Journal of Molecular Liquids 317 (2020) 114014

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Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

A sandwich electrochemical immunosensor based on antibody

functionalized-silver nanoparticles (Ab-Ag NPs) for the detection of
dengue biomarker protein NS1
Maryam Awan a,1, Sajid Rauf b,1, Azhar Abbas c, Mian Hasnain Nawaz a, Changping Yang b,
Shakir Ahmad Shahid c, Naima Amin d,⁎, Akhtar Hayat a,⁎
a
Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS University Islamabad Lahore Campus, Lahore, Pakistan
b
Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Faculty of Physics and Electronic Science, Hubei University, Wuhan, Hubei 430062, China
c
Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan
d
Department of Physics, COMSATS University Islamabad Lahore Campus, Lahore, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Dengue, a viral disease caused by a flavivirus belonging to family. Flaviviridae, is a serious threat to human health
Received 15 May 2020 in tropical and subtropical regions worldwide. Herein, we design a sandwich-type immunosensor based on a
Received in revised form 27 July 2020 nano-probe maintaining antibody functionalized‑silver-nanoparticles (Ab-Ag NPs) as a trace tag for simple, sen-
Accepted 6 August 2020
sitive, and selective detection of dengue biomarker NS1. Diazonium assisted immobilization of antibody on
Available online 7 August 2020
graphite pencil electrodes provided a selective and specific platform for analyte (NS1) recognition, sandwiched
Keywords:
by Ab-Ag NPs. Well defined and sharp electrochemical signals by Ag NPs, characteristics of their oxidation,
Electrochemical sandwich immunosensor probed the specific detection of NS1. A proportional increase in the faradic current for the oxidation of Ag NPs
Graphite pensile electrodes silver nanoprobes is observed with an increase in analyte NS1 concentration. This sandwiched based immunosensor facilitated to
Dengue virus achieve a linear range of 3–300 ng/mL with a detection limit of 0.5 ng/mL for NS1 detection. The studies evalu-
NS1 biomarker ating possible interference with analog analytes are carried to demonstrate the specificity of the designed
Human serum sample immunosensor. The applicability of the method is also evaluated to monitor NS1 in spiked human serum samples
analysis. The designed sandwich-type immunosensor strategy integrating nano trace tag may find wide-spread
clinical applications for early and rapid detection of biomarkers.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction community of investigation and medicine are trying to develop new

The world is facing various severe diseases that particularly effected
the third-world developing countries [1,2]. However, one of the most
severe and commonly caused diseases is dengue, which is considered
to be an infectious viral disease caused by a flavivirus (belongs to
Flaviviridae family) transmitted from person to person caused by the fe-
male contaminated mosquitoes [3]. Infection with one of the four den-
gue serotypes DENV 1–4 leads to dengue fever (DF) [4,5]. DENV
specific NS1 is only secreted upon infection with DENV. The DENV par-
ticle has a diameter of 500 Å and comprised of a positive-sense RNA ge-
nome with ~10,700 nucleotides and 3 structural proteins: capsid (C, 100
amino acids), precursor membrane (prM, 75 amino acids), and enve-
lope (E, 495 amino acids). Similarly, DENV NS1 is a 48-kDa glycoprotein
which is specific to the infection of DENV [6,7]. However, the scientist
⁎ Corresponding author.
E-mail address: akhtarhayat@cuilahore.edu.pk (A. Hayat).
1
These authors contributed equally.
techniques to diagnose and medicate the dengue at early stages [8].
Few known symptoms of dengue include high fever, nausea, severe
headache, vomiting, and pain in the joints [9]. The death ratio occurred
due to dengue virus is quite high 22,000, terribly in children (World
Health Organization WHO) [1,2,10,11].
Additionally, the flavivirus NS1 is a glycoprotein and an important
immunogen were secreted in the blood stream from infected cells and
can be accumulated in dengue patients at concentrations up to
50 μg/mL [12]. Recent studies have shown that several dengue diagnos-
tic tests involve the detection of NS1 protein [2,13,14]. At present, sev-
eral analytical methodologies have been adopted including virus
isolation and cultivation followed by an immune fluorescence assay
[11,15], a window for virus culturing, RNA detection by reverse
transcription polymerase chain reaction (RT-PCR) [11,15,16] and mono-
clonal antibody capture-enzyme linked immunosorbent assay (MAC-
ELISA) [11]. However, some techniques lack sufficient sensitivity [17]
some may inherently rely on expensive instruments [18,19] and some
are involved with complicated and time-consuming processes such as
purification, pre-concentration, and traditional isolation procedures.

https://doi.org/10.1016/j.molliq.2020.114014
0167-7322/© 2020 Elsevier B.V. All rights reserved.
2 M. Awan et al. / Journal of Molecular Liquids 317 (2020) 114014
NS1 can be determined through multiple different routes. Most im-
portant of existing routes are enzymatic and nanomaterials based. Out
of these two ways, nanomaterials-based detection is gaining more in-
terest due to material science, analytical science, and biomedical appli-
cations [20–22]. While there are certain limitations associated with the
enzymes such as less stability, low shelf life, high cost, and delicate han-
dling. Therefore, most suitable and relevant nanomaterials are noble
metallic nanoparticles such as silver nanoparticles (Ag NPs), platinum
NPS, and gold NPs etc are compatible and unique nanoscale properties
among different kinds of nanomaterials [23–27]. These nanoparticles
can be easily functionalized with nucleic acid, proteins, and various spe-
cific compounds/molecules without changing any surface properties to
achieve selectively and enhance the signal-output properties for bio-
sensing purposes [28,29]. Specifically, Ag NPs have been adopted to
use directly in electrochemical detection where the lower oxidation po-
tential and facile conditions enhance their electrocatalytic efficiency.
Moreover, Ag NPs have a high extinction coefficient, large interfacial
surface, high thermal, and electrical conductivity. In this context,
Mehrgardi et al. have reported Ag NPs for the amplification of electro-
chemical detection of single base mismatch [30]. Similarly, Miao et al.
have employed melamine modified Ag NPs (M-Ag NPs) nano-probe
for the detection of clenbuterol not only possesses a wide detection
range from 10 pM to 100 nM and a low detection limit (10 pM) but
also has antijamming properties [31].
The present work focuses on the applicability of a novel electro-
acitve nanoprobe in the construct of electrochemical sandwithcc
immunosensor to generate the electrochemical signal. The binding
event of biorecpetors on the transducer surfaces are mainly probed
with the labeling of biomolecules or bioreceptors. In the same context,
redox active species are employed to generate the output signal. The
commonly used labels in the affinity based biosensors are enzyme
such as Horseredish peroxidase and Alkaline Phosphatase, with require-
ment of additional dyes or substrates. These enzyme labels are
expensive, unstable being protein in nature, can't stand with harsh ex-
perimental conditions and involve complex modification process
which may decrease the binding affinity. Thus in order to overcome
these issues, there is a recent trend on the synthesis of electroactive
nanoprobes for further application as labels in the affinity based biosen-
sors. Nano-labels are cheap, highly stable and can retain the affinity of
bioreceptors. This work is first report on the synthesis of NS1 specific
electroactive AgNPs probe with subsequent application in the design
of electrochemical sandwich immunosensor for NS1 detection. The
present work was a proof of concept study which can be easily extended
to probe any type of antigen-antibody interaction in sandwich format
for diverse types of applications. Initially, Ag NPs were characterized
by different spectroscopic techniques such as powder XRD (XRD) and
photoluminescence (PL). (Ab-M-Ag NPs) nano-probe was prepared
through Ag NPs modified with melamine and further functionalized
with antibody via the inherent interaction between the carboxylic acid
group of proteinic antibody and amine of melamine modified Ag-NPs
using EDC/NHS chemistry. Consequently, the immunosensor was
established by covalently immobilizing the capture antibodies on 4-
carboxy phenyl diazonium salt-modified pencil-graphite electrode.
2. Materials and methods
2.1. Reagents and apparatus
Potassium ferrocyanide (K4Fe(CN)6) (99.0%) and potassium ferricy-
anide (K3Fe(CN)6) (96.0%) were purchased from UNI-CDHEM. Mela-
mine (C3H6N6) was purchased from DAEJUNG. Hydrochloric acid (HCl
31.5–33.0%) was purchased from AnalaR. Monohydrate (MES) (98%),
N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyle)-N′-
ethyle-carbodiimide hydrochloride (EDC) were purchased from Alfa
Aesar. 4-Amino benzoic acid, sodium nitrite (NaNO3), sodium borohy-
dride (NaBH4), silver nitrate (AgNO3), Dengue NS1 antibody, Bovine
Serum albumin (BSA, MW 66 KDa), Fetal bovine serum (FBA), and
human serum (male) were purchased from Sigma-Aldrich, France.
Phosphate-Buffered Saline (PBS) was prepared in deionized water
from ELGA PURELAB Ultra water deionizer.
2.2. Instrumentation
All electrochemical measurements were performed on GAMRY
potentiostat interface 1010E (USA). Pencil graphite electrodes (PGE)
were used as a working electrode (0.5 mm diameter), platinum as a coun-
ter electrode, and Ag/AgCl as reference electrode during complete exper-
imentation. Scanning Electron Microscope (SEM) (TESCAN VEGA3),
Photoluminescence (PL), Fourier-transform infrared spectroscopy (FTIR)
and Raman spectroscopy were carried out to study surface morphology,
optical properties, vibrational modes and structural changes and presence
of different functional groups in the immunosensor, respectively.
The phase crystallinity of Ag NPs was studied by employing X-Ray
Diffraction (XRD).
2.3. Synthesis of Ag NPs and modification with antibody nano-probe
Ag NPs were synthesized by mixing 100 mL of trisodium citrate
(0.25 mM), AgNO3 (0.25 mM) and 3 mL of NaBH4 (10 mM) solution
under stirring for 30 min. NaBH4 acted as a reducing agent. Ag NPs
thus formed were left overnight and then purified by centrifugation at
12000 rpm for 30 min. Furthermore, to achieve Ag NPs-antibody conju-
gation, firstly melamine modified Ag NPs were prepared. For this pur-
pose, Ag NPs (1 mg) and melamine (0.1 mM) were dispersed in 1 mL
of distilled water and left for a period of 24 h at room temperature.
The excess amount of melamine was removed via centrifuge at
12000 rpm to obtained suitable melamine modified Ag NPs (M-Ag
NPs). Afterward, the antibody was conjugated with as-prepared M-Ag
NPs aided with EDC/NHS chemistry. Antibody conjugation was per-
formed by stirring 200 μL of antibody (1/100 dilution from stock solu-
tion) in the presence of NHS (25 mM) and EDC (100 mM) with
melamine modified Ag NPs in the 800 μL Phosphate Buffer Saline
(pH - 7.4) for 45 min. Subsequently, the supernatants were removed
via centrifuge at 12000 rpm to obtain the antibody conjugated Ag
NPs. The antibody-conjugated M-Ag NPs were used directly or stored
at 4 °C for further use. The Ab tagged AgNPs did not show any de-
crease in the analytical performance in term of electrochemical re-
sponse and antibody binding affinity over an extended period of
time. This indicates that Ab tagging with Ag NPs not only served
as a novel nanoprobe but also provided a way to stabilize and main-
tain the Ab affinity over an extended period of time, which are
otherwise prone to destabilization with loss of binding affinity,
being protein in nature.
2.4. Immobilization of antibody on electrode surface
Prior to the modification, the surface of the pencil graphite elec-
trode was cleaned by 10 cyclic potential scans between 1.0 to
−1.5 V in 0.5 M H2SO4 . The surface of the electrode was modified
with a diazonium mixture. The diazonium cation mixture was pre-
pared by in situ reaction of 2 μL of NaNO 3 (10 mM) and 1 mL of
2 mM 4-aminobezoic acid prepared in 0.5 M HCl. The mixture was
left to react for 5 min at room temperature. Afterward, the linear
sweep voltammetry was performed in a potential range of 0.6 to
−0.8 V to electrochemically modify the surface of electrode with 4-
carboxyphenyl diazonium salt. After surface modification, the elec-
trode was thoroughly rinsed with distilled water to remove the pos-
sible excess content. Furthermore, the carboxylic group has the
capability to make a complex with antibodies, therefore, the anti-
body was conjugated to the carboxylic group on the electrode sur-
face by incubating a mixture of 20 μL NHS (25 mM), EDC (100 mM)
and antibody (1/100 dilution from stock) on modified electrode
 M. Awan et al. / Journal of Molecular Liquids 317 (2020) 114014
surface for 1 h. The electrode was rinsed with water and 1 mM mel-
amine was used as anti-fouling reagent onto the electrode surface for
45 min to block the electrode surface to avoid nonspecific adsorp-
tion. After performing the washing step, antibody modified elec-
trodes were used directly or stored at 4 °C for an extended time to
serve as ready to use transducer surface.
2.5. Functional assay for NS1
The dengue biomarker was quantitatively analyzed by employing
various known concentrations of NS1 on the designed transducer
surface. The detection was carried out in the potential range of
−0.2–0.5 V at the scan rate of 100 mV/s. Differential pulse
volatammetry (DPV) was performed to probe the electrochemical
signal. The method permitted to detect NS in the linear range of
3–300 ng/mL of OTA with a limit of detection (LOD) 0.5 ng/mL. Elec-
trochemical response of Ag Nps was proportional to the concentra-
tion of NS1. Higher the concentration of NS1, more will be the
binding of Ag NPs tagged NS1 specific antibody, thus increasing the
electrochemical response.
Moreover, the selectivity of the designed biomarker was studied by
considering various interfering biomolecules including fetal bovine
serum (FBA), bovine serum albumin (BSA) and mucin. NS1 protein
shows a very high electrochemical current signal where interfering bio-
molecules have portrayed negligible response.
2.6. Real sample analysis
To validate the practical application of the proposed sensor, real
sample analysis was carried out. The concentration of NS1 was calcu-
lated according to the calibration curve. Human serum sample was
spiked with three different concentrations of NS1 (6.0, 75.0, and
250.0 ng/mL) to study matrix effect. The human serum samples
were analyzed with standard reference method prior to spiking of
the samples. A level of 1 ng/mL was observed in the samples which
was deducted both from the spiked and obtained values.
3. Results and discussion
3.1. Spectroscopic and photoluminescence characterization of Ag NPs
The surface morphology of Ag NPs coated on the surface of
conducting carbon tape as shown in Fig. 1(A & B). The nanoparticles
are presented in different amplications of 500 nm and 200 nm. The
morphology realized the same particle size distribution within a
range of 45–60 nm calculated with the help of Image J software
(mean value of 10 readings). Besides, to have an in–depth insight,
Raman analysis was performed using InVia Raman Microscope by
RENISHAW UK shown in Fig. 1B. Before Raman analysis, the samples
were directly deposited on glass slide via drop-casting. There is a
band stretching at 175 cm −1 of silver–oxygen mode (Ag\\O) due
to ionic species on the metal surface observed in spectra. The peak
at 1510 cm−1 is attributed to the amide Igroup. Moreover, the at-
tached functional groups with Ag NPs after modification are studied
employing FTIR recorded by Thermo Scientific Nicolet FTIR 6700 at a
scan range of 500–4000 cm −1 at a resolution of 256 cm−1. Fig. 1D
shows FTIR spectra of functionalized Ag NPs with five broad peaks
obtained in the spectra at 2231, 1737, 1618, 1323, 931.26 cm −1 .
The broad bump at range of 1700–1300 cm−1 is attributed to the car-
boxylic group. Carbonyl group and hydroxyl group has direct inter-
action by the formation of a stable hydrogen-bonded structure
[32]. The hydroxyl group OH remains on the adsorbed surface or
unreacted because in the oxidation some of it remains unreactive.
The (CH) stretching band peak at 2370 cm−1 confirmed the presence
of aldehyde groups in the sample. Therefore, FTIR spectra have
clearly inferred us for the successful modification of Ag NPs with
3
carboxy phenyl and other important functional groups. Furthermore,
the diffraction pattern of Ag NPs powder was studied through XPert
PANlytical Powder machine Cu Kα (l = 1.54 Å) radiation shown in
Fig. 1E. It shows that XRD patterns of Ag NPs confirming that
as-prepared Ag NPs have pure phase having no other secondary
phase with no detectable impurities. The major four characteristic
peaks for Ag were observed at 2θ = 39°, 44°, 64°, 77°, which were at-
tributed to (111), (200), (220), and (311) make sure the formation of
face-centered cubic structure of silver according to JCPDS Card Num-
ber 87-0717 [33].
The photoluminescence spectra of Ag NPs are recorded via InVia
Raman Microscope by RENISHAW UK depicted in Fig. 2. There are
two distinguished two bands centered at 490 and 530 nm observed
individually with different intensity and profile at high concentra-
tion. The emission wavelength of Ag was red-shifted as the loading
content of smaller luminescent silver nanoparticles increased from
492 to 527 (Fig. 2). The gradual red-shift and decrease of fluores-
cent emission intensity can be attributed to the interplay of radia-
tive and non-radiative energy transfer between luminescent Ag
NPs pair.
3.2. Cyclic voltammetry and Electrochemical Impedance Spectroscopy (EIS)
characterization
The electrode surface can be characterized using cyclic voltam-
mograms (CV) after each step of immunosensor fabrication, there
is a change in peak-to-peak separation and peak current after each
step of sensor construction which can be related to the electron
transfer rate constant and electron transfer resistance. Herein, we
have employed a CV as a marker to study electrode/electrolyte inter-
facial properties [34,35]. In general, the presented CV data at each
step in Fig. 3A (a–f) in redox medium 5 mM of [Fe(CN)6]3−/4− at a
scan rate of 100 mV/s.
The bare pencil electrode showed a quasi-reversible redox peak
having 0.8 V redox couple peaks with cathodic and anodic current
signals maintain the ratio of unity shown in Fig. 3A (a), while at
every step of fabrication of immunosensor, there was a change in
peak current and peak-to-peak separation observed. After the elec-
trode surface modification with carboxy phenyl, the anodic and ca-
thodic peaks were absent maintaining high resistance to the flow of
electron flow between deprotonated COO − ions and negative
charge on the redox probe (Fig. 3A (b)). Afterward, the subsequent
immobilization of electrode surface with antibody, anodic and ca-
thodic peaks appeared again because negatively charged carboxyl-
ate ions were neutralized due to formation of the ester amide bond
and barrier/resistance in electron transfer became relatively re-
duced (Fig. 3A (c)). Although steric hindrance due to bulky anti-
body molecule might offer some resistance in the transfer of
electron but former process appeared to dominate as was evi-
denced by relatively higher peak currents. Subsequently, the mod-
ification of the electrode was carried out via melamine in order to
block the non-specific cites and cover the remaining unused car-
boxylic group (COOH\\). Consequently, there is a prominent in-
crease in the anodic and cathodic peak currents generated shown
in Fig. 3A (d). This can be attributed to the conversion of NH 2 to
NH +3 shared additional electron as a result, the rate of electron
transfer will increase between electrode and redox probe. With
the subsequent immobilization of dengue virus NS1, there was a
dramatic decrease in anodic and cathodic peak current (Fig. 3A
(e)). This prominent change in the faradic current response after
the immobilization of analyte NS1 might be due to steric hindrance
because NS1 glycoprotein has a molecular weight of 40 kDa. Further
decrease in peak currents after immobilization of the nano-probe
(antibody modified Ag NPs) might be explained on the based on
steric hindrance caused by bulky antibody molecules (Fig. 3A (f)).
Similarly, a similar behavior to that of CV characterization can be
4 M. Awan et al. / Journal of Molecular Liquids 317 (2020) 114014
Fig. 1. Scanning electron microscope images (A–B); Raman spectra (C); FTIR spectra (D) and X-ray diffraction spectra (E) of Ag NPs.
observed in the EIS. The bare pencil electrode showed a small semi-
circle indicative of low electron transfer resistance (Fig. 3B a). The
electron transfer resistance significantly increased upon electrode
surface modification with carboxylic acid (Fig. 3B b) and then sub-
sequent immobilization of electrode surface with antibody, there
was a slight decrease in electron transfer resistance (Fig. 3B c). Fur-
thermore, the modification of the electrode was melamine in-
creased the flow of electrons and covered the additional unused
carboxylic group and blocked the non-specific sites which is clearly
observed in EIS spectra (Fig. 3B (d)). Afterward, the immobilization
of NS1, resulted in a clear increase in the electron transfer resis-
tance due to steric hindrance (Fig. 3B e).
To investigate the loading of AgNps on the surface of Ab, we moni-
tored the electrochemical response of AgNPs at various stages, bare
AgNPs, Melamine modified AgNPs and finally Ab tagged AgNPs. As can
Fig. 2. Photo Luminescence spectra of AgNPs.
be seen from the Fig. 4. Ab tagged AgNPs were characterized with a re-
markable electrochemical response which shows that modification pro-
cess did not alter the electro-activity of particles and retained a good
yield in term of initial concentration of AgNPs (1 mg/mL) at various
modification steps. The relative gradual decrease in electrochemical re-
sponse of tagged AgNPs can be linked to the physical hindrance caused
by the melamine and Ab.
3.3. Quantitative analysis of NS1 based on DPV signal output of electro-
active nanolabel
The process of sandwich-type electrochemical immunosensor in-
volving Ag NPs-linked antibodies as electroactive nanoprobe and
electrode-functionalized capture dengue NS1 antibodies as a transducer
surface is depicted in Scheme 1. Through a sandwich-type immunoreac-
tion, the antibody functionalized Ag NPs were captured on the
immunosensor surface by the formation of immunocomplex. Higher
the concentration of analyte NS1, the greater will be the amount of Ag
NPs captured on the sensor surface. DPV was used to probe the electro-
chemical response of Ag NPs on immunosensor surface. The generated
current was proportional to the concentration of NS1 and permitted
quantitative detection of the analyte.
3.4. Optimization of detection conditions
The electrochemical and analytical performance of an immunosensor
is influenced by concentration and incubation time of antibody be-
cause these parameters influence the selectivity and sensitivity of
immunosensor. Therefore, before performing the detection of a spe-
cific analyte, there is a need of optimal conditions at every step of de-
signing immunosensor. In this regard, lower concentration of
antibody could compromise the sensitivity owing to less available
recognition site and in case of higher concentration of antibody;
bulky antibody molecules could cause blockage of electrode surface
and steric hindrance thereby reducing the sensitivity of the device.
Hence, to determine the optimal concentration of antibody stripping
 M. Awan et al. / Journal of Molecular Liquids 317 (2020) 114014
Fig. 3. (A) Cyclic voltammograms (B) Electrochemical Impedance spectroscopy of Nyquist plots of 5 mM [Fe(CN)6]3−/4− at scan rate of 100 mV/s for; (a) bare Pencil Electrode, (b) COOH
modified electrode, (c) Antibody modified electrode, (d) Melamine, (e) NS1 modified electrode, and (f) AgNPs modified antibody.
voltammetric response of various concentrations of antibody (0.1,
0.2, 0.3, 0.4, and 0.5 U/mL) were involved in Fig. S1B. The peak cur-
rent increases with an increase in antibody concentration until it
trended up to a reasonable concentration of 0.5 U/mL. After the opti-
mization of antibody concentration, the incubation time for antibody
and analyte NS1 were optimized by considering different time inter-
vals (05, 15, 30, 45 and 60 min). Fig. S1A shows an electrochemical
response plot for antibody incubation and current response against
various time intervals. It can be observed that the peak current in-
creases with an increase in incubation time of antibody and after
30 min of incubation there are same straight trends that occurred
with a constant value of current. Therefore, an optimum incubation
time for the antibody of 30 min. Thus, the incubation time and con-
centrations of the antibody are important parameters, which influ-
enced the performance of immunosensor. Similarly, the incubation
time of analyte NS1 is important parameter acquired to be opti-
mized, therefore, electrochemical signals were recorded at different
time intervals (05, 15, 30, 45 and 60 min) with optimized incubation
60 min shown in Fig. S1C.
Fig. 4. DPV at three steps of pure Ag NPs (a); Ag NPs tagged melamine (b); and Ag NPs-
melamine tagged Ab (c).
5
3.5. Analytical performance
The successful integration of nano-probe in the immunoreaction on
the sandwich-type immunocomplex was recorded through DPV. The
quantitatively captured Ag NPs on the immunosensor could easily be
detected by DPV analysis. The basic mechanism of detection of NS1 in-
volves following steps to design a sandwiched immunosensor 1) surface
modification of electrode with NS specific antibody; 2) melamine mod-
ification to serve as blocking agent; 3) addition of various concentra-
tions of NS1; 4) incubation of nano-probe to sandwich the analyte.
NS1 in the following concentration range 3.14, 6.54, 8.9, 12.23, 15.18,
23.78, 32.15, 48, 60.32, and 86.8 ng/mL was employed to perform the
quantitative measurements. A proportional increase in the faradic cur-
rent for the oxidation of silver was observed with an increase in analyte
NS1 concentration and which was calculated to construct calibration
plot for the detection of NS1. The analytical optimized conditions
for various parameters were used condit to obtain the calibration
curve with the help of DPV. The obtained limit of detection was
0.55 ng/mL, with a linear range of 3–300 ng/mL for NS1 estimated
from signal to noise ratio depicted in Fig. 5(A & B). It is known that
after the upper limit of 300 ng/mL, saturation was achieved and re-
sponse of no more linear. The linear range can be further extended
by increasing the concentration of immobilized NS1 specific anti-
body, however this will increase the LoD of sensors. As we know
that higher concentration of capturing immobilized baroreceptor
provide wider linear range with high limit of detection, while
lower concentration is characterized with relatively narrow linear
range with low LoD.
3.6. Evaluation of cross-reactivity/interfering analysis
The important section of designing a biomarker is indeed the con-
firmation the biomarker response towards other analogous biomole-
cules, preventing correct medical treatment for the infected patients
[32,33]. Therefore, the selectivity and specificity study of analyte
during the fabrication of an immunosensor device is of utmost im-
portance to validate the proposed methodology. For this purpose,
several analog analytes such as BSA, FBS, and Mucin 1 were evaluated
for cross-reactivity using the same strategy as in the case of NS1. It
can be evidenced from Fig. 6 that there was a negligible response of
the immunosensor for all these analytes even at much higher
6 M. Awan et al. / Journal of Molecular Liquids 317 (2020) 114014

Scheme 1. (A) Preparation of AgNPs modified Antibody (B) Construct of electrochemical sandwich immunosensor for diagnosis of dengue biomarker.

concentrations as compared to NS1. These experiments proved that
these analytes could not cause observable interference and indicate
higher selectivity of the designed immunosensor for the NS1
detection.
3.7. Immunosensor performance for real sample analysis
To evaluate the practical application potential and analytical reliabil-
ity of the developed sensor, real sample analysis was carried out. The
performance of the proposed strategy was evaluated using the above
optimal assay protocol and three different concentrations of NS1 (6.0,
75.0, and 250.0 ng/mL) were spiked into serum samples. The concentra-
tion of NS1 was calculated according to the calibration curve. The results
of recovery percentages 96.6–97.3% are appreciable and acceptable pre-
sented in Table 1 [7]. There are less error than 2.7–3.5% with negligible
standard deviation.
The objective of present work was to demonstrate the applicability
of novel electro-acitve nanoprobe in the construct of electrochemical
sandwich immunosensor to generate the electrochemical signal. This
nanoprobe has eliminated the need of commonly used signal labels
such as enzyme, redox active species etc. Furthermore, as can be seen
from the cross reactivity and recovery studies, our proposed sensor
was able to detect NS1 with good recovery values in the serum samples
without any interference, thus demonstrating the potential for real time
applicability of the proposed immunosensor. This infers us that utilizing
such a novel strategy incorporating nano-probe in the design of

Fig. 5. (A) Voltammograms for different concentrations of analyte in PBS 7.4 pH: (a) 3.14, Fig. 6. Interference studies of different analytes signifying the selectivity of the sandwich
(b) 6.54, (c) 8.9, (d) 12.23, (e) 15.18, (f) 23.78, (g) 32.15, (h) 48, (i) 60.32, (j) 86.8 ng/mL immunosensor towards NS1 with the same amount of all interfering analytes as that of
and (B) Calibration curve for NS1 concentrations. NS1 (490 ng/mL) and 45 min incubation time.
 M. Awan et al. / Journal of Molecular Liquids 317 (2020) 114014 7

Table 1 Acknowledgment
Recovery percentages obtained with designed electrochemical immunosensor.

NS 1 added (ng/mL) NS 1found (ng/mL) R.S.D % R.E %
5.8 3.8 3.4
6 PERIDOT/2016.
73 4.4 2.7
75
242 4.9 3.2
250
R.S.D % = relative standard deviation percentage; R.E % = relative error percentage; R% =
recovery percentage.
sandwiched immunosensor for the detection of NS1 has presented a
new path for successful progress.
It is important to mention the ranking of the present immunosensor
with already known sensing techniques. Comparison with literature
showed that the present immunosensor finds a prominent position
among dengue detection protocols except for a few research reports
as is reflected in Table 2.
4. Conclusion
This sandwich-type immunosensor showed an excellent perfor-
mance towards the detection of NS1 protein. The developed strategy
have high specificity and very low limit of detection (0.5 ng/mL). An
antibody functionalized- silver-nanoparticles (Ag NPs) were de-
signed as a trace tag for dengue biomarker using a disposable
immunosensor. Electrochemical signals by the oxidation of Ag NPs
were translated into a highly specific tool for the detection of NS1.
The possible cross-reactivity with analog analytes did not cause ob-
servable interference, making this strategy highly selective for NS1
detection. The NS1 detection experiments with human serum sam-
ples showed a negligible error of less than 2.7–3.5%, showing a
good accuracy of the developed method. The obtained results also
showed that the designed sandwich-type immunosensor strategy
with extra-ordinary selectivity and sensitivity may find potential in
clinical applications in early and rapid detection of dengue virus.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.molliq.2020.114014.
Declaration of competing interest
There are no conflicts of interest to declare.
R% This work was carried out as a part of the project by HEC (Higher Ed-
96.6
ucation Commission), Pakistan and PERIDOT, France; No. 2-3/HEC/R&D/
97.3
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Table 2
Comparison of present sandwich type immunosensor with other reported NS1 based dengue biosensors.

Sr. Type of sensor Method Linear range LOD Ref

no. (ng/mL) (ng/mL)

1 Carbon nanotube-ink printed electrode CV 40–2000 12 [36]

2 Streptavidin/biotin system on screen printed carbon electrodes CV 500–2000 30 [37]
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7 An electrochemical lateral flow immunosensor developed by combining screen-printed gold electrodes (SPGE) EIS and CV 1–25 0.5 [5]
8 Electrochemical peptide sensor EIS, CV 25–3500 25 [42]
9 Dual marker label-free electrochemical assay EIS, CV 1–5000 0.340 [34]
10 Carbon nanotube deposits on electrodes EIS, CV 10,000–0.001 0.001 [35]
14 SPGE surface-modified AuNPs–PEG–Ab–TEMPO CV 0–1000 50 [43]
15 BSA modified SPCE EIS 1–200 0.3 [44]
16 Compact-disc chip-based electrode CV, EIS 1–100 0.33 [45]
17 Chitosan modified carbon fiber electrode CV 1–175 0.94 [46]
18 Antibody-Ag NPs as trace tag using sandwich-type immunosensor using Pencil graphite electrode DPV 3–300 0.5 Present
work

CV. Cyclic Voltamogramms, EIS. Electrochemical Impedance Spectroscopy, OCP. Open Circuit Potential, DPV. Differential Pulse Voltammetry.
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