator between Hb and gold electrode. Experimental conditions

influencing the biosensor performance such as pH and/or potential are

optimized and assessed. This sensor offered an excellent electrochemical

response for H

concentration below µmol level with high

sensitivity, selectivity and short time response.

1. Introduction

Electrochemical properties of a redox molecule incorporated in a

phospholipid monolayer, bilayer, or membrane have attracted much

D.-Y. Lee et al. 50

attention in the past several decades.

Controlled modification of an

electrode surface with a monolayer film of organic molecular gas has

attracted much attention because of its relevance to the construction of

molecular devices. The modified electrode is also useful for the

understanding of the electron transfer mechanism at electrode/electrolyte

interfaces at a molecular level. An oriented monolayer assembly can be

created by both the Langmuir-Blodgett and the self-assembling methods

using various alkane derivatives and substrates.

The self-assembled

monolayers of alkane derivatives with sulfur containing head groups on

gold substrates have been widely examined recently, since the binding

between S atoms and the Au surface is strong and the S-anchored

monolayers thus formed have a well-oriented structure.

3,4

To understand

the structure of the assembled monolayer and electron transfer

mechanism, it is essential to conduct quantitative in situ monitoring of

mass transport both in the assembly process and in the electron transfer

process so that we can quantify the number of adsorbed molecules and

the movement of the ion and solvent accompanying an electron transfer

process. A recently developed electrochemical quartz crystal micro-

balance (EQCM) offers one of the best ways to obtain this quantitative

information.

Viologen derivatives are actually desirable electron mediators.

They

have been widely studied for their chemical stability, their relatively

simple behavior in redox reactions, and their possible practical

applications due to their electrochemical properties.

The electrochemical

behavior of the alkyl viologen as an electron transfer mediator and a

redox-active linker on a gold substrate, confined on the self-assembled

monolayer (SAM) coated on Au electrodes in the presence of different

supporting electrolytes. The investigation of a viologen derivative,

SH, has been carried out by cyclic voltammetry which indicated that

anions adsorbed and desorbed during redox reaction. Viologen exists in

three main oxidation states, namely, V

↔ V

•+

↔ V

. These redox

reactions, especially the first one (V

↔ V

•+

), are highly reversible and

can be cycled many times without significant side reactions.

This well-

known electrochemical behavior has used in various studies, such as an

electron transfer mediator.

Self-Assembled Viologen Derivative and Its Application 51

The QCM technique is based on the tendency of a piezoelectric

crystal to change its resonant frequency when additional mass adsorption

or desorption on the crystal electrodes takes place.

The analytical

technology has been improved or newly developed as to the incremental

requirement for electrochemical phenomena at the interface. The QCM

combined with electrochemical technique, namely EQCM, is a powerful

tool in investigating the interfacial processes that occur on surface and

thin-films.

The EQCM method is used in situ experiments to measure

mass changes at electrode surfaces after electrochemical deposition of

metal.

12,13

Theory on cyclic voltammograms of such surface-confined redox

species has been developed with use of the Langmuir adsorption

isotherm and the Nernst equation.

The self-assembling molecules

possess a redox-active site, the adsorption process can be monitored in

real time by electrochemical techniques, such as cyclic voltammetry

(CV). A number of researchers have also employed various electro-

chemical techniques to study the stability, permeability, and uniformity

of self-assembled films. The electrochemical techniques are very useful

in monitoring the adsorption process; however, they can only monitor

the amount of the adsorbed species having an electroactive site.

The

redox reaction and EQCM response using viologen derivative showed

influence of the anion.

16,17

Because the viologen molecules were finished

in self-assembly on the gold electrode of the QCM, they became the

reduction states. In this case, the anions were only adsorbed and

desorbed, but the paper showed the property of the anion.

Since the first biosensor, based on enzyme, invented by Clark and

Lyons,

there has been growing interest in the biosensor fields.

Biosensor is usually an analytical device being used in biotechnology,

medicine, industries and environmental monitoring. The detection of

hydrogen peroxide (H

) is a great interesting object to the biochemists

because of its validity in pharmaceutical, clinical, industrial, food area.

Recently, an increasing number of redox-active proteins have been

used to design biosensor for detecting H

and some other

target

materials.

19,20

In this point of view, horse radish peroxidase (HRP) is

the most commonly used enzyme.

21,22

But the HRP is not so stable in

aqueous media and very expensive. Alternately, hemoglobin (Hb) is one

D.-Y. Lee et al. 52

of the promising biomolecules which can be used as a bioreceptor

element. Hemoglobin, the main component in red blood cells, is one kind

of protein that picks up oxygen in the lungs and delivers it to the body

tissues. It has a molar mass of 67,000 g/mol

and consists of four kinds of

electroactive iron hemes. Usually, Hb can give the long-term stability to

the H

sensor as compared to other proteins. In addition, Hb is cheaper

than commonly used HRP.

In order to make a biosensor with the protein, it is very important the

electrochemical contact between redox-active protein, such as Hb and the

transducer such as electrode. Normally, the fast electron transfer between

protein, such as Hb, and electrode surface is not possible due to some

reason. Among them, the deep entombing of the electroactive heme

group in the protein structure is the main reason. There are numerous

papers have been published on the basis of direct electrochemistry of Hb.

In these circumstances, it is an intriguing object for biochemists to

achieve efficient electrical contact between protein and electrode. There

are numerous papers which have been published on the basis of direct

electrochemistry of Hb. Among them, hemoglobin immobilized on egg-

phosphatidycholine,

kieselgubr,

zirconium,

carbon nanotube

and

DNA

have been described successfully.

Several methods such as chemical modification

and polymers

modification

have been applied to electrically communicate redox

protein with the electrode. Langmuir-Blodgett (LB) technique has also

been used intensively for depositing the protein onto the electrode

surface.

In addition, there are a lot of materials such as polyphenol,

polypyrrole, polyaniline

30-32

have been used electrochemically with wide

range to immobilize biomaterials. But these kinds of deposited films tend

to be thick. Nonetheless, in the methods mentioned above, aloofness of

the protein or conformational change may affect the functional activity of

protein. As a result, the designed biosensor shows a lower sensitivity and

short longevity. Therefore, researchers are paying their attention to

fabricate the thin-films of a wide variety of materials which is just single

molecule thick. Dong and Li

have published a paper based on viologen

monolayer as the electron mediator of HRP. Viologen, redox-active

material, plays an important role as electron relays in electrochemical

process. It can enhance the electron transfer between the redox protein

Self-Assembled Viologen Derivative and Its Application 53

and electrode surface. There are only a few papers of SAMs of viologen

derivatives for designing biosensor with redox protein, have been

published till date. Therefore, it needs more investigation to understand

the intrinsic redox behavior of the protein with SAMs.

The purpose of this study is to observe the electrochemical behavior

of self-assembled viologen monolayers as electron transfer mediator and

its application in a biosensor. An in situ quartz crystal microbalance

(QCM) measurement indicated a similar adsorption trend and frequency

change for the formation of self-assembled monolayers (SAMs) of the

viologen.

A recently developed electrochemical quartz crystal micro-

balance (EQCM) method is used in ex situ experiments to measure mass

changes at electrode surfaces after electrochemical deposition of a metal

and offers one of the best ways to obtain this quantitative information.

We used a thiol-functionalized viologen derivative [VC

SH] for increasing

rate of electrons transfer of Hb. The method for modification of the

electrode is very simple and convenient. We designed the sensor by self-

assemble of viologen derivatives. To the best of our knowledge, for the

first time, we have designed a hydrogen peroxide biosensor based on

Hb-modified gold electrode where the thiol-functionalized viologen has

been performed a very important role for enhancing electrons transfer

rate of Hb.

2. Principle of QCM Technique

2.1. Overview

Since all solid materials have natural resonance frequencies of vibration,

almost any solid made of an elastic material could be used like a crystal.

For example, steel is very elastic and transmits sound with high-speed.

It was often used in mechanical filters before quartz. The resonance

frequency depends on size, shape, elasticity, and the speed of sound in

the material. High-frequency crystals are typically cut in the shape of a

simple, rectangular plate.

When a quartz crystal is properly cut and mounted, it gets deformed

in an electric field by applying a voltage to both electrodes on the crystal.

That is, mechanical strain generates electric polarization and electric

D.-Y. Lee et al. 54

field generates mechanical stress. This property is known as

piezoelectricity. When the field is removed, the quartz will generate an

electric field as it returns to its previous shape, and this can generate a

voltage. Consequently, a quartz crystal behaves like a circuit composed

of an inductor, capacitor and resistor, with a precise resonance

frequency.

The quartz crystal has 32 symmetry groups of trigonal system.

According to different cut angles, there are different kinds of quartz

plates, for example, AT-, BT-, CT-, DT-, NT-, and GT-cut quartz plates.

Each type of quartz cuts, indicated by a set of Euler angles, has different

available elastic, piezoelectric and dielectric properties, which are the

basic parameters for designing a crystal device. In this chapter, we used

an AT-cut quartz crystal.

Quartz has the further advantage that its elastic constants and its

size do not change by the change of the temperature. The frequency

dependence on temperature is also very low. The specific characteristics

depend on the mode of vibration and the angle at which the quartz is cut.

Therefore, the resonance frequency of the plate, which depends on its

size and thickness, does not change much. An early use of this property

of quartz crystals was in phonograph pickup. One of the most common

piezoelectric uses of quartz today is as a crystal oscillator. The quartz

clock is a familiar device using quartz. The resonance frequency of a

quartz crystal oscillator is changed by mechanically loading mass, and

this principle is used for very accurate measurements of very small mass

changes in the QCM and in thin-film thickness monitors.

In this chapter, we introduce overall QCM, including the equivalent

circuit, theory of a QCM and measurement principle of resonance

parameters.

2.2. Theory and Equivalent Circuit of QCM

The frequency shift induced by mass loading to the quartz crystal or

the electrode surface, is described by the Sauerbrey equation (1).

40-42

From Eq. (1), the ideal sensitivity can be calculated that a 9 MHz

QCM has the mass sensitivity about 1.1 ng per 1 Hz change in the

resonance frequency, where

= 2.95 × 10

g/cm⋅s

= 2650 kg/m

Self-Assembled Viologen Derivative and Its Application 55

and A = 0.2 cm

. This sensitivity is the reason the QCM has been widely

used as a mass sensor.

q q

f m

µ ρ

∆ = − ⋅ ∆

⋅

(1)

where, ∆f is the frequency change, f

is the fundamental resonance

frequency of the quartz, ∆m is the mass change, A is the electrode area,

is the shear modulus of the quartz, and

is the density of the quartz.

Since the Sauerbrey equation is useful for rigid mass loading in air,

Kanazawa and Gordon developed Eq. (2), which indicates the QCM can

be performed in liquid.

1/2

3/2

l l

q q

f f

η ρ

πµ ρ

 

∆ = −

 

 

(2)

where,

is the viscosity of the liquid and

is the density of the liquid.

The viscosity change of loading material also affects the resonance

property of the QCM. To measure the resonance resistance, R

, is useful

to investigate the viscosity change on the QCM as shown in Eq. (3).

As the resonance resistance is unaffected by mass, this parameter is

useful to analyze the rheological property on the QCM. After development

of Eqs. (2) and (3), the QCM has progressed to biosensors.

37,38

2 1/2

(2 )

l l

R k A

πη ρ

= − ⋅

(3)

where, k

is the electromechanical compliance.

The electrical resonance and the mechanical resonance of the shear

oscillation of the QCM are coupled each other. An AT-cut QCM is

regarded to be electrically same as the equivalent circuit depicted in

Fig. 1. Quantitatively, the left arm of the equivalent circuit represents the

parasitic capacitance of the QCM while the right arm represents the

mechanical oscillation of the QCM. Based on electromechanical analogy,

, C

, and R

correspond to energy storage into kinetic energy (i.e.,

mass), energy storage into elastic energy (i.e., compliance), and energy

dissipation (i.e., coefficient of viscous friction), respectively. When

certain material, such as a film and/or viscous liquid, contacts with the

D.-Y. Lee et al. 56

QCM surface, the L

, C

, and R

change due to acoustic loading. For this

reason, a QCM can be used as physical and chemical sensors.

2.3. Measurement Principle of Resonance Parameters

The resonance frequency and its changes are measured using and

oscillator circuit. The electromechanical coupling provides a simple

method to detect the resonance frequency by electrical means, such as a

frequency counter and an impedance analyzer.

The resonance resistance is able to be measured by using the

equivalent circuit. When the QCM is resonance,

2 2

1 1

( )

2 2

G B C

R R

   

− + − =

   

   

(4)

where, G is conductance, B is susceptance and

is angular frequency.

According to Eq. (4), the curve of B versus G for various

can be

plotted. Based on this G-B curve, we can determine the serial resonance

frequency f = 2

/(L

)

1/2

and the resistance R

simultaneously and

separately. The resonance resistance is obtained as the reciprocal

expression of the maximum conductance value.

For the purpose of reliable measurement and the high-speed data

acquisition, we used QCM 922 (Seiko EG&G, Japan)

whose measuring

mechanism is based on Eq. (4).

Fig. 1. The mechanical model and the equivalent circuit of the QCM. Both models are

coupled electromechanically. In electrical model, the left arm stands for mechanical

oscillation of the QCM and the right arm stands for the parasitic capacitance of the QCM.

Self-Assembled Viologen Derivative and Its Application 57

3. Experimental

3.1. Reagents

SH (viologen derivative) was synthesized by Qian et al. (Fudan

University). Figure 2 shows the molecular structure of the viologen

incorporated with a thiol group. Hemoglobin was obtained from Sigma.

Their stock solutions were stored at a temperature of 4°C. Stock

solutions of H

were diluted from 30% solution. All other reagents

were of analytical grade. The rest of the reagents used in this experiment

were of analytical grade and were used without any purification. All

solutions were prepared using Milli-Q water.

3.2. Electrode Modification

Self-assembly monolayers were similar on the gold electrode of the

QCM which is AT-cut gold-coated quartz crystals with a resonant

frequency of 9 MHz (5 mm diameter, Seiko EG&G, Japan). Prior to

use, the gold electrode of the QCM was cleaned by piranha solution

= 3:1) subsequently cleaned by cycling potential windows

between 0 and 1.5 V in 0.05 M H

solution at a scan rate of

100 mV/s for nearly 25 min until stable scans were recorded. Then

the electrode was thoroughly rinsed with the distilled water. After

pretreatment, the electrode was immersed in ethanol-acetonitrile (1:1)

solution containing 2 mM thiol-functionalized viologen with pure

Ar gas.

Fig. 2. Molecular structure of viologen derivative.

D.-Y. Lee et al. 58

After self-assembly, the electrode was removed from the deposition

solution and rinsed with ethanol and water to remove weakly adsorbed

viologen. And then, the viologen-modified electrode was immersed

into phosphate buffer solution containing 3 mg/ml hemoglobin for 5 h.

The modified electrodes will be abbreviated as VC

SH/Au and

Hb/VC

SH/Au, respectively.

3.3. Apparatus and Measurement System

Figure 3 shows the measurement system of electrochemical 3-electrode

set-up using the QCM. The frequency change and electrochemical

behavior were operated by WinEchem software and measured using

QCM 922 (Seiko EG&G, Japan) and potentiostat 263A (PerkinElmer,

USA). For conventional CVs, a three-compartment electrochemical

cell was employed using argon-purged electrolyte solutions. The self-

assembled gold electrode onto the QCM was used as the working

electrode. The Pt wire and Ag/AgCl (saturated KCl) were used as the

Fig. 3. Measurement system of electrochemical 3-electrode set-up using QCM.

QCM922