Talanta 250 (2022) 123737

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Talanta
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MIP-based electrochemical sensor for direct detection of hepatitis C virus

via E2 envelope protein
Mariia Antipchik, Jekaterina Reut, Akinrinade George Ayankojo, Andres Öpik, Vitali Syritski *
Department of Materials and Environmental Technology, Tallinn University of Technology, Ehitajate tee 5, 19086 Tallinn, Estonia

A R T I C L E I N F O A B S T R A C T

Keywords: Hepatitis C is the most common liver disease caused by Hepatitis C virus (HCV), and can evolve into serious
Electrochemical sensor health problems e.g. cirrhosis and hepatocellular carcinoma. Nowadays, the initial stage of the disease cannot be
Hepatitis C virus (HCV) detection practically diagnosed, representing thus an extremely important problem of modern public health care. This
HCV envelope Protein E2
study is aimed at the development of a sensor for direct detection of HCV. The sensor utilizes a synthetic
Screen printed electrode (SPE)
Molecularly imprinted polymer (MIP)
recognition element prepared by the technology of molecular imprinting and representing a molecularly
imprinted polymer (MIP) having molecular recognition sites of HCV envelope protein E2 (E2-MIP). E2-MIP in­
tegrated into an electrochemical sensor platform allows quantitative evaluation of binding of free E2 protein as
well as HCV-mimetic particles (HCV-MPs) in human plasma with LOD value of 4.6 × 10− 4 ng/mL (for HCV-MPs).
The developed electrochemical HCV sensor represents a simple, fast and inexpensive alternative for the existing
methods of HCV detection and paves the way for the point-of care diagnostics of Hepatitis C.

1. Introduction testing platforms could be enzyme immunoassays (EIAs), chem­

Despite the availability of highly effective, well-tolerated, short-term
and direct-acting antivirals with a high cure rate, hepatitis C virus (HCV)
is widespread throughout the world, and has a high mortality, making it
a global health problem [1]. The World Health Organization (WHO)
goals for ending hepatitis C require a significant increase in access to
testing (from <20% in 2015 to 90% in 2030) and treatment (from <10%
in 2015 to 80% in 2030) [2]. There is no vaccine against HCV, and
therefore early diagnosis is important to control the spread of the virus
[3]. In addition, the lack of well-equipped laboratories is a problem,
especially in low-income countries [4]. Thus, there is an urgent need to
develop new HCV detection methods and devices with an economic cost.
Modern methods of HCV diagnostics are based on molecular and
serological assays. The molecular assays are RT-PCR based nucleic acid
amplification tests for detection of HCV RNA in samples, which has
excellent sensitivity and specificity, and therefore is considered as the
gold standard for the HCV detection [5–7]. On the other hand, PCR is a
laborious and expensive test. It requires a well-functioning infrastruc­
ture and therefore has limitations in terms of point-of-care testing,
especially for screening of HCV in a large population for rapid diagnosis
[8]. The serological tests are suitable for the mass screening of HCV in
the general population as well as for treatment monitoring [9]. The
iluminescence (CLIA) or rapid tests [10] that can detect the presence of
anti-HCV antibodies, HCV antigen or both simultaneously [11–13]. In
addition, a wide range of HCV serological assays employing the different
sensing platforms are currently under development [14–18]. Among
them, electrochemical sensors have been shown to be a prospective HCV
detection approach due to its remarkable sensitivity, and simplicity as
well as the possibility of fast analysis [19–21].
As mentioned, the HCV detection mechanisms can vary depending
on the target analyte: antibody, antigen or whole viral particle. Anti-
HCV antibodies testing is most popular [22,23]. However, this strat­
egy has some drawbacks. Firstly, the results may be negative in the early
acute phase of hepatitis C and in profoundly immunosuppressed pa­
tients. Moreover, after the treatment, the antibodies may persist in the
absence of HCV RNA, but may decline and finally disappear in some
individuals [24]. Thus, the detection of HCV core antigen, which is
present in plasma of an infected person at the early stages of the disease,
represents a promising approach for HCV diagnostics [25–27]. It was
shown that HCV core antigen levels correlate well with HCV RNA,
however, the major limitation is its lower sensitivity as compared to
RT-PCR that can contribute to a false negative response [28]. Another
potentially promising method for HCV diagnostics is the detection of
HCV surface antigen E2, which is considered a marker of the presence of

* Corresponding author.
E-mail address: vitali.syritski@taltech.ee (V. Syritski).

https://doi.org/10.1016/j.talanta.2022.123737
Received 4 February 2022; Received in revised form 7 July 2022; Accepted 11 July 2022
Available online 13 July 2022
0039-9140/© 2022 Elsevier B.V. All rights reserved.
M. Antipchik et al.
active infection in the human body, since its level correlates with an
infectious state [29–31]. In this method, the detection of antigen can be
realized through the molecular recognition between the antigen, which
is located on the virus surface, and the complementary natural or syn­
thetic receptors [14].
In our previous work, by showing the structure and mechanism of
HCV function, we made an assumption about the possibility of early
detection of HCV by the electrochemical method where the surface
protein of HCV - E2 could be detected through molecular interaction
with the complementary cell receptor CD81 immobilized on the sensing
surface of the biosensor [32]. The biosensor demonstrated a rather high
LOD that limited its application only to the acute stage of the disease,
when the viral load on the body could be quite high. The replacement of
the biological receptor by an artificial analogue may overcome the
limitation associated with a labile nature of a biological molecule, thus
providing a robust and reproducible recognition element and improving
the sensitivity and stability of the sensor.
Molecular imprinting technology is one of the promising methods for
the design of the synthetic receptors so-called molecularly imprinted
polymers (MIP) containing artificially-created binding sites, or finger­
prints, of the target molecules. Molecular imprinting consists of poly­
merization of a mixture of functional monomers in the presence of a
target molecule that acts as a template. Subsequent removal of templates
from the formed polymer leaves behind binding sites that are comple­
mentary to the target molecule in size, shape and arrangement of
functional groups and, thus, are able to selectively recognize these
molecules. The main advantages of MIPs as synthetic receptors are
excellent chemical and thermal stability in combination with their
reproducible and cost-effective fabrication [33–35]. Thus, the combi­
nation of MIPs and electrochemical methods may represent a valuable
approach to the development of inexpensive, sensitive, fast and portable
sensor systems [36–39]. The detection principle of these sensors is
mainly based on monitoring the charge transfer carried by the redox
probe through the MIP thin film [40]. MIPs integrated with various
sensor platforms have been extensively studied in recent decades to
detect various biomarkers of diseases [41–45]. There are few reports on
the electrochemical MIP-based sensor for detection of Hepatitis A (HAV)
and B (HAB) viruses [46–50]. The aptamer-MIP strategy for the detec­
tion of HCV core antigen has also been reported [51]. However, to the
best of our knowledge, there is no report on the electrochemical
MIP-based sensor for detection of HCV surface antigen, E2.
Thus, in this work, we report for the first time on the development of
Fig. 1. Schematic representation of HCV diagnostics principle by HCV sensor.
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Talanta 250 (2022) 123737
a MIP-based electrochemical sensor for detection of HCV through its
surface protein E2 (Fig. 1). MIP was prepared by an electrochemical
surface imprinting approach using m-phenylenediamine (mPD) as a
functional monomer and E2 as a template protein. The method allowed
the direct integration of MIP as a synthetic receptor to screen-printed
electrode (SPE) as an electrochemical sensor transducer. The perfor­
mance of the prepared HCV sensor was tested against the binding of free
E2 protein as well as HCV-mimetic particles (HCV-MPs).
2. Materials and methods
2.1. Materials
4-aminothiophenol (4-ATP), 2-mercaptoethanol (2-ME), m-phenyl­
enediamine (mPD), dithiothreitol (DTT), bovine serum albumin (BSA),
sodium dihydrogen phosphate dihydrate, disodium hydrogen phos­
phate, sodium dodecyl sulfate, phosphate buffered saline (10 mM
Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4 (PBS)),
human plasma albumin (HSA) and immunoglobulin G (IgG) were ob­
tained from Sigma-Aldrich. Ethanol 96% was purchased from Estonian
Spirit OÜ (Estonia). 3,3′ -dithiobis [sulfosuccinimidyl propionate]
(DTSSP) was purchased from Thermo Fisher Scientific Inc., sulfuric acid,
hydrogen peroxide, potassium chloride (KCl), and acetic acid were
purchased from Lach-ner, S.R.O. Sodium chloride (NaCl) was obtained
from Fluka analytical. Potassium ferricyanide and ferrocyanide were
purchased from Riedel-de Haen. CD81 was purchased from ServiceGen
(St. Petersburg, Russia). Screen printed electrodes (BT220, Metrohm
DropSens, Spain) consisted of the working and counter electrodes made
of gold and the reference electrode made of silver.
All chemicals were of analytical grade and were used as received
without any further purification. Ultrapure Milli-Q water (resistivity
18.2 MΩ cm at 25 ◦ C, Merck KGaA, Darmstadt, Germany) was used for
the preparation of all aqueous solutions.
HCV viral envelope protein E2 was prepared according to the pro­
tocol described earlier [14]. In order to avoid the agglomeration of
protein and stabilize its structure E2 was used in complex with a
superfolder green fluorescent protein (GFP). The molecular mass of the
prepared E2-GFP complex was 54 kDa as determined by SDS-PAAG
electrophoresis and confirmed by mass spectrometry analysis showing
the presence of the peptides of the E2 sequence. The E2-GFP complex
was used as a template for preparation of MIP, in the rebinding exper­
iments, and for modification of HCV-MPs.
M. Antipchik et al.
HCV-MPs were prepared according to the protocols described earlier
based on the poly(D,L-lactic acid)-b-poly((ethylene glycol)-5000 methyl
ether) PLA-b-PEG copolymer [14]. The details of HCV-MPs preparation
are provided in SI, section S1. The prepared HCV-MPs had an average
hydrodynamic diameter and polydispersity of 75 ± 8 nm and 0.23,
respectively. Amount of E2 immobilized onto the surface of nano­
particles was 30 μg/mg as determined spectrophotometrically.
The human plasma samples were kindly provided by Dr Sirje Rüütel
Boudinot from the Department of Chemistry and Biotechnology of Tal­
Tech. The short description of the plasma samples preparation is pro­
vided in section S2 of SI.
2.2. HCV sensor preparation
The HCV sensor was prepared by modification of the sensing elec­
trode of SPE with the thin film of E2-MIP generated from PmPD by
adapting the electrochemical surface imprinting approach developed by
Syritski’s group [52,53]. Before modification SPE was cleaned in the
Novascan Digital UV Ozone System (Novascan Technologies, USA) for
15 min. The protocol for synthesis of E2-MIP consisted of several steps.
Firstly, the working electrode of SPE was modified with 4-ATP by
incubating it in 100 mM 4-ATP ethanolic solution for 30 min and 3 times
washing in ethanol to remove the non-bound 4-ATP. Then the cleavable
linker monolayer was generated by the covalent attachment of DTSSP to
4-ATP-SPE via incubation in 10 mM DTSSP solution in PBS for 30 min
and subsequent washing with PBS. For E2 immobilization,
DTSSP-modified SPE was incubated for 30 min in PBS containing the
optimal concentration of E2 selected in the range of 1–25 μg/mL and
subsequently washed with plenty of PBS. The electrodeposition of PmPD
on E2-modified SPE was performed with an electrochemical workstation
(Reference 600TM, Gamry Instruments, USA) from PBS containing 10
mM mPD at 0.65 V vs Ag/AgCl/KCl. The thickness of PmPD film was
optimized by applying different charge densities in the range 1–3
mC/cm2. Finally, the generated polymer film was treated with PBS
containing 50 mM DTT for 30 min to cleave the S–S bond of DTSSP and
facilitate the release of E2, followed by washing with a 10% acetic acid
solution on vortex for 30 min resulting in the formation of molecular
imprints of E2 at the surface of polymer film (E2-MIP).
Reference sensor having a polymer film with non-eluted template-a
reference polymer-was prepared in the very same manner as E2-MIP
with the exception of treating the polymer film with DTT to save the
covalent attachment of E2 in the polymer matrix and to avoid formation
of the molecular imprints of E2 in this film.
Differential pulse voltammetry (DPV) was used to characterize each
step of the E2-MIP film preparation. The DPV measurements were per­
formed with the aid of an electrochemical workstation (Reference 600,
Gamry Instruments, USA) using a special connector for SPE electrodes. A
drop of a solution containing 1 M KCl and 4 mM redox probe (K3[Fe
(CN)6]/K4[Fe(CN)6]) was placed on SPE surface in the way to cover all
its electrodes and the signal was recorded in the potential window be­
tween 0 V and 0.4 V with a pulse amplitude of 0.025 V, a pulse width of
0.05 s, a step potential of 0.005 V and sample period 0.1 s. Every
measurement was performed in triplicates to confirm its reproducibility.
2.3. Binding and selectivity studies
The rebinding of E2 (free and as part of HCV-MP) on the HCV sensor
was measured by DPV. Before DPV measurement the sensor was incu­
bated and stabilized in PBS for 15 min. Then the sensor was incubated in
the testing solution containing E2 or HCV-MPs in the range of 0.01–50
ng/mL in PBS and measured by DPV. In the case of experiments in
biological samples, human plasma was used instead of PBS for stabili­
zation and for the preparation of analytical samples.
The DPV peak currents were normalized according to Eq. S1 (section
S3 of SI) to obtain response signals (In) of the sensor. The binding iso­
therms were plotted and fitted using Langmuir-Freundlich adsorption
3
Talanta 250 (2022) 123737
model (Eq. S2, section S3). The binding parameters (dissociation con­
stant KD, response at saturation Isat), limit of detection (LOD) and limit of
quantification (LOQ) were calculated according to the equations pro­
vided in the section S3.
The optimal incubation time of the sensor with E2 or HCV-MPs was
initially chosen as 15 min based on the previous experience of the
research group [43] and confirmed experimentally for the prepared HCV
sensor (see Fig. S1 in section S4). The DPV measurements were per­
formed similarly to that in section 2.2.
The selectivity of the HCV sensors was assessed by comparing the
responses against the target (E2) and interfering proteins analytes (HSA
(66.5 kDa, pI 4.7), IgG (153 kDa, pI 8.8) and CD81 (26 kDa, pI 5.4)) at
equivalent concentrations. More detailed characteristics of the proteins
can be found in the section S5 of SI.
The stability of HCV sensors was assessed by comparing responses of
the freshly prepared sensors with those stored for 7, 14, 21 and 30 days
at room temperature. The responses were recorded against 5 ng/mL of
E2 in PBS.
3. Results and discussion
3.1. Preparation of HCV sensor
The HCV sensor represents a SPE modified with E2-MIP as a recog­
nition element and connected with potentiostat that is controlled by
software in a tablet computer. The sensing concept is based on so-called
“gate effect” [40] that relies on the inhibition of the charge transfer of
the redox probe ([Fe(CN)6]3-/[Fe(CN)6]4-) through thin E2-MIP film to
the gold electrode surface of SPE upon the selective binding of target - E2
(Fig. 1). It is supposed the inhibition is correlated with the concentration
of E2 in the sample. The intensity of the current resulting from the redox
reaction of [Fe(CN)6]3-/[Fe(CN)6]4- at the HCV sensor surface was
monitored. When the imprinted cavities of E2-MIP were not occupied by
the bound protein molecules, the redox probe ions could freely move at
the electrode/solution interface and current peak intensity was the
highest (current peak I0 in Fig. 1). If the film had rebound its target
protein (E2) after incubation of the sensor in a sample solution, the
charge transfer was efficiently hindered causing a current decrease
(current peak I in Fig. 1) that correlated with the concentration of the
virus protein in the solution.
All stages of SPE modification during the preparation of E2-MIP were
monitored by DPV measurements (Fig. 2). The redox activity decreased,
when the linker and the protein were subsequently immobilized on the
SPE sensing surface, and almost disappeared after PmPD electrodepo­
sition indicating non-conducting polymer film formation. However, the
treatment of the modified electrode in dithiothreitol and acetic acid
caused a prominent increase in the peak current indicating supposedly,
elution of E2 from the PmPD matrix and formation of molecular
Fig. 2. The DPV peak current of bare Au, and after the subsequent fabrication
steps of E2-MIP: modification by 4-ATP, DTSSP, E2, electrodeposition of PmPD
and treatment in DTT and acetic acid.
M. Antipchik et al. Talanta 250 (2022) 123737

imprints, which tunneled the charge transfer across the ultra-thin non-
conducting PmPD layer to the electrode.
The choice of polymer film thickness is of great importance for the
formation of protein imprinted polymer stipulated by the fact that the
film undergrowth or overgrowth can either lead to inferior imprinting or
irreversible entrapment of the surface-immobilized protein, respec­
tively. In this work, the thickness of the electrodepositing PmPD film
needed to form the optimal E2-MIP was determined through the com­
parison of the responses of HCV sensors having different thicknesses of
E2-MIP and reference polymer upon E2 rebinding (Fig. 3A, Fig. S2). As it
was seen, all three HCV sensors decorated with E2-MIP had the greater
response than the sensors having the similarly thick reference polymer.
However, the most pronounced difference (2.41 times) showed the pair
of the sensor with E2-MIP and reference polymer synthesized by 2 mC/
cm2 (Fig. 3A). Apparently, in this case, the polymer generated at the
surface of the electrode confines the anchored E2 to the extent that the
proteins still can be successfully extracted at the washing stage. It was
previously found that charge of 2 mC/cm2 generated about 4.5 nm thick
PmPD film that was optimal to prepare a MIP selective towards the
protein having the molecular weight close to E2, namely SARS-CoV-2
nucleoprotein (MW 49.5 kDa) [43]. Thus, it is not surprising that
E2-MIP generated by 2 mC/cm2 was also the most optimal to imprint E2.
To optimize further, we presumed that the amount of E2 immobi­
lized on the sensor surface before the polymer generation could also
affect the rebinding capacity of the resulting E2-MIPs. In order to
elucidate this matter, the various concentrations of E2 (0.1–25 μg/mL)
were applied at the immobilization stage and the responses of the
resulting HCV sensors towards E2 (5 ng/mL in PBS) was subsequently
studied (Fig. 3B). It can be seen that E2 bound the activated SPE and
saturated the surface at about 7.5–10 μg/mL, however incubation at 5
μg/mL was already sufficient to endow the resulting HCV sensor with
the highest response. Thus, this concentration was used afterwards for
preparation of E2-MIP.
It should be noted that since the E2-GFP complex was used as a
template for preparation of MIP (see section 2.1), the resulting MIP
likely had molecular imprints of E2-GFP, but we still called it E2-MIP.
Moreover, the same E2-GFP complex (alone or as a part of HCV-MP)
was later used in the rebinding experiments to confirm the imprinting
effect of E2-MIP (see section 3.2). Thus, we believe that, at least, in the
frame of this study the use of E2-GFP complex instead of pure E2 did not
significantly affect the validity of the experimental results.
3.2. Performance of HCV sensor
3.2.1. Interaction with E2
The HCV sensor exposed to PBS solution or human plasma contain­
ing E2, demonstrated a saturated binding isotherm that could be accu­
rately fitted to the Langmuir-Freundlich model of adsorption (Fig. 4).
This model gave an accurate fit to the experimental data yielding close
to unity the coefficients of determination, R2, values (Table 1). The KD
and Isat values derived upon testing the sensor in PBS and human plasma
were found to be rather equal, indicating the binding affinity of E2 to E2-
MIP was not compromised by plasma components and supposedly whole
sensor performance will remain also consistent to detect E2 in real
samples.
The selectivity of the HCV sensor was examined through a compar­
ison of its sensor responses towards E2 against HSA, IgG and CD81
applied at the same concentrations. The rationale for selection of the
specified proteins was their molecular weights, pIs and possibly to be
present in real samples along with E2. Thus, as compared with E2, HSA
(66,5 kDa) has approximately similar molecular weight, IgG (153 kDa)
is a bigger molecular substance, but with similar pI, and CD81 (26 kDa)
is selected as a smaller molecular weight substance (see section S4). As it
can be seen, the sensor showed much superior response towards E2 than
those towards the other interferents applied (Fig. 5A). It should be noted
that the sensor readily discriminates between E2 and CD81 proving the
hypothesis that the molecular imprints in the E2-MIP sensing layer are
obviously complementary to E2 not only in size but also in arrangement
of the functional groups. The latter does not allow other compounds to
be easily fixed on the surface of E2-MIP, even if they would have smaller
dimensions than the target molecule.
Moreover, since HSA and IgG constitute the most abundant proteins
in human plasma, further selectivity study was executed to determine
the sensor’s response to plasma samples spiked with equimolar con­
centration each of HSA and IgG as against E2. For this purpose, an
optimal plasma dilution was determined by subjecting the plasma
sample to a serial dilution (from 5 to 640 fold) in PBS. The responses
received upon incubating a fresh HCV sensor in each diluted sample
were used in plotting an adsorption isotherm (Fig. 5B) that was fitted to
the Langmuir equation. The optimal dilution suitable for further analysis
was estimated as the dilution giving 50% of the response at saturation
(KD value). The fitting indicates a KD of 0.002 a.u corresponding to a 500
fold plasma dilution. However, since the response induced by this
diluted plasma is still significantly high to be used as background
(Fig. S4), a further twice dilution giving reasonable response within a

Fig. 3. (A) Effect of charge densities applied during synthesis of PmPD on the saturated responses Isat of E2-MIP and NIP-modified sensors. The right Y axis shows the
ratio of Isat(MIP)/Isat(reference polymer). The Isat(MIP) and Isat(reference polymer) were derived from fitting the corresponding binding isotherms to the Langmuir-
Freundlich adsorption model (Fig. S2). (B) Effect of E2 concentration applied at the immobilization stage on HCV sensor response towards E2 (5 ng/mL in PBS).
The black rectangular dots are normalized DPV peaks recorded on the activated working electrode of SPE as a function of applied E2 concentration, while the line
represents a fit to the Langmuir-Freundlich adsorption model. The bars represent the responses of HCV sensors prepared at the respective concentration of E2 chosen
from the adsorption isotherm and having E2-MIP generated by 2 mC/cm2. The responses were measured by DPV as described in Section 2.2. The error bars on the
plots represent the standard deviation (SD) of 3 measurements of three sensors.

4
M. Antipchik et al.
Fig. 4. Adsorption isotherm of E2 as measured by HCV sensor in (A) PBS and (B) human plasma. The error bars represent the standard deviation (SD) of 3 mea­
surements by three separate sensors.
Table 1
The parameters derived from fitting the binding isotherms to the Langmuir-
Freundlich adsorption model (Fig. 4).
PBS (0.01 M, pH 7.4) Human plasma
Isat 0.78 ± 0.07 0.63 ± 0.11
KD (ng/mL) 3.6 ± 0.5 2.1 ± 0.7
m 0.45 0.5
R2 0.998 0.982
certain limit was considered optimal and used for the selectivity
experiment. Although spiking with either HSA or IgG shifts their initial
plasma concentration to a level difficult to ascertain within the scope of
this experiment, the sensor still demonstrates a discriminatory recog­
nition for E2 against HSA or IgG whose induced responses are either
comparable to or below the zero analyte response (Fig. 5C). However,
for a quantitative determination of E2 in plasma samples of HCV pa­
tients, a competitive experiment at a fixed level of the interferent vs
increasing concentration of E2 is planned, to obtain a calibration plot
suitable for subsequent quantitative analysis. In addition, the sensor’s
response to plasma solutions containing mixtures of E2, HSA and IgG at
different molar ratios, 1:1, 1:10, 1:100 (See section S7 for details) was
studied (Fig. 5C). As observed, although the increasing amount of HSA
and IgG in the solution mixture causes a slightly higher response than
sole E2, even at a 100 times higher concentration of the interferents, the
difference in the response (about 16%) may not be sufficient to conclude
that either HSA or IgG would significantly affects E2 recognition.
The stability of the HCV sensors was also addressed in this work
(Fig. 5D). During twenty days the readings of the sensor deviated from
the initial value in the range of 10%, followed by the significant upward
shift in the next 10 days. From this it can be speculatively assumed that
PmPD swells and contains water molecules immediately after synthesis,
which evaporate during storage. As well the reproducibility of the sensor
manufacture should not be underestimated. Thus, further research is
needed to confirm the phenomenon of the increase of instability of
PmPD-based E2-MIP and to refine the process of MIP preparation.
However, maximal deviation of sensor response was less than 20%,
which exhibits promising applicability of the sensor even after long-term
storage.
3.2.2. Interaction with HCV-MPs
HCV-MPs were used to evaluate the applicability of the sensor to
detect HCV. The rationale for use of VMPs in validation of the perfor­
mance of the sensor is similarity of their physico-chemical characteris­
tics with biological viruses, while they are cheaper and safer. The sensor
showed the saturated binding isotherm signal against HCV-MP presence
in human plasma. The isotherm can be fitted well to the Langmuir-
5
Talanta 250 (2022) 123737
Freundlich model (Fig. 6A). The calculated KD and Isat values were
found to be 1.1 ± 0.4 ng/mL and 0.75 ± 0.08, respectively, which are
close to the values for free E2 binding (Table 1). Thus, it can be
concluded that the prepared E2-MIP sensing layer was capable of
effective binding of E2 as a part of HCV-MPs. It should be noted, how­
ever, that the sensor gave a somewhat enhanced response against HCV-
MPs as compared to that which appeared after binding of sole E2. Likely,
the larger size of HCV-MPs may create a more pronounced hindrance to
charge-transfer via E2-MIP surface than the smaller sized E2 proteins.
Apart from the smaller size, the abundant amount of charged residues in
a protein may also facilitate electron transfer.
The pseudo-linear response was observed up to 0.1 ng/mL of HCV-
MPs with LOD and LOQ (Eqs. S3 and S4) values of 4.6 × 10− 4 ng/mL
and 15.3 × 10− 4 ng/mL, respectively (Fig. 6B). In an attempt to compare
the obtained LOD value, with that of the PCR method, the theoretical
calculations were made. Considering the LOD of PCR as 15 IU/mL or
37.5 HCV RNA/mL [24,54] and assuming mass of the HCV-MP particle
as 2.76 × 10− 7 ng, the calculations resulted in the value of 10− 3 ng/mL
HCV-MPs. Thus, the LOD of the HCV sensor is small enough and can
compete with existing methods for determining the disease at an early
stage.
Several studies demonstrated the capability of MIP-based sensors for
detecting hepatitis viruses in blood samples (Table 2). In most cases
MIPs were prepared for HAV and HBV detection using a whole virus
particle as a template [47,48,50,55,56] and in one work an aptamer-MIP
for detecting HCV core antigen was reported [51]. Thus, according to
our best knowledge, this study reports for the first time on the MIP-based
sensor capable of detecting HCV surface antigen (E2) and indicates a
significant improvement in terms of LOD, detection duration, and line­
arity, to existing reports (Table 2). The obvious advantage of this
method is the capability to detect both a free antigen E2 and a whole
virus particle via E2. Since E2 level correlates with active infection state
in the human body [30,57], the developed HCV sensor represents a
promising diagnostic method. The analytical parameters of the HCV
sensor are also comparable with electrochemical biosensors intended for
the detection of HCV core antigen (Table 3). Moreover, the HCV sensor
demonstrated improved results compared to the HCV surface antigen
sensor developed in our previous work [32], where the selected peptide
fragments of the complementary cell receptor CD81 were used as
recognition elements. We assume that the remarkable improvement of
LOD value by ca 5 order of magnitude in the case of the developed HCV
sensor is more likely due to the better compatibility of the MIP-based
synthetic receptor to the electrochemical readout of the sensor.
4. Conclusion
This work demonstrates for the first time the electrochemical HCV
M. Antipchik et al. Talanta 250 (2022) 123737

Fig. 5. The responses of HCV sensor (A) after incubating in PBS containing 5 ng/mL of protein (E2, HSA, IgG or CD81), (B) plotted as adsorption isotherm for
different dilutions of plasma samples and fitted to the Langmuir adsorption model (Eq. S5), (C) after incubating in 1000 fold diluted plasma containing equimolar
concentration (0.093 nM) of proteins (E2, HSA, or IgG) and mixture of different molar ratio of E2, HSA and IgG, (D) stored for 7, 14, 21 and 30 days and after
incubating in solutions of E2 (5 ng/mL), the initial response is graphed against 0. The error bars on the plots represent the standard deviation (SD) of 3 measurements
on three separate sensors.

Fig. 6. (A) Adsorption isotherm of HCV-MPs as measured by the HCV sensor in the human plasma. (B) The calibration plot obtained at low concentration of HCV-
MPs in human plasma (incubation time - 15 min). The solid line shows a linear regression fit. The error bars represent the standard deviation (SD) of 3 measurements
of three separate sensors.

sensor detecting HCV through capturing its envelope protein (E2) by the binding of free E2 as well as E2 as a part of HCV-MPs in human plasma.
synthetic receptor (E2-MIP). Notable selectivity of E2-MIP towards E2 The sensor showed a linear response to HCV-MPs up to the concentra­
was achieved by adopting the surface imprinting approach. After opti­ tion 0.1 ng/mL, with LOD value of 4.6 × 10− 4 ng/mL, which was small
mization, the HCV sensor was capable of quantitative evaluation of enough to compete with existing methods determining the early stage of

6
M. Antipchik et al. Talanta 250 (2022) 123737

Table 2
Comparison of MIP-based sensors for detection of different hepatitis viruses.
Target Template for MIP Detection time LOD Linear range Method Reference
a
HAV HAV 150 min 8.6 pmol/L 0.04–6.0 nmol/L RLS [50]
HAV HAV 30 min 1.1 pmol/L 0.02–2.0 nmol/L RLS [55]
HAV HAV 20 min 0.1 pmol/L 0.02–2.0 nmol/L RLS [47]
HAV, HBV HAV, HBV 20 min 3.4 pmol/L 0.3–95 nmol/L fluorescence [48]
5.3 pmol/L 0.5–90 nmol/L
HBV HBV 20 min 1.8 pmol/L 10–3500 pmol/L ratiometric fluorescence [56]
HCV core antigen Apt [HCV core antigen] complex 40 min 0.08 fmol/L 0.24–47.62 fmol/L DPV, CVb, EISc [51]
HCV E2 HCV E2 15 min 8.52 fmol/L 0.1852–925.93 pmol/L DPV This work
a
Resonance light scattering.
b
Cyclic voltammetry.
c
Electrochemical impedance spectroscopy.

Table 3
Comparison of electrochemical biosensors for HCV antigen detection.
Object Receptor Detection time LOD Linear range Method Reference

5-type hepatitis virus antigens 5-type hepatitis virus antibodies 5 min 0.8 ng/mL 1.5–350 ng/mL potentiometry [58]
HCV core antigen HCV core antigen antibody – 3 fg/mL 0.05 pg/mL–60 ng/mL DPV [59]
HCV core antigen aptamer against core protein – 3.3 pg/mL 10–400 pg/mL EISa, CVb, DPV [60]
HCV core antigen monoclonal HCV core antibody – 0.01 pg/mL 0.25–300 pg/mL SWVc, CV, EIS [61]
HCV envelope protein E2 selected peptide fragments of cell receptor CD81 3.5 h 0.02 μg/mL 1–300 μg/mL DPV [62]
HCV envelope protein E2 E2-MIP 15 min 0.46 pg/mL 0.01–50 ng/mL DPV This study
a
Electrochemical impedance spectroscopy.
b
Cyclic voltammetry.
c
Square wave voltammetry.

disease. Moreover, the MIP-based receptor shows advantage over the
biological counterparts, used in our previous work - recombinant LEL
loop of CD81 and its short peptide fragments - in delivering binding
events of E2 under the very same conditions [32].
The simplicity and fast analysis provided by the electrochemical
sensor platform along with the selectivity endowed by E2-MIP makes the
developed HCV sensor a promising tool for point-of-care testing of
Hepatitis C at the early stage as well as for chronic infection. Further
research could be performed, however, to shed light on stability of E2-
MIP after long-term storage.
Credit author statement
Mariia Antipchik: Formal analysis, Funding acquisition, Investiga­
tion, Methodology, Validation, Visualization, Writing – original draft,
Writing – review & editing. Jekaterina Reut: Methodology, Writing –
original draft, Writing – review & editing. Akinrinade George Ayan­
kojo: Investigation, Formal analysis, Writing - review & editing. Andres
Öpik: Resources. Vitali Syritski: Conceptualization, Formal analysis,
Data curation, Funding acquisition, Methodology, Project administra­
tion, Resources, Supervision, Visualization, Writing – original draft,
Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by the European Regional Development
Fund and the programme Mobilitas Pluss (grant MOBJD489) and Esto­
nian Research Council (grant PRG307). The authors thank Dr. Dmitry
Polyakov personally and Institute of Experimental Medicine of St
Petersburg for kindly providing the E2 viral protein as well as Dr. Sirje
Rüütel Boudinot from the Department of Chemistry and Biotechnology
of TalTech for kindly providing human plasma samples.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.talanta.2022.123737.
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