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DOI: 10.1002/elan.201900410
Fabrication of the Enzyme-less Voltammetric Bilirubin
Sensor Based on Sol-gel Imprinted Polymer
Maedeh Akhoundian,[a] Taher Alizadeh,*[a] and Guoqing Pan[b]
Abstract: Measuring the serum bilirubin (BR) level is part
of the common test for newborns, since high concentra-
tions of bilirubin in the blood can cause brain damage and
even death for them. Therefore, achieving a fast, accurate
and cost effective method for bilirubin assay in medical
diagnosis and Medical management of patients with
jaundice, especially neonates, is very important. In this
study, an electrochemical sensor was developed to detect
low amounts of bilirubin in serum samples. The basis of
the work is the BR imprinted polymer synthesis and the
use of this polymer as a recognition element in the
production of the bilirubin voltammetric sensor. For this
Keywords: Bilirubin · Carbon paste electrode · Sol-gel imprinted polymer · Voltammetric sensor · Nanoparticles
1 Introduction
Bilirubin (also known as hematoidin), a yellow com-
pound, is a result of catabolic fraction of hem. It is also
one of the main compounds of hemoglobin found in red
blood cells. BR is called uncondjugated bilirubin before it
reaches the liver as a red cell product. Most bilirubins are
bound to certain sugars in liver and produce conjugated
BR. The normal concentration of BR in a healthy
person‘s blood is in the range of 0.3 to 1.9 mg/dL [1]. Iron
deficiency or coronary artery disease can lead to decrease
the BR lower than normal level; while its high serum
concentrations (higher than 2.5 mg/dL) results in hyper-
bilirubinemia which is known as jaundice. In fact, about
50 % of normal babies and 80 % of premature infants
have hyperbilirubinemia at their early stages of life [2].
Bilirubin is excreted in bile and urine and its high levels in
the serum can indicate jaundice, thalassemia, Gilbert
syndrome, hemolytic uremic syndrome and sickle cell
anemia [3]. Neonatal jaundice is a common condition,
especially in infants born before the 38th week of
pregnancy (early infants), and a number of breastfeeding
babies. It is usually occurs because the liver is not mature
enough to remove the bilirubin produced in the blood
from the bloodstream [4]. Given the above, it is really
necessary to achieve a fast, accurate and cost effective
method for measuring the amount of bilirubin for medical
diagnosis and management in the case of patients with
jaundice.
Several methods have been proposed for bilirubin
assay in the blood so far, including the Diazo method. BR
reaction with diazo sulfanilic acid is known as a diazo
reaction. In this reaction, two azo isomer dyes with a
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purpose, bilirubin imprinted polymer was synthesized
using the simple sol gel method and the prepared polymer
is characterized by SEM and FT-IR. In the next step, this
polymer was applied to modify the carbon paste electrode
and provide a sensitive and selective sensor for the BR
molecule. The proposed sensor was able to measure the
bilirubin in the concentration range of 1 to 100 μM after
optimizing the operating conditions. The limit of detec-
tion (LOD) and limit of quantification (LOQ) was
calculated 0.75 μM and 2.5 μM respectively. It is also
applicable to bilirubin assay in serum samples.
maximum absorption of 530 nm are formed. Glucuronized
Bilirubin (direct BR) reacts directly with the diazo
reagent, due to its higher solubility in serum and less
tendency to albumin. On the other hand, a non-glucuron-
ized bilirubin necessitates an accelerator for solubility,
and therefore, is called indirect bilirubin [5]. Another
method is the vanadate oxidase reaction which is based
on the BR oxidation to biliverdin using vanadic acid as an
oxidizing agent. It has a good correlation with diazo
method and has superiority for samples containing
interferences due to its ease and rapidity [6]. Of course,
there are some problems with the spectrophotometric
bilirubin meters even in blood tests. Including that
spectroscopic measurement method is affected by the
interference of other hem proteins; and the diazo reaction
is also pH-dependent. On the other hand, rapid changes
in water concentration of body during therapy, can lead
to fluctuations in the BR measurement and thus disrupt it
[7–9].
Different fluorescence methods have been widely
studied for BR assay. Fluorescence quantum yield in
[a] M. Akhoundian, Prof. T. Alizadeh
Department of Analytical Chemistry, Faculty of Chemistry,
University College of Science, University of Tehran, P.O. Box
14155-6455, Tehran, Iran
Tel: + 98-021-61112788
Fax: + 98-021-61112788
E-mail: talizadeh@ut.ac.ir
t_alizadeh.ut@yahoo.com
[b] G. Pan
Institute for Advanced Materials, School of Material Science
and Engineering, Jiangsu University, Zhenjiang 212013, China
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aqueous solution is very low for BR. However, when
bonded to its natural carrier, albumin, the quantum yield
increases in aqueous solution. The bilirubin albumin
complex has a fluorescence emission of 520 nm [10].
Several reports of bilirubin measurements are available
with fluorimetric methods [11–17].
Electrochemical techniques are other methods used to
bilirubin determination. Despite the fact that, there are
several studies on BR so far, the quantitative assay of this
compound by electrochemical methods, still has a great
way to proceed. Electrochemical sensors have ease,
rapidity and sensitivity in comparison with other techni-
ques and often do not need to complex pre-treatment of
samples. There are three basic approaches in these
methods: the first one is the direct oxidation of bilirubin
at the electrode surface [18–23]. The second one is an
enzymatic method that utilizes the bilirubin oxidase
enzyme for electrochemical measurement of BR. Electro-
chemical methods which use the bilirubin oxidase enzyme
to detect and quantify the BR may have the limitations,
including the high detection limit of the sensor due to the
weak electron transfer between the active centre of
enzyme and the electrode surface; Also, the short life
span and low storage stability of the enzymatic electrodes
[24–28].
The third approach is the use of molecularly imprinted
polymers (MIPs) in bilirubin assay. MIPs can be utilize to
provide these sensors due to their high selectivity as an
alternative for the enzyme. Molecular imprinting techni-
que enables us to prepare the materials with semi-specific
sites for a target species in the dimensions of a molecule
[29–34]. For this aim, sites or cavities are created in very
small sizes in an appropriate matrix which can be based
on organic matter, inorganic or a combination of both. In
order to create such cavities, the target molecule is often
used as a template and the monomers are placed around
it according to its functional groups. The next step is the
template removing out and thus the selective cavities
remaining. MIPs, exhibit high resistance to high acidic or
alkalic environments, as well as high temperatures and
high pressures, unlike the bio-receptors. In addition, their
storage life is significantly longer than biological receptors
and can maintain their diagnostic characteristics even
after many years [35, 36]. Molecular imprinting techni-
ques, have been used for quantitative assay of BR, given
the fact that, the bilirubin oxidase-based method is faced
with many limitations and challenges [37, 38].
Bilirubin photoelectric sensor based on TiO2/polypyr-
role conducting polymer [39] and preparation of sol-gel
imprinted polymer for in situ micro-extraction of bilirubin
as a lung cancer biomarker [40] are the examples of these
reports. Also, the BR imprinted polymer is used to
provide a piezoelectric sensor (QCM) to detect this
compound [41, 42]. The preparation of BR imprinted
polymer based on cryogels is another set of reports in this
regard [43].
According to our best knowledge, a number of these
reports relate to the BR electrochemical sensors based on
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MIPs, which include amerometric BR sensors using a gold
electrode [44, 45] and a voltammetric BR sensor with
modified glassy carbon electrode [46]. However, the
amprometric method does not possess the sensitivity and
accuracy of advanced voltammetric techniques and the
only report presented for voltammetric measurement of
the BR using MIP has relatively complex and time
consuming steps in modification of the electrode surface.
In this study, bilirubin measurement was carried out in
a simple, precise and cost effective way, using a modified
carbon paste electrode. The electrochemical behaviour of
BR at the electrode surface was investigated via cyclic
voltammetry method and it was found that, the proposed
electrode is efficient enough in BR assay. This sensor
(Carbon paste Electrode Modified with MIP and carbon
nanotube), was able to measure the BR in a concentration
range of one to 100 μM after optimization of working
conditions of the sensor in a bufferic environment. Also,
the experiments showed that the proposed sensor could
be used to determine the bilirubin in the blood serum
samples.
2 Materials and Methods
2.1 Chemicals and Apparatus
Triethoxy silyl propylamine (TESPA) and Tetraethyl
ortho silicate (TEOS), obtained from Sigma-Aldrich
(Munich, Germany). Graphite powder was purchased
from Fluka (Buchs, Switzerland). Bilirubin and all other
chemicals were of analytical grade and purchased from
Merck (Darmstadt, Germany).
Electrochemical data were obtained with a three-
electrode system using a potentiostat/galvanostat model
Vertex, Ivium. The MIP involved sensor was used as a
working electrode. A platinum wire and an Ag/AgCl
electrode were used as the counter and reference electro-
des respectively. Scanning electron microscopy (SEM)
images are provided using the instrument (S-4160,
Hitachi, Japan). The UV-Vis determinations were re-
corded by UV-Vis spectrophotometer equipped with
quartz cell with a 1-cm path length was used for recording
absorbance spectra (Lambda 25, Perkin-Elmer, USA).
2.2 Preparation of Bilirubin-imprinted Polymer
Bilirubin-imprinted polymer (MIP) was synthesized using
a simple sol-gel polymerization method. For this aim,
0.02 mmol (11 milligram) of bilirubin was dissolved in
5 mL of ethanol. Then, 4.3 mmol (1 mL) of TESPA and
4.5 mmol (1 mL) of TEOS were added to this solution
under mild stirring. Then, 1 mL of water was added. The
mixture was placed in a dark place for 12 hours and the
polymer was formed under room temperature.
After polymerization, the template (bilirubin) was
removed by multiple washing of the polymer by NaOH
(3 M) solution. UV-Vis spectroscopy (absorbance at λ =
416 nm) was used to ensure the complete removal of the
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template from the polymer. The non-imprinted polymer
nanoparticles (NIP) was also synthesized using the same
method but in the absence of bilirubin.

2.3 Fabrication of the Modified Carbon Paste Electrode

(Bilirubin Sensor)
The modified MIP/CNT-CP and NIP/CNT-CP electrodes
were fabricated by mixing graphite, carbon nanotube,
melted n-eicosane (binder) and MIP/NIP with a ratio of
75.5 : 4 : 20 : 0.5 weight percent. The obtained paste was
then used to fill a hole (4.0 mm in diameter, 3 mm in
depth) at the end of a Teflon electrode body. The excess
of solidified material was removed from the surface of
electrode by polishing on a smooth paper sheet. The
polymer-free electrode (CNT-CPE) was also prepared in
the same way, but the difference was that the graphite,
carbon nanotube and binder ratio was changed to 76 : 4 : 20
weight percent.

2.4 Bilirubin Determination Procedure

The measurements were performed by a three-electrode
system: working electrode (the modified carbon paste
electrode), counter electrode (Pt) and reference electrode
(Ag/AgCl). Electrochemical measurements at optimum
conditions for different concentrations of BR were carried
out according to the following instruction:
The modified carbon paste, counter and reference
electrodes were placed in 20 mL of buffer solution
(phosphate buffer 0.05 M, pH = 5), containing the specific Fig. 1. Scanning electron microscopy images of BR-imprinted
concentration of BR, while the solution was stirring for polymer in the scale of 1 μm (A) and 500 nm (B).
5 sec. Afterwards, the stirring was stopped and voltammo-
gram was recorded after 2 sec (as equilibrium time). The
analytical current signal of BR was obtained at a potential 3 Results and Discussion
of about + 0.5 V. Cyclic voltammograms were recorded in
3.1 Characterization of Nano-sized Bilirubin Imprinted
step potential and scan rate of 0.01 V and 100 mV/s,
Polymer
respectively.
Figure 1 shows the images of the scanning electron micro-
scopy (SEM) of the bilirubin imprinted polymer in two
2.5 Real Sample Analysis
different magnifications. As can be seen, polymer par-
In order to measure BR in serum samples, a drop of ticles have a uniform and spherical morphology with
concentrated HCl was added to 2 mL of serum sample particle size of 60 to 90 nm.
(for precipitation of proteins) and centrifuged (2700 rpm FT-IR spectra related to the MIP, and readsorbed
for 5 min). Then, 17 μl of the bilirubin (0.01 M) was added MIP is shown in Figure 2, which have been recorded to
to 1 mL of the supernatant solution to reach the confirm the interaction between BR and the polymer. As
concentration of bilirubin at (10 mg/dL) and diluted two can be seen in this figure, two main bands for the silica
times using PBS (0.1 M, pH = 2) nanoparticles are recognizable. The band in wave number
In the next step, the bilirubin content was extracted to of 1050 Cm 1, which is attributed to the asymmetric
organic phase by adding 1 mL of KCl (2 M) and 5 mL of vibration of the Si O Si, and is deformed in the presence
chloroform. Afterwards, the chloroform containing bilir- of the analyte (BR). Another band in 794 Cm 1 794
ubin was transferred to a 10 mL beaker and the solvent relates to the Si O Si symmetric vibration. Also, the
was evaporated at 45 °C. Finally, 5 mL of analysis buffer appearance of 1390, 1412 and 1640 Cm 1 bands in the
(PBS 0.05 M pH = 5) was added to this beaker and the presence of BR is related to the bending vibration of the
voltammogram was recorded due to the section 2.4. in =C H group.
optimum condition.

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Fig. 2. Comparison of the FT-IR spectra for washed MIP and readsorbed (unwashed) MIP.

Fig. 3. Predicted Products in the oxidation process of bilirubin and obtained voltammogram using a MIP (1 % w/w) modified carbon
paste electrode. Analysis conditions: 0.1 M phosphate buffer (pH = 5) BR concentration = 50 μM, extraction time = 5 seconds, scan
rate = 100 mV/s, step potential = 10 mV.

3.2 Electrochemical Behaviour of Bilirubin on
Modified-carbon Paste Electrode
In this part, electrochemical behavior of bilirubin on MIP/
CNT- modified carbon paste electrode surface was
investigated using cyclic voltammetry. The BR oxidation
mechanism, can be described by a three-step process,
according to previous reports [47], in which the bilirubin
is converted to the biliverdin in first step, followed by the
oxidation of biliveridin to purpurin. Finally, purpurin can
be oxidized to choletelin. What has been observed, using
our modified carbon paste electrode in cyclic Voltammo-
gram of BR (Figure 3), is a relatively broad peak in the
potential range of 0.5 V, which seems to be achieved by

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Fig. 4. Cyclic voltammograms obtained from MIP-CNT-CP electrode in different scan rates (A), Anodic current signals vs scan rate (B)
and square root of scan rate (C); Analysis conditions: 0.1 M phosphate buffer (pH = 5) BR concentration = 100 μM, extraction time =
5 seconds, step potential = 10 mV.
the combination of two neighbour peaks according to its
appearance. In other words, what is apparent from the
voltammogram appearance, is the combination of the two
first stages of oxidation; the formation of biliverin and its
immediate conversion to purpurin.
Cyclic Voltammetry is one of the efficient methods for
studying electron transfer processes, stability studies,
electrocatalytic properties and the effect of chemical
reactions on electrode reactions. In order to investigate
the voltammetric scan rate effect, the modified carbon
paste electrode was placed in analysis solution containing
100 μM bilirubin in PBS 0.1 M, and cyclic voltammetry
was recorded in scan rates of 10 to 300 mV/s. Figure 4 A
is presented the obtained voltammograms. As it is clear,
anodic peak has increased with the increase in scan rate.
In Figure 4 B and C, current signal dependence to the
scan rate and square root of the scan rate are plotted
respectively. The results showed that, the current signal
changes to scan rate is fitted to linear regression more
regularly, which can indicate the electrode process
controlled by the absorption of bilirubin in the specific
positions of the electrode surface.
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3.3 Application of Bilirubin-imprinted Nanopolymer for
Voltammetric Sensor Preparation
In order to study the recognition ability of imprinted
polymer, electrodes of MIP-CNT-CP, NIP-CNT-CP and
CNT-CP were prepared and used in separate experiments
to measure bilirubin. The results are presented in Figure 5
A. As can be seen, there is a significant difference
between the signals obtained from the modified electro-
des with MIP and NIP, indicating the presence of selective
bonding sites as recognition elements in the MIP, which,
absorb the analyte from the test solution more effectively.
In other words, unlike the NIP (control polymer), which
only acts through the non-selective surface adsorption
process, the complementary cavities formed in the MIP
structure are capable of selective pre-concentration of the
target (BR) from the test solution.
Also, the current signal for the MIP-CNT-CP elec-
trode is clearly higher than the current for the CNT-CP
electrodes, which shows the role of imprinting polymers
present at the electrode surface for BR accumulation and
signal enhancement.
The interesting point is that the MIP modified
electrode, shows a higher current signal than CNT-CP
electrode, despite the presence of the polymer with
insulating property. This observation is due to the
important role of the MIP selective cavities in analyte
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Fig. 5. Comparison of cyclic voltammograms obtained from MIP/CNT/CPE, NIP/CNT/CPE and polymer-free electrode (A),
optimization of MIP percent in electrode composite (B), analysis buffer concentration (C) and extraction time (D); Analysis conditions:
BR concentration = 50 μM, scan rate = 100 mV/s, step potential = 10 mV.

absorption and these results demonstrate the proper
performance of synthesized imprinted polymer.
3.4 Optimization of Electrode Fabrication and
Measurement Conditions
In this step, different parameters affecting the sensor
efficiency were optimized. In order to find the optimum
amount of polymer content in the modified carbon paste
electrode, the polymer weight percent was changed from
1 to 0.2 %, while other parameters were kept constant.
The results of Figure 5 B show that the maximum signal is
obtained when the weight percentage of polymer is equal
to 0.5 %. An increase in the MIP content of the electrode
(up to 0.5 %), can lead to an enhancement in the
electrode signal because of increasing the number of
selective sites in the electrode surface. However, further
increment in the MIP content may lead to a destructive
effect on the sensor response due to the insulating effect
of the MIP particles. Therefore, 0.5 % polymer was
selected as optimal amount.
Also, the optimal electrolyte concentration (phosphate
buffer) was obtained. For this purpose, measurements
were made at various concentrations of the electrolyte
while other parameters were kept constant. The results
showed that the most suitable signal in terms of intensity
and peak form was obtained with 0.05 M PBS (Figure 5
C). Therefore, the concentration of 0.05 M PBS was
selected as the optimal concentration of the analysis
solution.
The extraction time was another under study parame-
ters. For this aim, the prepared electrode was used to
extract BR from sample solution in various periods of
time, and voltammetric responses were recorded. In each
of these experiments, all conditions, other than extraction
time, are kept constant. The obtained results obtained in
Figure 5 D showed that increasing of extraction time
more than 10 seconds results in a decrease in the sensor

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Fig. 6. Comparison of cyclic voltammograms obtained from modified carbon paste electrodes at different pH values of 3 (A), 5 (B) and
7 (C), and column graphs related to each; Analysis conditions: 0.1 M PBS, BR concentration = 100 μM, extraction time = 30 seconds,
scan rate = 100 mV/s, step potential = 10 mV.

response, while there is not significant differences be-
tween the 5 and 10 seconds. It seems that the placement
of modified electrode in the aqueous solution more than
this optimal time, causes the polymer to deform and swell
due to the sol-gel type of the imprinted polymer, which
results in the reduction of selective cavities’ efficiency.
Thus, 5 second was selected as the optimal time for
extraction.
In order to evaluate pH effect on sensor performance,
various pH values were studied in the range of 3 to 7.
Figure 6 shows the obtained voltammograms at different
pHs and column graphs for each current signal. As can be
seen, the highest response was obtained from the
electrode modified with MIP at pH = 7, and the best
signal (in terms of peak appearance) was obtained at
pH = 3.
But there are also other tip to choose the optimal pH.
Including that, we have tried to choose a pH, in which,
the difference between the signals obtained from the MIP
and NIP based electrodes was maximum. In other words,
in this situation, there is a certainty about that the major
part of the signal is related to the specific bonding of the
analyte in selective cavities, and only a small amount
derived from non-specific adsorption. According to this

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view, pH = 5, in which the MIP modified electrode signal
was 4 times the NIP modified electrode, was selected as
optimal pH. While, this ratio is about 1.5 times for the
pHs of 3 and 7.

3.5 Calibration Curve and Analytical Characteristics of

the Sensor
One of the most important features of an electrochemical
sensor is the ability to detect a particular analyte in the
presence of other chemical and biological compounds. In
this study, the effect of some interference compounds
such as creatinine, uric acid, glucose and dopamine have
been evaluated, on sensor performance. In order to aim
this, the MIP modified electrode was placed in solutions
containing 30 μM of bilirubin, which separately contained
each of the interference compounds, and in each case,
electrochemical measurements were carried out in opti-
mal conditions. The results have shown that the creatinine
does not interfere, even up to 10 times the concentration
of BR. Also, uric acid and glucose do not interfere with
Fig. 7. Selectivity experiment; CV responses of the sensor in the
one-fold concentrations of BR; and just the dopamine presence of BR, testosterone and cholesterol; [tested com-
compound have shown interference at a concentration pounds]: 50 μM, extraction time: 5 sec, Analysis condition: 0.05 M
equal to the analyte (30 μM). In these experiments the PBS (pH = 5), scan rate: 100 mV/s, step potential = 10 mV.
tolerance limit was established as the maximum concen-
tration of foreign species that caused a relative error of
5 % in the analytical signal. However, due to the fact that,
the presence of dopamine with such a concentration in
infant serum is almost impossible (the maximum concen-
tration of dopamine in the normal person’s serum is about
200 pM), the effect of dopamine interference on the
function of the proposed sensor can be ignored.
In order to investigate the selectivity of the proposed
sensor, some of the important biological compounds such
as testosterone and cholesterol was considered as com-
petitive molecules. For this aim, the solutions of each
compound and also a mix of them was prepared in
phosphate buffer 0.05 M (pH 5) and voltammetric re-
sponses was recorded. The stoke solutions (1 mM) of
testosterone and cholesterol was prepared in DMSO and
ethanol respectively and it was then diluted to the
micromolar concentrations using analysis buffer. Figure 7
is depicted the obtained voltammograms. As expected,
the provided sensor did not tend to absorb the cross-
reactants and indicated no signal to them. On the other
hand the signal for BR in the presence of two other
compounds is intact. This behavior of the sensor is
demonstrating the formation of the selective cavities of
imprinted polymer for bilirubin.
Figure 8 shows the calibration curve and voltammo-
grams of different concentrations of BR for the proposed
sensor under optimum conditions. As can be seen, there is
a linear relationship between the oxidation current signal
and the BR concentration in the range of 1 to 100 μM. Fig. 8. Cyclic voltammograms obtained from different concentra-
This sensor shows a linear region in the mentioned tions of BR using proposed sensor (A) and calibration curve (B)
concentration range with the desired value of the in optimum conditions; scan rate = 100 mV/s, step potential =
correlation coefficient. The detection limit obtained for 10 mV.

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Table 1. Comparison of some previously reported electrochemical sensors for bilirubin and the present work.

Electrode Technique Linear range (μM) LOD (μM) Ref.

a b
MWCNT/CPE CV 200–500 9.40 [18]
FcAI/GN/MWCNTs/GCEc Amperometry 1–100 0.12 [19]
Box/GMEd SWVe 0.01–500 0.005 [20]
NG/PtMEf SWV 100–500 56.0 [21]
Box/GrONPs/NiNPs/ITOg Amperometry 0.01–600 0.15 × 10 3 [22]
RGO/PSS/GCEh Chronoamperometry 0–450 2.00 [23]
MWCNT-COOH/GN/Box/GCE Amperometry 1.33–71.56 0.34 [24]
BOx/GONP/Ppy/FTOi Amperometry 0.01–500 0.1 × 10 3 [25]
Box modified membrane Amperometry 100–200 8.00 [26]
Box/Pt Amperometry 1–300 0.70 [27]
BOx/GN/Au electrode Amperometry 1–5000 1.4 × 10 3 [28]
MIPj-Au electrode Amperometry 0–17 – [44]
MIP-Au layer Amperometry 1.7–17 1.7 [45]
MIP/POM/CNk/GCE SWV 1 × 10 4–1 × 10 6
1 × 10 7 [46]
Nafion/RGOl/GCE SWV 2–70 0.84 [47]
Nafion/Mn Cu Amperometry 1.2–420 25 × 10 3 [48]
Sol-gel IPm/MWCNT/CPE CV 1–100 0.75 This work
a: Multiwall carbon nanotubes/carbon paste electrode, b: Cyclic voltammetry, c: Ferrocene carboxamide/gold nanoparticle/Multiwall
carbon nanotubes/glassy carbon electrode, d: Bilirubin oxidase/gold microelectrode, e: Square wave voltammetry, f: Nanographite-
based Pt microelectrode, g: Bilirubin oxidase/graphene oxide nanoparticles/nickel nanoparticles/ITO, h: Reduced graphene oxide/poly
styrene sulfonate/glassy carbon electrode, i: Bilirubin oxidase/graphene oxide nanoparticles/polypyrrole/fluorine doped tin oxide glass
plate, j: Molecularly imprinted polymer, k: Polyoxometalate/Carbon Nitride Nanotubes, l: Reduced graphene oxide, m: Imprinted
polymer.

Table 2. The results of bilirubin measurement in serum samples

this method was 0.75 μM and the limit of quantification (repeat = 3).
was 2.5 μM.
Table 1 is presenting some previous reported electro- Sample Blank sam- Added BR Found Recovery RSD
name ple (μM) (μM) % %
chemical methods for BR determination. Due to the some
limitations of enzymes such as their short lifespan and Serum (1)ND 170 166 98 1.87
irresistibility to pH changes, the specific detection of Serum (2)ND 85 81.5 96 3.15
biological species without the use of an enzyme is an ND: Not Detected
advantage. The present study did not use the Box enzyme.
But significant selectivity has been achieved using MIP
and the simplicity of sensor construction compared to 4 Conclusions
previous methods is evident.
On the other hand, the molecularly imprinted polymer In this study, an electrochemical voltammetric sensor was
used in the proposed sensor, is not synthesized at the developed to detect the bilirubin in neonatal blood serum.
electrode surface despite previous MIP reports. Therefore For this purpose, the bilirubin imprinted polymer was
the thorough washing of the polymer without concerning synthesized via the sol-gel method, and was used as a
about its loss and the reproducibility is another benefit of recognition element in the production of bilirubin sensor.
this method. Also, the detection limit for this sensor is This sensor (carbon paste electrode modified with MIP
appropriate and applicable due to the normal range of and carbon nanotubes), was able to measure the bilirubin
bilirubin in the blood serum (in the range of 1 to 10 μM). in a concentration range of one to 100 μM after
optimization of the working conditions of the sensor.
Also, the experiments showed that the proposed sensor is
3.6 Application of the Proposed Sensor for Bilirubin
useful to bilirubin determination in the blood serum
Determination in Plasma Sample
samples. One of the most important strengths of this
After optimization of all conditions, analytical efficiency sensor, was the replacement of the enzyme commonly
of the sensor was evaluated for real serum samples. For used in this type of sensor (as receptor of the sensor),
this purpose, two serum samples were dilluted and with molecularly imprinted polymer. Another advantage
prepared in buffer solution, according to the materials of this method was the cost-effective preparation of the
and methods section and voltammetric measurements proposed electrodes, which allows the provision of
were performed in optimal conditions. The results are disposable sensors. Also, the method for producing these
presented in Table 2. Low RSD values and favorable sensors has the potential to use in disposable screen
recovery percent, indicate the repeatability and reliability printing electrodes.
of the proposed sensor.

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Full Paper
Aknowledgements
This work was supported by the Center for International
Scientific Studies and Collaboration (CISSC), Ministry of
science research and technology, Iran.
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Received: July 2, 2019
Accepted: September 25, 2019
Published online on ■■■, ■■■■
Electroanalysis 2019, 31, 1 – 11 10
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FULL PAPER
M. Akhoundian, Prof. T. Alizadeh*,
G. Pan
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