Chapter 6

GC/styrene/NH4(ηηηη5555-C5H5)2FeNCS-M

n+ (M

n+ = Ag

+ or Hg

2+)

electrode: a non enzymatic probe for detection of cholesterol

in solution

6.1 Introduction

Cholesterol and its fatty acids esters are essential structural constituents of cell

membranes and balanced quantity of cholesterol in blood provides durability and

integrity to the cell architecture [1]. They are also precursors of a number of biological

materials like bile acid and steroid hormones. But abnormal values of cholesterol may

also lead to various complicacies in our body. Higher cholesterol concentration is

associated with cardiovascular diseases which include heart attack, stroke, peripheral

vascular disease etc. Moreover, hypertension, arteriosclerosis, coronary artery disease,

cerebral thrombosis, etc. are also related to abnormal levels of cholesterol in blood. It

also appears to boost the risk of Alzheimer’s disease. High cholesterol level leads to

build up of plaque that narrows the arteries. Besides these, low levels of cholesterol

(less than 40 mg/dL for men and less than 50 mg/dL for women) have also been shown

to increase the risk of heart disease. The alarming rise in the rate of clinical disorders

associated with various heart diseases motivated scientists to develop methods which

are cheap, reliable and sensitive to estimate cholesterol in blood.

Various methods such as colorimetric [2], spectrophotometric [3] and high

performance liquid chromatography (HPLC) [4] for detection of cholesterol are known

from long back but are expensive and complicated; inspiring scientists to develop

electrochemical biosensors for cholesterol. The recent developments in cholesterol

biosensors and their importance have been nicely reviewed by the group of B D

Malhotra [5]. In most of the electrochemical biosensors for cholesterol, cholesterol

oxidase (COx) is immobilized on a suitable matrix such as conducting polymers[6],

carbon nanotubes (CNTs) [7], nanoparticles (NPs)[8], sol–gel/hydrogels[9,10] and self-

assembled monolayer (SAM) [11,12]. COx catalyzes the oxidation of cholesterol in

presence of di-oxygen, into 4-cholesten-3-one and hydrogen peroxide [13]. The

electrooxidation current of hydrogen peroxide is detected after applying a suitable

potential to the system. Moreover, other enzymes such as cholesterol esterase (CEs) and

Chapter 6

124

peroxidise (PEx) are also used for simple, sensitive and specific routine analysis of

cholesterol [14-26]. CEs belong to a large family of proteins called the �/�-hydrolase

fold, and share the same catalytic mechanism in the hydrolysis of lipid substrates. In

addition to these enzymes horse-radish peroxidase (HRP) is also used in the fabrication

of photometry based biosensors.

But the main problem with enzyme based biosensors is their less than

desirable performance and high cost [27]. Piletsky and his colleagues proposed

cholesterol sensing method based on molecular imprinting technique such as

hexadecylmercaptan as film medium on gold electrode surface and potassium

ferricyanide as mediator [28]. Ji-Lai Gang et al reported poly (2-

marcaptobenzimidazole) imprinted gold electrode using potassium ferricyanide as

mediator [29]. In molecular imprinting method, the mediator molecules come in contact

with the electrode surface through the channels created by the template molecule,

cholesterol. The redox current of mediator decreases on addition of cholesterol into the

electrolytic solution as they can block the channels. Hence, molecular imprinting

method is an indirect one and estimation of cholesterol is based on decrease in redox

currents.

From the literature we have known that no such methods have been developed

where cholesterol is made to come in direct contact with the electrode surface. In this

chapter, we report that cholesterol imparts a significant change in redox potential of a

new ferrrocene derivative in presence of soft metal ions silver (Ag+) and mercury

(Hg2+

). This shift in redox potential could be utilized in estimation of cholesterol in

solution.

6.2 Experimental

6.2.1 Synthesis of [(�5-C5H5)2Fe(NCS)]NH4

0.187 g ferrocene (1mmol) was dissolved in 20 mL acetonitrile and to that

solution added 0.076 g (1 mmol) of ammonium thiocyanide. The mixture was refluxed

at 60 oC for 2 hours, cooled to room temperature and allowed to stand overnight. Dark

brown colure crystalline compound obtained which was filtered, washed with methanol

and dried. The synthesis process of the new ferrocene derivative is shown in scheme

6.1.

Chapter 6

125

Scheme 6.1 Synthesis of [(�5-C5H5)2Fe(NCS)]NH4

The compound was characterized by CHN analysis, FTIR and 1H NMR

spectroscopy. Elemental analysis shows C 46%, H 5.84% and N 10.33% corresponding

to the formula NH4 [Ferrocene-NCS] (the calculated values are C 45.80%, H 5.34%, N

10.68%).

FTIR spectrum shows peaks (i) corresponding to ferrocene at 3087cm-1

(νCH),

1102 cm-1

and1399 cm-1

(νCC), 1000 cm-1

(δCH), 814.8 cm-1

and 856 cm-1

(πCH); (ii)

corresponding to NCS at 2050 cm-1

(νCN), 814 cm-1

(νCS), 476 cm-1

(δCH); and (iii)

corresponding to NH4 at 3440cm-1

(νNH4), 1646.3 cm-1

(δa HNH), 1170 cm-1

(δs HNH), 674

cm-1

(ρr NH4). The assignments of the peaks have been made from reported literature

[30].

Fig.6.1 FTIR spectra of of [(�5-C5H5)2Fe(NCS)]NH4 in KBr

Chapter 6

126

1H nmr spetrum of the compound was recorded in CDCl3 (Fig.6.2). Two sharp

peaks were observed at 1.597 ppm and 4.164 ppm. The peak at 1.597 may be assigned

to the four equivalent protons of ammonium and the peak at 4.164 is due to ten

equivalent protons of the two cyclopentadienyl rings of ferrocene. The ratio of the peak

areas is found to be 2.5 corresponding to proton ratio of ammonium and ferrocene

(4:10).

Fig.6.2 1H NMR spectra of of [(�

5-C5H5)2Fe(NCS)]NH4 in CDCl3

On the basis of above spectral data analysis, the probable structure of the

compound is shown in scheme 6.2.

Scheme 6.2 Probable structure of [(�5-C5H5)2Fe(NCS)]NH4

Chapter 6

127

6.2.2 Preparation of GC/styrene/[(�5-C5H5)2Fe(NCS)][NH4] Electrode

GC electrode was polished firmly on micro-cloth using fine 0.05 �M alumina

powder followed by sonication for 2-3 minutes in double distilled water and then rinsed

thoroughly with water. 0.1 g of [(�5-C5H5)2Fe(NCS)]NH4 was dissolved in 10 mL

dichloromethane and 1.0 �L of the solution was placed on the tip of the pre-cleaned GC

electrode surface using a Hamilton micro-syringe. The electrode was dried under

nitrogen environment for 5 minutes. 1.0 �L of a styrene solution (prepared by

dissolving 0.1 g styrene in 10 mL of dichloromethane) was dropped over the above

modified electrode and again dried under the blanket of nitrogen for 10 minutes. The

modified electrode is now designated as GC/styrene/[(�5-C5H5)2Fe(NCS)][NH4]

henceforth in this chapter

6.3 Results and Discussion

6.3.1 Determination of Surface Coverage of the GC/styrene/[(�5-

C5H5)2Fe(NCS)][NH4] Electrode

An approximate estimation of the surface coverage of the electrode was made by

adopting the method used by Sharp et al [31]. According to this method, the peak

current is related to the surface concentration of the electroactive species, �, by the

following equation:

Ip = n2F

2 A��/4RT

Where n represents the number of electrons involved in the reaction, A the

geometric surface area (0.09 cm2) of the electrode, � (mol m

−2) the surface coverage and

� the scan rate and other symbols have their usual meanings. For the anodic peak current

at scan rate 0.100 Vs-1

, the calculated surface concentration of [(�5-C5H5)2Fe(NCS)]NH4

is 4.4142 × 10-5

mol m-2

.

6.3.2 Electrochemistry of [(�5-C5H5)2Fe(NCS)]NH4

Cyclic voltammogram of [(�5-C5H5)2Fe(NCS)]NH4 was recorded in acetonitrile

using GC as working electrode at scan rate 0.100 Vs-1

(Fig.6.3). A quasi reversible

cyclic voltammogram with an oxidation peak at potential value 0.597 V ± 0.005 V with

peak current -1.259 × 10-5

A and a reduction peak at potential value 0.495 V ± 0.005 V

Chapter 6

128

with peak current 1.494 × 10-5

A was observed. The redox potential obtained was 0.546

V ± 0.005 V.

.Fig.6.3 Cyclic voltammogram of [(�5-C5H5)2Fe(NCS)]NH4 in acetonitrile at scan rate

0.100 vs-1

(Ag-AgCl is the reference electrode)

It was observed that the redox peak current was directly proportional to the scan

rate � over the range 10-100 mV s-1

(Fig.6.4A). The electrochemical reversibility of the

compound was examined by plotting the redox peak currents against square root of scan

rate. The linearity of the plot (Fig.6.4B) reveals the reversible electrochemical reaction

that takes place on the surface of the electrode. Moreover, the ratio of the cathodic to

anodic peak current, Ipc/Ipa is found to be almost unity (1.18).

The reported redox potential of ferrocene in acetonitrile is + 0.470 V versus Ag-

AgCl as reference electrode. This redox potential value is due to ferrocene/ferrocenium

couple. By comparing our results with the redox potential value of ferrocene it can be

said that in case of [(�5-C5H5)2Fe(NCS)]NH4, also the redox process observed by cyclic

voltammogram must be due to FeII/Fe

III couple of ferrocene.

Chapter 6

129

Fig.6.4A Cyclic voltammogram of [(�5-C5H5)2Fe(NCS)]NH4 in acetonitrile at different

scan rate (range of 10-100 mVs-1

) (WE: GC and RE: Ag-AgCl)

Fig.6.4B Plot of cathodic and anodic current as a function of scan rate for [(�5-

C5H5)2Fe(NCS)]NH4 in acetonitrile (WE: GC and RE: Ag-AgCl)

Chapter 6

130

6.3.3 Voltammetric Detection of Cholesterol using GC/styrene/[(�5-

C5H5)2Fe(NCS)][NH4] Electrode

The cyclic voltammogram of the GC/styrene/[(�5-C5H5)2Fe(NCS)]NH4 electrode

in phosphate buffer solution (PBS) at pH 7.0 is shown in Fig.6.5. The redox potential

was found to be + 0.410 V ± 0.005 V with peak separation value 0.200 V. Inside styrene

film the redox potential of [(�5-C5H5)2Fe(NCS)]NH4 was found to undergo a 0.136 V

cathodic shift compared to that in solution. The electron rich phenyl groups of styrene

film relatively stabilized the FeIII

state over FeII state which made the reduction of Fe

III

relatively difficult (compared to that in acetonitrile) and a negative shift in redox

potential resulted.

Fig.6.5 Cyclic voltammogram of GC/styrene/[(�5-C5H5)2Fe(NCS)]NH4 electrode in

PBS at pH 7.0 (Ag-AgCl is the reference electrode, scan rate: 0.100 Vs-1

)

Now on gradual addition of AgNO3 into the electrolytic solution both the

oxidation and reduction peaks were found to undergo cathodic shift till the redox

potential became + 0.260 V ± 0.005 V. This value of redox potential was obtained when

Chapter 6

131

final concentration of Ag+ ion in the electrolytic medium was 4 × 10

-4 M. The peak

separation value was narrowed down to 0.080 V (Fig.6.6, curve 2). Hence, addition of

Ag+ ion into the electrolytic medium caused a cathodic shift of 0.150 V to the modified

electrode and shrinks the peak separation value by 0.120 V of the modified electrode.

Fig.6.6 Cyclic voltammogram of GC/styrene/(η5-C5H5)2FeNCS (1), GC/styrene/(η5

-

C5H5)2FeNCSAg (2) and GC/styrene/(η5-C5H5)2FeNCSAg in presence of 0.2 mM

cholesterol (3). Cyclic voltammograms were recorded in PBS at pH 7.0 (Ag-AgCl is the

reference electrode, scan rate: 0.100 Vs-1

)

The added Ag+ ions are likely to coordinated to the S atom of NH4(η

5-

C5H5)2FeNCS molecules coated onto the electrode surface. This was evident from the

fact that the colour of the modified electrode surface was dark brown before addition of

Ag+ which became white after addition of Ag

+ into the electrolytic medium. This white

coating, observed by naked eye, must be of Ag on electrode surface. The modified

electrode attained a new composition GC/styrene/(η5-C5H5)2FeNCSAg.

Co-ordination of NCS to Ag through S resulted in an excess in electron density

on Ag as Ag+ has 3d

10 configuration. Therefore, electron density entered N=C=S

Chapter 6

132

through back bonding and as N is more electronegative than C and S most of the gained

electron density was centred on N. This stabilized ferrocenium ion due to electrostatic

reason leading to a negative shift in redox potential of (η5-C5H5)2FeNCSAg compared

to that of NH4(η5-C5H5)2FeNCS.

When Hg2+

ion was gradually added into the electrolytic solution in place of

Ag+, the redox potential of GC/styrene/ NH4(η

5-C5H5)2FeNCS electrode was found to

shift to + 0.270 V from + 0.410 V (Fig.6.7, curve 2). The net shift in redox potential

was 0.140 V which was 0.010 V less compared to GC/styrene/(η5-C5H5)2FeNCSAg

electrode.

Fig.6.7 Cyclic voltammogram of GC/styrene/(η5-C5H5)2FeNCS (1), GC/styrene/(η5

-

C5H5)2FeNCSHg+ (2) and GC/styrene/(η5

-C5H5)2FeNCSHg+ in presence of 0.1 mM

cholesterol (3). Cyclic voltammograms were recorded in PBS at pH 7.0 (Ag-AgCl is the

reference electrode, scan rate: 0.100 Vs-1

)

Chapter 6

133

Hg2+

was bound to the S of the GC/styrene/NH4(η5-C5H5)2FeNCS electrode and

the electrode became GC/styrene/(η5-C5H5)2FeNCSHg

+. When Hg

2+ was associated to

S end of the modified electrode, like Ag+, the back electron density donation by Hg

2+

(5d10

configuration) caused the cathodic shift. As Hg+

had a single positive charge in the

modified electrode the back electron density donation into N=C=S was less compared to

electrode modified with neutral Ag. This accounts for the 0.010 V more positive redox

potential of GC/styrene/(η5-C5H5)2FeNCSHg

+ electrode (+ 0.270 V) compared to

GC/styrene/(η5-C5H5)2FeNCSAg electrode (+ 0.260 V).

Effect of addition of cholesterol into the electrolytic solution on the redox

potential of GC/styrene/(η5-C5H5)2FeNCSAg and GC/styrene/(η5

-C5H5)2FeNCSHg+

electrode are shown in Fig6.6 curve 3 and Fig6.7 curve 3, respectively. A net cathodic

shift can be clearly observed in both the cases but for GC/styrene/(η5-C5H5)2FeNCSHg

+

this is 0.020 V more than the shift observed when cholesterol interacts with

GC/styrene/(η5-C5H5)2FeNCSAg electrode. Net comparisons of the results of both the

electrodes are shown in Table 6.1.

Table 6.1 Comparison of electrode potential values for GC/styrene/(η5-

C5H5)2FeNCSAg and GC/styrene/(η5-C5H5)2FeNCSHg

+ electrode

Electrode E1/2 �E

[(�5-C5H5)2Fe(NCS)]NH4 in CH3CN 0.546 V 0.102 V

GC/styrene/[ferrocene-NCS][NH4] 0.410 V 0.200 V

GC/styrene/(�5-C5H5)2FeNCSAg 0.260 V 0.080 V

GC/styrene/(�5-C5H5)2FeNCSAg in 2.025 × 10-5

M

Cholesterol

0.240 V 0.081 V

GC/styrene/(�5-C5H5)2FeNCSHg+ 0.270 V 0.083 V

GC/styrene/(�5-C5H5)2FeNCSHg+ in 12.0 × 10-5

M

Cholesterol

0.230 V 0.085 V

Chapter 6

134

Addition of different amount of cholesterol into the electrolytic solution shifts

the redox potential of GC/styrene/(η5-C5H5)2FeNCSAg and GC/styrene/(η5

-

C5H5)2FeNCSHg+ electrode linearly as shown in Fig.6.8 (A and B).

Fig.6.8 Effect of cholesterol concentration on the redox potential of (A) GC/styrene/(η5-

C5H5)2FeNCSAg and (B) GC/styrene/(η5-C5H5)2FeNCSHg

+ electrode in PBS at pH 7.0

A

B

Chapter 6

135

The linear range was found to be 0.025 × 10-5

to 2.025 × 10-5

M (correlation

coefficient, R = 0.9941) for GC/styrene/(η5-C5H5)2FeNCSAg electrode and 1.0 × 10

-5

to 12.0 × 10-5

M (correlation coefficient, R = 0.99701) for GC/styrene/(η5-

C5H5)2FeNCSHg+ electrode. The sensitivity was calculated to be 0.0722 V/mM for

GC/styrene/(η5-C5H5)2FeNCSAg electrode and 0.696 V/mM for GC/styrene/(η5

-

C5H5)2FeNCSHg+

electrode. The detection limit found was 0.2 × 10-5

M and 0.4 × 10-5

M respectively for the electrodes GC/styrene/(η5-C5H5)2FeNCSAg and GC/styrene/(η5

-

C5H5)2FeNCSHg+. As there is not any reported voltammetric sensors for cholesterol

based on redox potential versus cholesterol concentration, it is not possible to make a

comparative study. All the observations obtained from the above discussion are

summarised in the form of Table 6.2.

Table 6.2 Comparison of experimental data of GC/styrene/(η5-C5H5)2FeNCSAg and

GC/styrene/(η5-C5H5)2FeNCSHg

+ electrode

GC/styrene/(η5-C5H5)2FeNCSAg GC/styrene/(η5

-C5H5)2FeNCSHg+

Sensitivity 0.0722 V/mM 0.696 V/mM

Linear range 0.025 × 10-5

to 2.025 × 10-5

M 1.0 × 10-5

to 12.0 × 10-5

M

R 0.9941 0.99701

Detection limit 0.2 × 10-5

M 0.4 × 10-5

M

A response time of 5 s was obtained after each addition of cholesterol. The

response time of the biosensor was defined as the time after analyte addition for the

biosensor response to reach 95% of its final value. The response time in this study is

better than the methods for immobilization of COx in carbon nanotube-chitosan-

platinum composite (8 s) [7], chitosan hybrid composite (13 s) [32], poly(2-

hydroxyethyl methacrylate) polypyrrole composite film (30 s) [21], layer-by-layer

assembling polymer films (30–40 s) [33], poly(1,2-diaminobenzene) film (51 s) [34],

and silicic sol–gel matrix (60 s) [35].

The most favourable site in cholesterol to be targeted by the modified electrode

for interaction is its lone double bond (Scheme 6.3).

Chapter 6

136

Scheme 6.3 Structure of cholesterol

Ag and Hg+ are prone to form complexes containing double bond. The

association of Ag and Hg+ with cholesterol will enhance electron density on them which

will be diffused through N=C=S, finally augmenting electron density on N due to its

high electronegativity. The impact is a further cathodic shift in redox potential of the

respective modified electrodes on interaction with cholesterol. It is obvious that

GC/styrene/(η5-C5H5)2FeNCSHg

+ electrode will interact more strongly with cholesterol

compared to GC/styrene/(η5-C5H5)2FeNCSAg because of the presence of positive

charge on Hg which results in a 0.020 V more cathodic shift in redox potential.

6.3.4 Interference Study

Interference by ascorbic acid, uric acid and glucose are generally studied for

voltammetric sensors for cholesterol [1, 13]. We too studied the effect of these three

compounds on the cyclic voltammetric response of the modified electrodes-

GC/styrene/(η5-C5H5)2FeNCSAg and GC/styrene/(η5

-C5H5)2FeNCSHg+. The redox

potential and redox currents were not affected by any one of the interfering compounds

at their 10-2

M concentration.

6.4 Conclusions

In this work we establish that a new derivative of ferrocene, [(η5-C5H5)2Fe-

SCN], synthesized and characterized, is a good electrode modifying agent to sense

Chapter 6

137

cholesterol. Glassy Carbon electrode surface has been modified with [(η5-C5H5)2Fe-

SCN] impregnated styrene film. Ag+

and Hg2+

ions have been attached to the S atoms of

the ferrocene derivative on the electrode surface resulting in a modified electrode of the

type GC/Styrene/(η5-C5H5)2Fe-SCNAg and GC/Styrene/(η5-C5H5)2Fe-SCNHg+.

Presence of cholesterol in the electrolytic medium shifts the redox potential of these two

electrodes cathodically by 0.020 V and 0.040 V respectively. The modified electrodes

have very low response time of 5 s; detection limit 0.2 × 10-4

M and 0.4 × 10-5

M;

sensitivity 0.0722 V/mM and 0.696 V/mM and linear range 0.025 to 2.025 × 10-5

M and

1.0 to 12.0 × 10-5

M respectively. These electrodes are easy to prepare and of handle

compared to enzyme modified electrodes.

This work has been presented as poster in 13th

Biennial National Symposium on

Modern Trends in Inorganic Chemistry (MTIC-XII) held at IISc, Bangalore, India

during December 7 – 10, 2009.

Chapter 6

138

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