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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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