Electrochemical biosensor for detection of 17β-estradiol using semi-conducting polymer and horseradish peroxidase

Article abstract of DOI:10.1039/c9ra09902f

A convenient electrochemical sensing pathway for 17β-estradiol detection was investigated. The system is based on a conducting polymer and horseradish peroxidase (HRP) modified platinum (Pt) electrode. The miniature estradiol biosensor was developed and constructed through the immobilization of HRP in an electroactive surface of the electrode covered with electroconducting polymer-poly(4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole). The detection strategy is based on the fact that 17β-estradiol (E2) and pyrocatechol (H₂Q) are co-substrates for the HRP enzyme. HRP, which does not react with E2, in the presence of H₂O₂ catalyses the oxidation of H₂Q to o-benzoquinone (Q). With the optimized conditions, such constructed biosensing system demonstrated a convenient level of sensitivity, selectivity in a broad linear range-0.1 to 200 μM with a detection limit of 105 nM. Furthermore, the method was successfully applied for hormone detection in the presence of potential interfering compounds (ascorbic acid, estriol, estrone, uric acid and cholesterol).

Full text of DOI:10.1039/c9ra09902f

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PAPER
Electrochemical biosensor for detection of 17b-
estradiol using semi-conducting polymer and
horseradish peroxidase
Cite this: RSC Adv., 2020, 10, 9079
Kamila Spychalska, Dorota Zaj a˛ c and Joanna Cabaj
*
A convenient electrochemical sensing pathway for 17b-estradiol detection was investigated. The system is
based on a conducting polymer and horseradish peroxidase (HRP) modified platinum (Pt) electrode. The
miniature estradiol biosensor was developed and constructed through the immobilization of HRP in an
electroactive surface of the electrode covered with electroconducting polymer – poly(4,7-bis(5-(3,4-
ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole). The detection strategy is based on the fact
that 17b-estradiol (E2) and pyrocatechol (H
not react with E2, in the presence of H
2
Q) are co-substrates for the HRP enzyme. HRP, which does
O
2 2
catalyses the oxidation of H Q to o-benzoquinone (Q). With
2
Received 26th November 2019
Accepted 23rd February 2020
the optimized conditions, such constructed biosensing system demonstrated a convenient level of
sensitivity, selectivity in a broad linear range – 0.1 to 200 mM with a detection limit of 105 nM.
Furthermore, the method was successfully applied for hormone detection in the presence of potential
interfering compounds (ascorbic acid, estriol, estrone, uric acid and cholesterol).
DOI: 10.1039/c9ra09902f
rsc.li/rsc-advances
ion. HRP catalyzes the oxidation of many substrates, however, it
is very oen used for the oxidation of phenolic compounds.
Introduction
6
Many studies conducted in recent decades have shown that a lot HRP belongs to the class of oxidoreductases in which the elec-
of chemical substances – both those of natural origin as well as tron acceptor is hydrogen peroxide. This enzyme is used on
man-made ones – can interfere with the proper functioning of a large scale due to commercial availability in high purity, which
the endocrine system and cause health- and life-threatening allows to study biological behaviors that catalyze the oxidation
effects on animals and humans. These compounds have been of substrates in the presence of H
referred to as endocrine disrupting compounds (EDCs). These the production of amperometric sensors detecting H
compounds can be found in many commercial products, such small organic and inorganic substrates.
2
O
2
. It is used, for example, in
1
2
O
2
and
7
as food, medicines, cosmetics, detergents, food metal cans,
toys, plastic bottles, pesticides and many others.
An extremely important task in the production of ampero-
metric sensors is the efficient and effective immobilization of
2
1
7b-Estradiol (E2) (Fig. 1) and other naturally occurring the biolm on the surface of the electrode. Conducting poly-
hormones, natural chemicals, man-made chemicals and phar- mers deserve particular attention due to their redox, optical,
maceutical products are classied as endocrine active mechanical and electrical properties. Due to their high dura-
compounds due to their ability to mimic endogenous bility and stability, they are a suitable material for the
3
8,9
hormones. Due to their unfavourable effect, they can interfere construction of sensor devices. The performance of the
with the hormonal, immune and nervous systems of biosensor depends mainly on the structure of the surface, the
4
mammals. The concentration of EDC in the environment is interaction between the enzyme and the surface of the electrode
small, however, chronic low exposure can cause harmful bio- and the protection of the three-dimensional structure of the
5
logical effects on animals and humans. To solve these prob- protein. Therefore conductive polymers have proven to be one
lems, it is necessary to use simple, fast, sensitive and accurate
methods for the determination of these combinations.
Horseradish peroxidase (HRP) is one of the most widely
studied and most frequently used enzymes in the construction
of enzyme biosensor systems, due to its easy availability and low
cost. This enzyme contains heme as a prosthetic group, which is
thus the active site of proteins with a resting state of iron (Fe)
Faculty of Chemistry, Wrocław University of Science and Technology, Wybrze ˙z e
Wyspia n´ skiego 27, Wrocław, Poland. E-mail: joanna.cabaj@pwr.edu.pl
Fig. 1 Chemical structure of 17b-estradiol.
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of the most useful transducers due to their simplicity of
production. Conducting polymers act as a three-dimensional
10
matrix for deposition of biomolecules.
Different methods are used for hormone determination
which are reported in the literature such as high-performance
1
1
12
liquid chromatography (HPLC), capillary electrophoresis, UV
13
spectrophotometry or gas chromatography combined with
14
mass spectroscopy (GC-MS). However, commonly used tech-
niques for determination of EDCs, such as HPLC or GC-MS are
time-consuming, very expensive and oen not accurate. Elec-
trochemical biosensors have recently gained popularity because
of a number of advantages such as low cost, fast response, ease
15
of use and real-time analysis. Electrochemical biosensors for
7b-estradiol detection are widely described in the literature,
however, only few items deal with electrochemical biosensors
Fig. 2 Schematic representation of the 17b-estradiol enzyme-based
biosensor.
1
16,17
with the enzyme as a biologically active element.
Wang et al.
proposed an electrochemical biosensor for the detection of 17b-
estradiol based on electropolymerized L-lysine molecules on
a glassy carbon electrode (GCE) modied with citric acid and
graphene (CA-GR) and cross-linked with laccase. This biosens-
ing system could effectively determine the concentration of 17b-
transferred into an electrochemical cell containing pH 7.00
phosphate buffer, where given amounts of HRP, pyrocatechol
(
H
2
Q), H
both enzyme co-substrates. In the presence of H
catalyzes the oxidation of H Q to benzoquinone (Q) and E2 to
2
O
2
and 17b-estradiol (E2) were added. H
2
Q and E2 are
2 2
O
, HRP
ꢁ
13
18
2
estradiol in tested sample with LOD 1.3 ꢀ 10
M. Another
20
given product. The electrochemical response of the system is
detection system based on laccase was proposed by Povedano
et al. For this purpose they used glassy carbon electrode coated
with nanocomposite based on graphene oxide with rhodium
nanoparticles. On the surface of such prepared electrode, the
enzyme laccase was successfully anchored to construct a vol-
tammperometric biosensor for 17b-estradiol determination.
Such constructed system was able to measure the concentration
proportional to H Q concentration and inversely proportional
2
to the E2 concentration in the test samples. Therefore, the
maximal electrochemical response was obtained with the
minimum concentration of E2 in the sample analyzed (Fig. 2).
Materials and methods
Reagents and materials
19
of 17b-estradiol in the 0.9–11 pM range with LOD of 0.54 pM.
Herein, we present a simple and very sensitive electro-
chemical enzyme – based biosensor for determination of 17b- Horseradish peroxidase (HRP) (EC 1.11.1.7), 17b-estradiol (E2),
estradiol (E2). Horseradish peroxidase (HRP), due to the low uric acid (UA), ascorbic acid (AA), estriol (E3), estrone (E1),
cost and high availability, is one of the most extensively inves- cholesterol (CH), tetrabutylammonium hexauorophosphate
tigated enzymes in the case of the development of biosensors. (TBA-HFP), n-butyllithium (2.5
M
in hexane), bis(-
HRP-based biosensors present the highest sensitivity for triphenylphosphine)palladium(II) dichloride (99%), 3,4-ethyl-
a number of phenol derivatives since they can act as electron enedioxythiophene (97%) as well as 4,7-bis(5-bromothiophen-2-
donors for enzyme regeneration. The use of the enzyme in the yl)benzothiadiazole (99%) were purchased from Sigma-Aldrich
system signicantly reduces the time of analysis. In the case of Co. Dichloromethane, KH PO , NaH PO , ethanol and anhy-
2
4
2
4
biosensors in which antibodies are a biologically active drous tetrahydrofuran were purchased from POCH. Monomer
element, very oen before proceeding to the proper measure- 4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzothia-
ment, prior incubation with the analyte to form antibody– diazole was synthesized according to the method described
antigen complexes is required. The regeneration of the elec- below. All buffers, which were needed to wash an unbounded
trode aer the measurement is also signicant. In the case of protein and to prepare the measurement solution were
enzymatic biosensors, measurements can be carried out prepared according to generally known, obligatory standards.
continuously, very oen only rinsing the electrode in a medium All chemicals were analytically pure and used without any
containing no analyte is required. In the case of immuno- further purication. Preparative column chromatography was
sensors, it is necessary to use more radical reagents that will performed on the glass column with Acros Organics silica gel
1
˚
break the antibody–antigen binding while preserving the anti- for chromatography, 0.035–0.075 mm 60 A. 600 MHz H NMR
1
3
bodies on the electrode surface. Each subsequent radical and C NMR spectra were recorded in deuterated chloroform
treatment of the electrode may cause detachment of antibodies (CDCl ) on Bruker Avance II 600 Instruments, respectively.
3
from the electrode surface, thereby shortening the lifetime of Chemical shis were locked to chloroform dH 7.26 (s) and dC
the constructed system.
The sensor was constructed through the immobilization of determined using a Bruker micrOTOF-Q spectrometer, FWHM-
the enzyme – horseradish peroxidase (HRP) on the surface of 17500, 20 Hz. The 2-(tributylstannyl)-3,4-ethyl-
thin polymer layer based on poly(4,7-bis(5-bromothiophen-2-yl) enedioxythiophene (2) were synthesized according to previously
77.16 (t) signals. The molecular weight of the nal product was
17
benzothiadiazole). Such prepared layered electrode was published procedures.
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Apparatus and electrodes
In the next step, HRP was immobilized on the surface of the
modied electrode by physical adsorption and covalent cross-
The platinum electrode (Pt, diameter 3 mm, produced by BASi)
was polished before the experiment with 3 mm ne diamond
polish and rinsed thoroughly with double distilled water. Then,
ꢁ
1
linking with glutaraldehyde. HRP solution (3.0 mg mL ) was
prepared in a pH 7.00 phosphate buffer. The immobilization
process was carried out for 24 hours, by the immersion of
electrode in glutaraldehyde solution (2%) for 1.5 h. Then elec-
trode was immersed in the solution of enzyme for 22 h. The
unbound protein was washed by repeatedly plunging the elec-
trode in pH 7.00 phosphate buffer. The prepared enzyme-
modied electrode was stored in a phosphate buffer at an
it was immersed in a solution of H
2 4 2 2
SO + H O (3 : 1 v/v) during
5
min. Finally, it was rinsed with water and ethanol, and air
dried. The counter electrode (CE) was a platinum wire. Ag/AgCl
electrode saturated in 4 M KCl was used as a reference elec-
trode. The measuring system for performing DPV, CV and
chronoamperometry was Autolab PGSTAT 128 potentiostat run
with the GPES soware. All DPV and CV measurements were
performed in the potential range from ꢁ0.2 to 0.7 V (step
potential 0.00495 V, modulation amplitude: 0.04995 V).
ꢂ
optimal pH at 4 C.
Optimal enzyme working conditions
In order to determine the optimal pH of enzyme work, a number
of buffers were prepared – acetate with a pH of 4.0 and 5.0 and
phosphate with a pH of 6.0, 7.0 and 8.0. Catalytic activity was
measured by spectrophotometry method. The substrate for the
enzyme was a solution of ortho-phenylenediamine in acetate or
phosphate buffer and hydrogen peroxide. The reaction was
carried out on a magnetic stirrer for 15 minutes for each buffer
at 470 nm, taking 1 mL of absorbance measurement solution
every 30 seconds for the rst 5 minutes of the reaction and then
every 1 minute.
Synthesis and characterization of monomer 4,7-bis(5-(3,4-
ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole
To a mixture of 4,7-bis(5-bromothiophen-2-yl)benzothiadiazole
(
1) (1.00 g, 2.18 mmol) and 2-(tributylstannyl)-3,4-ethyl-
enedioxythiophene (2) (2.07 g, 4.80 mmol) in anhydrous THF
80 mL) was added bis(triphenylphosphine)palladium(II)
dichloride (Pd(PPh Cl ) (0.306 g, 0.436 mmol) at room
(
3
)
2
2
temperature under nitrogen atmosphere. The resulting mixture
was reuxed with stirring for 48 h. Then the reaction mixture
was concentrated under reduced pressure, diluted with water,
and extracted with EtOAc. The extract was washed with brine,
Electrochemical measurements
1
7b-Estradiol detection was performed using CV and DPV
method with a potentiostat/galvanostat AUTOLAB PGSTAT
28N with GPES soware. The measurements were conducted
dried over MgSO , and concentrated. The residue was puried
4
by silica gel column chromatography (hexane–EtOAc) to give 3
1
(
1.075 g, 84.6%) as a purple solid state.
Mp: 143–145 C.
ꢂ
with typical three-electrode system in 10 mL cell. Platinum
electrode (modied with monomer and enzyme) was used as
working electrode, together with a coiled platinum wire as the
auxiliary electrode and an Ag/AgCl reference electrode. CV and
DPV were carried out by repeated potential scanning in range
1
H NMR (400 MHz, CDCl
3
), d (ppm): d 8.07 (d, J ¼ 4.0 Hz, 2H),
7
4
.83 (s, 2H), 7.30 (d, J ¼ 4.0 Hz, 2H), 6.27 (s, 2H), 4.41–4.39 (m,
H), 4.28–4.26 (m, 4H).
1
3
3
C NMR (151 MHz, CDCl ), d (ppm): d 150.15, 146.93,
ꢁ
1
ꢁ
0.2 to 0.7 V with scan rate: 50 mV s in the presence of
1
1
32.46, 132.24, 130.89, 130.52, 128.80, 128.48, 123.76, 114.70,
01.66, 90.06, 68.72.
MS (m/z): [M ] 580.9799.
different concentration of 17b-estradiol dissolved in 0.1 M
phosphate buffer pH ¼ 7.0 (0.1–200 mM). All electrochemical
measurement were carried out in room temperature and air-
opened conditions.
+
Modication of electrode
The Pt electrode was modied with a thin layer of poly(4,7-bis(5-
Results
(
3,4-ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole)
and HRP. The electropolymerization process of monomer was
carried out using potentiostat/galvanostat AUTOLAB
Synthesis and characterization of monomer 4,7-bis(5-(3,4-
ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole
a
PGSTAT128N with GPES soware in a typical three-electrode The detail of synthetic route for new benzothiadiazole derivative
electrochemical cell (10 mL) appointed with a working platinum is shown in Scheme 1. The designed macrostructure 3 was
electrode, Ag/AgCl reference electrode (saturated in 4 M KCl), synthesized by palladium catalyzed Stille coupling reaction of
and a coiled platinum wire as the counter electrode. In order to 4,7-bis(5-bromothiophen-2-yl)benzothiadiazole (1) and 2-(trib-
synthesize the polymeric layer onto the surface of the clean utylstannyl)-3,4-ethylenedioxythiophene (2) with excellent yield
electrode, 4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl) (85%).
benzothiadiazole (1 mM) was dissolved in a dichloromethane
solution containing 0.1 tetrabutylammonium tetra- creating matrices in biosensor systems. Due to the presence of
uoroborate (TBA-TFB). The electrodes were dipped in 8 mL of conjugated p electrons (a system having coupled C]C bonds)
Conducting polymers (CPs) are an interesting alternative to
M

the monomer solution. The polymer lm electrodeposition was CP are characterized by unique electronic properties, such as:
performed using chronoamperometry method (potential 1.5 V, high electron affinity, low optical transition energy or low
duration 600 s). Then, the polymer layer was washed gently with ionization potential. Importantly, CPs serve as electron media-
dichloromethane and pH 7.00 phosphate buffer.
tors to improve the ow of electrons between the enzyme's
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3 2 2 2
Scheme 1 The synthetic route of the 4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole (3). (i) Pd(PPh ) Cl , THF, N , 48 h.
active center and the electrode surface. They create an appro- of biosensors. The enzymatic activity depends on the chemical
priate microenvironment to immobilize the protein and act as species used, which are a matrix for the immobilization of
9,21
a transducer during the transfer of electric charge. In fact, CP biocatalyst. During the process, an increase in current values is
is easy to manipulation of their electrical and physicochemical visible during the passage of time. This demonstrates an
characteristics through reduction or oxidation process (n-type effective
electropolymerization
of
4,7-bis(5-(3,4-ethyl-
22
or p-type materials). Furthermore, conductive polymers have enedioxythiophene)thiophen-2-yl)benzothiadiazole – a p-type
a hydrophobic backbone that facilitates stackable p–p mole- polymer. As a result, a thin layer of 50 nm polymer lm was
cules, making the interaction between the protein and the obtained on the surface of Pt electrode. The thickness of the
polymer matrix stronger. The use of conducting polymers as layer was calculated using the eqn (1):
matrix molecules allows to increase the stability, speed and
Q
23
d ¼ a A
(1)
sensitivity of constructed sensor devices. In addition to elec-
tronic properties, CPs are also characterized by low synthesis
price and universality – their properties can be modied by
physical changes (such as pH or temperature), even aer the
where: d – thickness of the polymer layer [nm], a – constant
characteristic for a given polymer [cm nm mC ] (for poly-
thiophene-based polymers a ¼ 2,5), Q – charge [mC], A – elec-
2
ꢁ1
24,25
synthesis process.
Moreover, we can investigate the effects of
2
trode surface [cm ].
modications in chemical structures of CP. Furthermore, one of
the most attractive p-type semiconductors, 3,4-ethyl-
enedioxythiophene (EDOT) has been demonstrated as a neutral
The study of the stability of the polymer covering the surface
of the working electrode with cyclic voltammetry was also
carried out. For this purpose, 15 measurement cycles were
carried out in the potential range 0–1.7 V, as the scanning speed
was set at 50 mV s . The test was carried out in a 0.1 M TBA-TFP
solution in dichloromethane. Along with the subsequent
measurement cycles, no signicant changes in the current
intensity were observed (Fig. 4). This conrms the stability of
the formed polymer.
26
electrode or electroactive scaffold for protein and have
advanced stability due to the exclusion of oxidative-damage-
ꢁ
1
27
based inactivation.
Electrodeposition of the monomer was provided with the
chronoamperometric method (Fig. 3), which allows to obtain
controlled thin layer of polymer lm on the surface of the
platinum electrode. The Ag/AgCl electrode was used as a refer-
ence electrode. Surface modication with enzyme and platform
for anchoring the protein is a key step during the construction
The electropolymerization process and effective deposition
poly[4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)
benzothiadiazole] on the surface of Pt electrode was also
of
Fig. 3 Chronoamperogram of electrochemical deposition of poly Fig. 4 Stability of the polymer (potential range: 0–1.6 V, scan rate 50
ꢁ
1
[
4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzothiadia-
mV s , number of scans: 10, supporting electrolyte: TBA-TFP solution
zole] on the surface of Pt electrode (potential: 1.5 V, duration: 600 s). in dichloromethane).
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Fig. 5 SEM images for poly[4,7-bis(5-(3,4-ethylenedioxythiophene)
thiophen-2-yl)benzothiadiazole] 10 mm (A) and 5 mm (B).
Fig. 7 Representative CV-scans of the bare Pt electrode, Pt electrode
modified with polymer and Pt/Pol/HRP system in the presence of 17b-
conrmed using scanning electron micrographs (SEM) dis-
played in Fig. 5. Micrographs conrm the formation of a poly-
mer with unobservable surface defects and a granular structure
characteristic of benzothiadiazole derivatives. The diameter of
the resulting grains is about 1 mm. Such a pore diameter is able
estradiol 10(mM) under applied potential range (potential range ꢁ0.4 to
ꢁ1
1
.0 V, scan rate 100 mV s , via Ag/AgCl).
buffer and the measurements will be carried out at room
temperature.
˚
to allow a much smaller enzyme molecule (resolution 1.57 A to
0.000157 mm) to anchor freely in this structure while main-
Fig. 7 presented the cyclic voltammograms (CVs) of different
electrodes: a bare platinum electrode (Pt), a poly(4,7-bis(5-(3,4-
ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole) (Pol)
modied Pt electrode and the Pt/Pol/HRP complex, recorded in
the presence of a 10 mM solution of 17b-estradiol. As shown in
Fig. 7, evident electrochemical redox peak of the bare Pt elec-
trode and Pt/Pol in the PBS solution couldn't be observed.
While, a reduction peak at about 0.05 V and an oxidation peak
at 0.4C appeared at the Pt/Pol/HRP electrode. The HRP is a kind
of common electron transfer protein, and HRP immobilized on
the surface of poly[4,7-bis(5-(3,4-ethylenedioxythiophene)thio-
taining its catalytic activity due to the lack of formation of
covalent bonds and possible rotation to direct the active site of
the enzyme towards the substrate. The above information
conrms that semiconducting polymers can be successfully
used as a basic element in the construction of biosensor devices
–
not only due to semi-conductive properties – but also as
a matrix for anchoring the enzyme.
Electrochemical measurements
For the measurements, 17b-estradiol was dissolved in ethanol phen-2-yl)benzothiadiazole] in a certain extend can promote
and then in pH 7.00 phosphate buffer for two reasons. First, the electron transfer between semi-conductive polymer and the
buffer creates an optimal environment for real sample testing, electrode. The good electron transport material based on poly
and secondly – the buffer is the most optimal for horseradish
peroxidase activity. As shown in Fig. 6, optimal conditions for
horseradish peroxidase activity is pH 7.0, where the enzyme
[
4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzo-
thiadiazole] retrieved a very important role in promoting direct
electrochemistry of HRP. At the same time, the synthesized
ꢂ
28
activity is the highest at 60 C. However, due to the fact that material is a good support material and can load a large number
the sensor is intended to be used in point of care diagnostics, of HRP molecules and keep its activity, so the electro-
ꢂ
where it is oen impossible to raise the temperature to 70 C, it synthesized polymer inuences positively on the electronic
was decided that the solutions will be prepared in the given transfer of HRP.
The detection basis in the constructed system is the Q
reduction reaction to H
2
Q according to the reaction equation: Q
+
+
2e + 2H / H
2
Q. 17b-estradiol and H Q are a phenolic
2
compounds with a hydroxyl group located at carbon 3 and both
are co-substrates for HRP. Research conducted by Molina et al.
conrmed that HRP is able to recognize 17b-estradiol as a co-
substrate in a homogenous system. In this article, higher net
peak currents were observed at lower E2 concentrations, indi-
20
cating that HRP catalyzes the oxidation of H
concentration increases, a decrease in peak current is observed,
conrming that HRP reacts with both H Q as well as with E2.
2
Q to Q. As E2
2
The current values corresponding to the enzymatically
produced Q are inversely proportional to the amount of E2 in
test samples. Therefore, the maximal electrochemical response
Fig. 6 Activity of the immobilized horseradish peroxidase at various
pH values (at room temperature).
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Fig. 8 Variation of the Ip,n for 100 mM of E2 solution in constructed
ꢁ
3
system as a function of: (A) C*H
2
O
2
(C*H
2
Q ¼ 1.0 ꢀ 10 M, C*HRP ¼
ꢁ10
ꢁ3
1
1
.5 ꢀ 10
M); (B) C*H
2
Q (C*H
2
O
2
¼ 1.0 ꢀ 10 M, C*HRP ¼ 1.5 ꢀ
ꢁ
10
0
M).
was obtained in the absence of 17b-estradiol in the tested
sample.
Optimization of the concentrations of species involved in the
reaction of the biosensor
Fig. 8A shows the effect of H
2 2
O concentration change at a given
ꢁ
3
ꢁ10
concentration of H
2
Q (1.0 ꢀ 10 M), HRP (1.5 ꢀ 10
M) and
ꢁ
4
concentration of 17b-estradiol 1 ꢀ 10 M. The optimal H O
2
2
ꢁ
3
concentration was found as 5 ꢀ 10 M. The effect of H Q
2
concentration at given concentrations of HRP, H O and 17b-
2
2
2
estradiol was also analyzed (Fig. 8B). The optimal H Q
ꢁ3
ꢁ10
concentration was 3 ꢀ 10 M for HRP (1.5 ꢀ 10
M), H
10 M) and 17b-estradiol (1 ꢀ 10 M), respectively. These
optimal H and H Q amounts were used for further
experiments.
2
O
2
(5 Fig. 9 (A) The curve of Ip,n/I0p,n corresponding to the concentrations
of E2 (where Ip,n is the net peak current in the presence of E2, I0p,n is
the net peak current in the absence of E2). (B) The linear relationship
between Ip,n/I0p,n and the logarithm of E2 concentration. (C) Typical
DPV responses for E2 with different concentrations (from a to j: 1 mM, 2
mM, 3 mM, 4 mM, 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, respectively).
ꢁ3
ꢁ4
ꢀ
2
O
2
2
Calibration curve for 17b-estradiol. Analytical performance
Electrochemical character of 17b-estradiol was investigated
employing DPV method (potential range ꢁ0.2 to 0.8 V) in
oxygen-saturated conditions. The higher net peak currents were
observed at lower E2 concentrations, indicating that HRP
inversely proportional to the amount of E2. As the concentra-
tion of estradiol increases, the H Q oxidation peak practically
2
disappears on the modied electrode, which was the expected
effect. A titration curve was performed for various concentra-
tions of 17b-estradiol (0.1–200 mM) (Fig. 9A). The amperometric
catalyzes the oxidation of H Q to Q. As E2 concentration
2
increases, a decrease in peak current is observed, conrming
that HRP reacts with both H
2
Q as well as with E2. The current
response for the 17b-estradiol detection showed to be effective
values corresponding to the enzymatically produced Q are
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Table 1 Reported electrodes for estrogens determination
Biological
Electrode
Fe @Au-GSH/MIPs/GCE
MIPs/PtNPs/GCE
MIPs/PtNPs/rGO/GCE
CuThP/rGO/GCE
BPIDS/GCE
AuNPs/ceramic
biosensor
CuO/CPE
component
Method
LOD
2.76 ꢀ 10 mM
Linear range [mM]
Sensitivity
Recovery
Ref.
ꢁ
3
ꢁ1
ꢁ1
3
O
4
ꢀ
DPV
DPV
DPV
DPV
DPV
CV
0.025–10
0.03–50
0.04–50
0.1–1
0.1–10
0.001–0.032
0.58 mA mM
0.79 mA mM
ꢀ
>85%
>90%
>90%
>95%
>95%
>90%
29
30
31
32
33
34
ꢁ2
ꢀ
1.6 ꢀ 10 mM
ꢁ3
ꢀ
2.0 ꢀ 10 mM
ꢁ3
ꢁ1
ꢁ1
ꢀ
5.3 ꢀ 10 mM
0.39 mA mM
0.19 mA mM
ꢀ
ꢁ
2
ꢀ
5.0 ꢀ 10 mM
ꢁ2
ER-a
2.6 ꢀ 10 mM
ꢁ
2
ꢁ1
ꢀ
SWV
DPV
2.1 ꢀ 10 mM
0.06–0.8
0.1–5
3.409 mA M
ꢀ
>90%
>90%
35
36
ꢁ3
GNR-FS-Au-CA/CPE
E2-specic
aptamers
ꢀ
7.4 ꢀ 10 mM
ꢁ
2
ꢁ1
ꢁ
rGO-DHP/GCE
Pt/Pol/HRP
CV
DPV
7.0 ꢀ 10 mM
0.4–5.0
0.1–200
1.65 mA M
>95%
>90%
37
This
work
4
ꢁ1
ꢁ2
HRP
105 nM
1.16 ꢀ 10 A mM cm
Table 2 Analytical performance
2
Linearity
.1–200 mM
LOD
LOQ
R
Slope
SD of slope
Intercept
SD of intercept
Sensitivity
3.65 ꢀ 10ꢁ
6
8.52 ꢀ 10ꢁ8
1.65 ꢀ 10ꢁ5
1.20 ꢀ 10ꢁ7
1.16 ꢀ 10ꢁ4
0
105 nM
159.57 nM
0.99
in the range form 0.1–200 mM and a linear calibration curve was stability of both the polymer matrix covering the electrode
obtained by plotting the ratio of I (in the presence of estradiol)/ surface as well as the enzymatic protein immobilized on its
p
Ip0 (in the absence of estradiol) to the logarithm of the 17b- surface and repeatability of results.
estradiol concentration added. The linear interval can be The limit of quantication (LOQ) was also determined using
expressed by the equation y [I/A] ¼ ꢁ0.1525[log[E2]] + 0.6858 eqn (3) and it equalled 159.57 nM.
(
Fig. 9B and C). Limit of detection for constructed survey (LOD)
23
LOQ ¼ 5s /b
(3)
has been calculated as (2):
B
In which s
blank response, b is the slope of the regression line. Further-
B
is the standard deviation of the population of blank more, sensitivity of the proposed biosensor was found to be 1.16
B
is the standard deviation of the population of
LOD ¼ 3.29s
B
/b
(2)
23
where: s
ꢁ
4
ꢁ1
ꢁ2
response, b is the slope of the regression line. In this way LOD ꢀ 10 A mM cm . All parameters demonstrating an analyt-
was calculated and found to be 105 nM. In Table 1, the char- ical validation are shown in Table 2.
acteristic of proposed electrode is highlighted with those pre-
sented in the literature for estrogens determination.
The increase in 17b-estradiol concentration in the
measurement system led to saturation (plateau) of the system
Compared with the detection methods described in Table 1, on the calibration plot. Because of this, Michaelis–Menten
the enzyme-based biosensor presented has good sensitivity and kinetics analysis was also performed. The apparent Michaelis–
a relatively wide linear range. The detection limit of the current Menten
m
K constant characterizing the enzyme-analytics
sensor is good enough and has many advantages such as low- kinetics was determined using the Lineweaver–Burk equation
cost materials – all reagents necessary for the construction of (eqn (4)), described as:
the biosensor are rather cheap and easily available compared to
1
1
K
m
1
¼ I þ I
(4)
systems using a larger number of biologically active elements
e.g. systems containing antibodies or receptors), relatively easy
I
max
max
C
(
where I is the steady-state current reached aer addition of the
analyte (substrate), Imax is the maximum current measured
under saturated conditions, and C is the analyte concentration.
synthesis of semi-conductive material – properly optimized
method allows obtaining material with excellent yield (around
85%) in a short time, and easy manufacturing. Moreover, the
ꢁ
5
The Km constant was found to be 6.83 ꢀ 10
M. The Km
use of the enzyme in the constructed system signicantly
reduces the time of analysis by skipping oen time-consuming
steps, such as the incubation of the electrode with the target
analyte (in the case of immunosensors and systems based on
receptors) or regeneration of the electrode to free places of
interaction with the analyte before proceeding to the next
measurement. Such created system is characterized by high
constant indicates that horseradish peroxidase immobilized
onto the Pt/Pol platform retains its bioactivity towards 17b-
estradiol.
The analysis of the lifetime of the constructed system was
also carried out. The lifetime of enzymatic biosensors is limited
by the loss of enzyme activity over time. For this purpose, the
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over time, which at the turn of the 5th and 6th week exceeded
5
5
%. Therefore, the lifetime of the system can be determined for
weeks.
Determination of 17b-estradiol in the presence of interfering
substances
The selectivity and specicity of a biosensor are very important
parameters in the context of correct design bio-devices. It
means, that the biosensor should not be subjected to interfer-
ence by other hormones or biomarkers while in use. In this
project, we were studied ve different substances (ascorbic acid
(
(
AA), estriol (E3), estrone (E1), uric acid (UA) and cholesterol
CH)) as a potential interference compounds during the detec-
tion of 17b-estradiol. The justication of selecting previously
mentioned substances in this investigation was based on the
similar chemical structure between 17b-estradiol and potential
interference substances, and also the presence of such
compounds in the human body was taken into account. All
presented specimens were added in a concentration equalled to
50 mM to different E2 samples (10, 50 and 100 mM) to check the
inuence of these compounds in large excess, equilibrium and
deciency of interfering substances. Each tested reagent has an
insignicant effect (<10%) on the peak current of the samples
compared to the blank (0% AA, 6% E3, 9% E1, 5% UA and 2%
CH).
Fig. 10 Lifetime analysis of constructed system.
Presented results (Fig. 11) affirm insignicant impact on the
selectivity of constructed system, and convict that checking
interferences does not disturb the work of proposed 17b-estra-
diol test. Furthermore, presence of HRP in the detection system,
allows the efficient examination of lower 17b-estradiol concen-
trations (>10 mM) and interfering species have insignicant
effect on the measurements, because of the enzymes selective
nature. As a result, the biosensor exhibited adequate selectivity
for 17b-estradiol determination.
Pharmaceutical sample analysis
To evaluate the practicability of the presented procedure, elec-
trochemical analysis was used to determine the estradiol level
in the labelled pharmacological product (Estradiolum, 2 mg,
Novo Nordisk A/S). The analytical results for testing samples are
Fig. 11 Effect of interfering substances (50 mM) on the determination
of E2 level.
system response to 1uM E2 concentration was measured at 1 given in Table 3. A very promising recovery value (ratio of the
week intervals. The system was considered stable until the determined concentration to the actual concentration of 17b-
change in peak current did not exceed 5%. As can be deduced estradiol in the sample, expressed in %), clearly conrms the
from the graph presented in Fig. 10 the system lost its activity efficiency of the proposed method for the useful detection of
17b-estradiol.
Table 3 Detection effect of 17b-estradiol based on the method presented
Real sample
C
detected
C
calculated
Recovery (%)
RSD
Estradiolum, 2 mg, Novo Nordisk A/S
0.97 mM
1 mM
25 mM
50 mM
102.69
96.37
93.12
ꢃ2.43%
25.94 mM
3.69 mM
5
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1
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