The development of an ultra-sensitive electrochemical immunosensor using a PPyr-NHS functionalized disposable ITO sheet for the detection of interleukin 6 in real human serums

Article abstract of DOI:10.1039/d0nj03183f

A label-free impedimetric immunosensor based on a conjugated poly(pyrrole N-hydroxy succinimide) (PPyr-NHS) polymer covered disposable indium tin oxide electrode (ITO) was fabricated for the ultrasensitive determination of interleukin 6 (IL 6) antigen. In this sensing platform, the PPyr-NHS polymer, which carried a number of succinimide groups on its end side, was used as a matrix material for the first time. This synthesized polymer had excellent biocompatibility and good electrical conductivity. In addition, the utilization of this material as a matrix material provided direct immobilization of the IL 6 receptor (IL6R), which was employed as a biorecognition element. The preparation of the working electrode, the successful attachment of IL 6R and specific interaction between IL6R and IL 6 antigen were confirmed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. Moreover, the working electrode surface was characterized by Scanning electron microscopy (SEM), Atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy during its construction. An impedimetric method was employed for the quantification of IL 6 antigen. Under optimized experimental conditions, the designed sensing platform could detect IL 6 antigen with a wide detection range from 0.03 pg mL-1 to 22.5 pg mL-1 with a relatively low detection limit of 10.2 fg mL-1. In addition, the developed biosensor had outstanding sensitivity and specificity, good repeatability and reproducibility, high stability and reusability. Additionally, the designed sensing tool was successfully used in human serum samples. Consequently, the suggested immunosensor was clinically useful in the early detection of prostate cancer by direct determination of the serum IL 6 antigen level after simple dilution.

Full text of DOI:10.1039/d0nj03183f

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View Article Online
DOI: 10.1039/D0NJ03183F
Development of an ultra-sensitive electrochemical immunosensor
using PPyr-NHS functionalized disposable ITO sheet for detection
of interleukin 6 in real human serums
Received 00th January 20xx,
Accepted 00th January 20xx
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Elif Burcu Aydına*, Muhammet Aydına, Mustafa Kemal Sezgintürkb
DOI: 10.1039/x0xx00000x
www.rsc.org/
A label-free impedimetric immunosensor based on conjugated poly(pyrrole N-hydroxy succinimide) (PPyr-NHS) polymer
covered disposable indium tin oxide electrode (ITO) was fabricated for ultrasensitive determination of interleukin 6 (IL 6)
antigen. In this sensing platform, PPyr-NHS polymer, which carried a number of succinimide groups on its end side, was used
as a matrix material for the first time. This synthesized polymer had excellent biocompatibility and good electrical
conductivity. In addition, the utilization of this material as a matrix material provided direct immobilization of IL 6 receptor
(IL6R), which was employed as a biorecognition element. The preparation of working electrode, the successful attachment
of IL 6R and specific interaction between IL6R and IL 6 antigen were confirmed by electrochemical impedance spectroscopy
(EIS) and cyclic voltammetry (CV) techniques. Moreover, the working electrode surface was characterized by Scanning
electron microscopy (SEM), Atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and Raman
spectroscopy during its construction. Impedimetric method was employed for quantification of IL 6 antigen. Under
optimizing experimental conditions, the designed sensing platform could detect IL 6 antigen with a wide detection range
from 0.03 pg/mL to 22.5 pg/mL with a relatively low detection limit of 10.2 fg/mL. In addition, the developed biosensor had
outstanding sensitivity and specificity, good repeatability and reproducibility, high stability and reusability. Additionally, the
designed sensing tool was successfully used in human serum samples. Consequently, the suggested immunosensor was
clinically useful in the early detection of the prostate cancer by direct determination of the serum IL 6 antigen level after
simple dilution.
operation, rapid response, low-cost and compatibility with
minizaturation technology 9-11
.
Introduction
Interleukin 6 (IL 6) is a pro-inflammatory cytokine, which has a
significant role in immunity. It stimulates growth and differentiation
of B cells, secretion of immunoglobulins, differentiation and
activation of T cells and macrophages 12. In addition, it acts as an
anti-inflammatory myokine in the inflammatory process 13. IL 6 is a
circulating protein, which is present in healthy human plasma
around 6 pg/mL. The overexpression of IL 6 is a sign of tumor genesis,
tumor growth, and cancer cell differentiation14. The high
concentrations of IL 6 biomarker are related to the prostate cancer
15. Until now different analytical methods including bioassays,
immunoassays, molecular biology techniques and flow cytometry
have been used for IL 6 detection12, 15, 16. Among them, enzyme-
linked immunosorbent assay (ELISA) is the most common method.
The sensitivity of ELISA is high, but it suffers from some
interferences. Because of this, new approaches, which can increase
the sensitivity and selectivity of the analytical techniques should be
found to detect some biomarkers found at ultra-low levels in the
earlier diagnosis of prostate cancer 17. Different studies for IL 6
biomarker detection have been reported in literature. Wang et al.
(2010) constructed an amperometric immunosensor based on
polydopamine-gold nanoparticles (AuNPs) modified glassy carbon
electrode (GCE), in which the linear detection range and detection
limit were found as 4-800 pg/mL and 10 pg/mL, respectively 18. Liu
et al. (2013) manufactured an electrochemiluminescence biosensor
based on graphene oxide/polyaniline nanowires/CdSe quantum dots
nanocomposite modified GCE, and the detection limit of this
biosensing system was found as 0.17 pg/mL 17. A sensitive
aptasensor was fabricated by Tertis et al. (2019) by using p-
aminobenzoic acid, p-aminothiophenol and AuNPs modified GCE. A
thiol-terminated aptamer, which was specific to IL 6 antigen was
utilized as biorecognition element and the immunosensor displayed
Cancer is one of the leading causes of death 1, with prostate cancer
being one of the most common type in man globally and its early
detection is the key to the high potential, easy, low-cost and
effective therapy 2. Several potential biomarkers such as protein
biomarkers, circulating tumor cells, circulating tumor DNA and cell
fragments are found in blood and utilized for disease diagnosis
3, 4
.
The usage of cancer biomarkers in disease diagnosis provides early
detection of cancer and increases the survival rate of patients 5 . To
date, several methods such as enzyme-linked Immunoassay (ELISA),
radioimmunoassay and fluorescence immunoassay methods have
been reported for cancer biomarkers detection. The traditional
ELISA is one of the most preferred method for quantification of
biomarkers in biological fluids based on spectrophotometric reading.
This method offers reproducible, sensitive, specific and reliable
results that makes it a suitable tool in scientific research and clinical
diagnosis. Besides these advantages, it has some disadvantages such
as time–consuming and tedious procedure, the short shelf life of the
labeled antibody, excess reagent consumption, high cost and the
narrow detection range6. Additionally, the detection limit of classic
ELISA method is barely less than the nanomolar concentration level,
which is insufficient to obtain the clinical threshold of many protein
biomarkers, which are important in the early stage of diseases7.
Furthermore, the ELISA method cannot be reused for many
measurements and its regeneration is not possible8. Taking into
account all of these disadvantages, electrochemical strategies have
gained interest owing to their intrinsic properties such as simple
a. Namık Kemal University, Scientific and Technological Research Center, Tekirdağ-
TURKEY. E-mail: ebbahadir@nku.edu.tr; Tel: +90 282 250 1137
b. Çanakkale Onsekiz Mart University, Faculty of Engineering, Bioengineering
Department, Çanakkale-TURKEY
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high sensitivity, wide linear detection range (5 pg/mL-100 ng/mL)
and ultra-low detection limit (1.6 pg/mL) 19
performed in this research was approved by the ethical committee
of non-invasive clinical researches of TDeOkiIr:d1a0ğ.103N9a/mD0ıkNJ0K3e1m83aFl
University (Ethic committee approval number 2013/86/07/05).
View Article Online
.
Indium tin oxide (ITO) is a conductive transparent oxide material and
ITO film coated substrates have been utilized as an electrode
platform for biosensors. The advantages of ITO substrate as working
electrode are high stability, ease of combination with electrode
fabrication process and lower cost than gold and platinum
electrodes 20. Immobilization of biorecognition molecules on the
Apparatus and measurements
Fourier transform infrared (FTIR) spectrum was collected Bruker
Company Vertex 70 spectrometer. Raman spectrum was recorded
with a Thermo Company DXR Raman spectrometer (780-nm
excitation laser). Proton Nuclear Magnetic Resonance spectra was
carried out via Bruker Avance II (400 MHz) using CDCl3 deuterated
solvents. Electrochemical measurements (EIS and CV) were
conducted on a Gamry workstation with a conventional three
electrode system composed of a platinum wire as a counter, Ag/AgCl
as a reference and an ITO sheet as a working electrode, respectively.
EIS analyses were performed in [Fe(CN)6]3−/4− solution within the
frequency range from 0.5 Hz to 50 kHz. CV analyses were performed
in [Fe(CN)6]3−/4− solution at the range of −0.5 V - +1 V at a scan rate
of 100 mVs−1. Electrochemical impedance measurements were
performed by applying an alternating potential of 5 mV to the
working electrode. The formal potential applied in the impedance
studies was 0 V. Scanning electron microscope (SEM) images and
energy dispersive X-ray (SEM-EDX) analysis were taken using Field
Emission Scanning Electron Microscope QUANTA FEG-250. Atomic
force microscopy (AFM) images were performed on an ambient AFM
Nano-Magnetic Instrument in dynamic AFM mode. The scan rate
was 5 µm/s with a resolution of 256 pixels per line. Three-
dimensional (3D) AFM images were prepared via Nano-Magnetics
image analyzer software.
working electrode is a crucial step in the designing of biosensors 21
.
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The physical and chemical features of the matrix materials utilized in
the fabrication of the biosensors have an important effect on their
sensitivity and stability 22. In order to increase the detection
performances of biosensors for determination of cancer biomarkers,
more and more electrochemical biosensors have been developed
and fabricated 23-25. In recent years, conjugated polymers are
introduced to modify the electrode surface due to their excellent
physical and chemical features 26, 27. They can transfer the electron
produced during the biochemical reaction and they are suitable
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matrices for biorecognition element immobilization
.
Poly(thiophene), poly(pyrrole) and poly(aniline) are traditional
conjugated polymers and up to now different conducting polymers
have been synthesized and utilized in the biosensor development 29-
32. Poly(pyrrole) is extensively studied matrix material because of its
advantages in biosensors development. Electrical conductivity, easy
functionalization, biocompatibility and stability make this material
an excellent component for electrochemical biosensor fabrication
14. In this study, PPyr-NHS polymer was synthesized and polymerized
for the first time to use as a suitable matrix for biorecognition
molecule immobilization and the developed immunosensor was
successfully applied to cancer biomarker monitoring in human sera.
This polymer had not only succinimide groups to anchor IL6R
through amide bound, but also it exhibited good conductivity. In
addition, the usage of PPyr-NHS polymer as a biosensor matrix
material provided a biocompatible surface for IL6R and did not
require the NHS/EDC activation step and IL6R covalently bound to
polymer modified ITO surface.
Synthesis of Monomer and polymer
Synthesis of acid-substituted Pyrrole Monomer (Pyr-Pac)
The starting monomer for the present synthesis was the known
compound N-pyrrolylpropanoic acid (Pyr-Pac), which was prepared
from 1-pyrrolepropionitrile. The synthesis was performed using a
modified version of the protocol of Khan et al. (2007)33 for the
conversion of the nitrile group in 1-pyrrolepropionitrile. In short, 2.5
g (2.08 mmol) of 1-pyrrolepropionitrile was mixed with 15 mL
potassium hydroxide solution (5 g, 8.9 mmol) and they were heated
and stirred at 1000 C under a reflux of dry nitrogen for overnight. The
obtained product was acidified to pH 4 with 8 M HCl and it was
extracted six times with diethyl ether. At the end of this process, the
organic phase was dried over anhydrous MgSO4 and solvent was
evaporated. Thus, pure product was produced as a beige crystalline.
(Yield: %87, 2.01g). FTIR (cm-1): 3500-3100; 2940-2884;
1695(carbonyl); 1600-1450; 1216; 931; 787, 724; 647; 613; 500(Fig.
SI-2B). Raman (λlaser=780 nm): 1710 (C=O); 1570; 1462; 1322;
Experimental
Chemicals and material
Unless otherwise noted, materials and solvents were obtained from
commercial suppliers and used without further purification. 1-
pyrrolepropionitrile (Pyr-CN), potassium bromide (KBr), potassium
hydroxide (KOH), 4-dimethylamino pyridine (DMAP), anhydrous
iron(III) chloride (FeCl3), N-Hydroxysuccinimide (NHS), 1-ethyl-3-
(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC),
nitromethane (CH3NO2), chloroform (CHCl3), tetrahydrofuran (THF),
hydrochloric acid (HCl), dimethylsulfoxide (DMSO), acetone, sulfuric
acid (H2SO4), potassium chloride (KCl), potassium dihydrogen
phosphate (KH2PO4), dipotassium phosphate (K2HPO4), potassium
ferrocyanide (K4[Fe(CN)6]) and potassium ferricyanide (K3[Fe(CN)6])
were from Sigma-Aldrich (USA). ITO coated polyethylene
terephthalate film (0.5 cm x 2 cm), human IL6R, human IL 6 antigen
and bovine serum albumin (BSA) were purchased from Sigma-
Aldrich (USA). IL 6R, IL 6 antigen and BSA solutions were prepared by
a pH 7.4 phosphate buffer solution (PBS, 50 mM containing 1 M KCl).
Interleukin 1α (IL 1α), p53 antigen (p53), neuron specific enolase
(NSE) and tumor necrosis factor α (TNF α) were selected as
interferences and they were purchased from Sigma-Aldrich (USA).
The national guideline stated in the National regulations on clinical
trials published on the issue of government gazette (Number: 28030,
August 19, 2011) was followed. Moreover, the experiments
1
1145;1167; 932; 780; 695; 653; 620(Fig. SI-3A). H-NMR (δH, ppm):
11.6 (Ha, 1H); 2.8 (Hb, 1H); 4.2 (Hc, 2H); 6.7 (Hd, 2H) and 6.2 (He, 2H)
(Fig. SI-4).
Synthesis of succinimide substituted Pyrrole Monomer (Pyr-NHS)
Adapting a literature procedure, we used the Steglich esterification
of Pyr-Pac with N-Hydroxysuccinimide. 34, 35. Briefly, Pyr-Pac (0.7 g, 5
mmol), DMAP (0.056 g, 0.5 mmol), EDC (1.917 g, 10 mmol) and NHS
(0.69 g, 6 mmol) were solved in dry tetrahydrofuran and were stirred
at room temperature under argon flow for 48 hours. After that,
reaction solvent was evaporated, and the organic layer in diethyl
ether was washed repeatedly with ultrapure water. Finally, the
product was dried over anhydrous magnesium sulfate and
concentrated in rotary evaporation. (Yield:1.32g, 56%). FTIR (cm-1):
2940-2926 (CH); 1810, 1776, 1732(C=O); 1201; 1060; 890; 807; 717;
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641 (Fig. SI-2C). Raman (λlaser=780 nm): 1816, 1779 (C=O); 1286;
1090; 995; 898; 815; 730; 665 (Fig. SI-3B).1H-NMR (δH, ppm): 6.17
ppm (Ha, 2H), 6.70 ppm (Hb, 2H), 4.31 ppm (Hc, 2H), 3.08 ppm (Hd,
2H) ve 2.85 ppm (He, 4H) (Fig. SI-5).
BSA molecules. In conclusion, the prepared ITO electrode was ready
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to measure of IL 6 antigen. After every step above mentioned, the
DOI: 10.1039/D0NJ03183F
modified electrodes were characterized by using EIS and CV
methods. The whole fabrication process is illustrated in Scheme 1.
Synthesis of N-Succinimidyl Ester Polypyrrole (PPyr-NHS)
Practical applicability of the IL 6 biosensor
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The succinimide functional group substituted polymer (PPyr-NHS)
was prepared by using the previously reported oxidative
polymerization method with minor adaptation 36. The monomer Pyr-
NHS (0.236 g, 1 mmol) was solved in chloroform (10 mL) in a two-
necked round bottom flask and it placed in an ice-salt bath and a
solution of 0.65 g (4 mmol) of anhydrous iron chloride (FeCl3) in 3 mL
of nitro methane was added dropwise over 20 min. After stirring for
100 min at room temperature under argon gas, the black color
reaction mixture was concentrated and precipitated in cold
methanol. Then, dark brown precipitate was obtained by filtration
and it was rinsed with ultrapure water and methanol. Finally, PPyr-
NHS was dried using vacuum oven. (Yield: 0.32g, 45%). FTIR (cm-1):
2958, 2933(C–H); 1813, 1781, 1733 (C=0); 1421; 1362; 1201; 1065;
997; 807; 646 (Fig. SI-2D). Raman (λlaser=780 nm): 1815, 1780 (C=O);
1576; 1330; 1130; 1019; 990; 825 668 (Fig. SI-3C).
The constructed biosensor was utilized for determination of IL 6 in
order to verify its practical applicability. The IL 6 antigen level in the
human sera was measured by the designed immunosensor, and
standard addition method was used to examine the accuracy of the
immunosensor.
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Results and Discussion
In this work, PPyr-NHS polymer modified IL 6 immunosensor was
constructed by simple 3 preparation steps. The construction strategy
of the label-free IL 6 immunosensor is depicted in Scheme 1. Since
PPyr-NHS polymer had free succinimide groups, it was easily used to
immobilize the IL6R biomolecules (Step 1). The free succinimide
groups were blocked by BSA (Step 2) and lastly, the biorecognition
event formation between IL6R and IL 6 antigen (Step 3).
For the synthesis of succinimide functional group substituted
poly(pyrrole) polymer, we needed a series of reactions which were
performed in three steps. The synthetic route of the PPyr-NHS
polymer is illustrated in Scheme 1. The first step was the conversion
reaction of nitrile group to acid group. In the second step, the
steglich esterification of N-pyrrolylpropanoic acid with N-
hydroxysuccinimide was utilized. In the final step, the succinimide
substituted pyrrole monomer (Pyr-NHS) was polymerized via
chemical oxidative coupling technique with anhydrous ferric
chloride. The chemical structures of monomer and polymer were
investigated by several spectral methods (FTIR and 1H NMR and
Raman spectroscopy) to indicate the success of the synthesis
method and results were illustrated in detail in the Supp. Inf. file.
Construction of the biosensing platform
Prior to use, the bare ITO electrode was successively rinsed in an
ultrasonic cleaner with ethanol, soap solution and ultrapure water,
and dried under an argon flow. Then, 10 µL of synthesized PPyr-NHS
polymer (0.24 mg/mL) was dropped on the ITO electrode and a thin
film was formed on the ITO electrode by using a spin coater.
Subsequently, the biorecognition element IL 6 Receptor was
accomplished by dipping PPyr-NHS modified ITO substrate into 100
µL PBS containing IL 6 Receptor (3 ng/mL) for 1 hour. After covalent
attachment, the modified ITO electrode was incubated in BSA (0.5%)
solution to block the free succinimide groups. Then, the ITO
electrode was rinsed with ultrapure water to remove unattached
Scheme 1. Schematic illustration of the composite preparation procedure and the fabrication process of the immunosensor.
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Chemical characterization of PPyr-NHS polymer coated ITO
electrode and IL 6R attached ITO electrode
Fig. 1. FTIR measurements of PPyr-NHS coated ITO electrode (A)
and IL 6R attached ITO electrode (B), Raman measurements of
PPyr-NHS coated ITO electrode (C) and IL 6R attached ITO
electrode (D).
The FTIR spectra of PPyr-NHS polymer (A) and IL6R modified ITO
electrode (B) are illustrated in Fig 1. As shown in Fig. 1A, , the FT-IR
spectra for polymer modified electrode showed two strong bands at
1814 cm-1 and 1782 cm-1 that proved characteristic carbonyl groups
of the NHS ester 37, 38. The strong peak at 1730 cm-1 was the evidence
of the typical C=O stretching vibration of carbonyl groups 39. When
two FTIR spectra were analyzed, in the FTIR spectra of the Fig 1B, the
disappearance of characteristic carbonyl bands of the NHS ester and
new absorption bands at 1652 cm-1 (amid 1) and 1551 cm-1 (amid 2)
proved the covalent bond formation of between succinimide groups
of polymer and amino group of receptor (Scheme 1). The presence
of polymer on the electrode surface and chemical interactions
between polymer succinimide groups and amino groups of receptors
were also examined with Raman spectroscopy.
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Electrochemical characterization of the immunosensing surface
EIS was utilized to characterize the fabrication process as it was one
of the most powerful tools to investigate the interfacial property of
the electrode surface. The impedance analysis results were fitted to
an equivalent circuit (inset 1E), which was composed of the
electrolyte solution resistance (Rs), the surface electron transfer
resistance (Rct), the Warburg impedance (Zw) and the constant phase
element (CPE) 42-44. Fig. 2A and 2B show the EIS and CV obtained in
ferri/ferrocyanide solution (5 mM) containing 1M KCl at different
electrode surface. The PPyr-NHS modified ITO electrode had a low
Rct because of the conductive property of PPyr-NHS polymer.
Compared to polymer modified ITO electrode, the IL 6R immobilized
ITO electrode displayed increased Rct because of the successful
immobilization of IL 6R. The IL 6R immobilization reduced the
conductivity of the ITO surface and obstructed the electron transfer.
Surface blocking with BSA led to a further increment in Rct due to the
insulativity of BSA protein. The specific biointeraction between IL6R
and IL 6 antigen caused a protein layer coating that prevented the
electron transfer. Moreover, the immunosensor was characterized
by CV measurements, which provided valuable information about
the variation formed on the modified electrode surface. The polymer
coated ITO surface had high peak currents with enhanced electron
transfer. After IL6R attachment onto the ITO surface, the peak
currents decreased significantly due to non-conductivity of protein
molecules. The BSA blockage step caused a hindrance effect to
electron transfer because of non-conductive property of BSA
molecules. When ITO surface was dipped in IL 6 antigen solution, the
redox peak currents of ferri-ferro were decreased due to capturing
of target IL 6 antigen by IL 6R. The results were well consistent with
EIS responses, confirming the successful preparation of the
immunosensor.
Raman spectroscopy can give knowledge about the specific
interactions in polymer blends, nucleic acids and large molecules.
The one of the biggest advantages of Raman spectroscopy is the ease
of sample preparation. Moreover, the relative intensity variation
and the shift of Raman band proved the chemical interaction
between polymer and receptors. The Raman spectra of PPyr-NHS
modified electrode before IL6R immobilization (red line) and after
IL6R immobilization (purple line) are displayed in Fig. 1C and 1D,
respectively. The Raman spectra in Fig. 1C demonstrated that
stretching vibrations of carbonyl bonds in succinimide groups were
monitored at 1815 cm-1 and 1770 cm-140. Amide I, amide II and amide
III bond bands of protein structure are usually monitored mostly
between 1650–1680 cm-1, 1480-1570 cm-1 and 1235-1300 cm-1,
respectively41. The peaks of 1656, 1530 and 1329 cm-1 were
attributed characteristic peaks in IL 6R attached electrode surface.
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The surface topography of the IL 6 immunosenVsioewr AfortircleeOacnlhine
construction stage was examined by SEM DaOndI: 1A0F.1M03.9F/iDg0. N3JA03a1n8d3F
3B show SEM-EDX images of bare ITO substrate and PPyr-NHS
polymer modified ITO substrate. EDX images were taken
simultaneously with the SEM shown in Fig. 3C. The spectrum of
bare ITO electrode shows the peaks that indium, tin, oxygen and
carbon elements (Fig. 3A). After spin coating of polymer on the
ITO substrate surface, the spectrum shows carbon, nitrogen,
oxygen, indium, tin peaks. The peak of “N” element belongs to
polymer, which has successfully coated on the bare ITO substrate
surface (Fig. 3B). As illustrated in Fig. 3C, PPyr-NHS polymer was
uniformly covered on the ITO substrate and the average
roughness (Ra) was measured as 17.8 nm over 5 x 5 µm2 area (Fig.
3D). After attachment of IL 6R on the polymer modified electrode,
the SEM and AFM images illustrated that round-shaped
macromolecules were immobilized on the electrode surface (Fig.
3E). The capturing of IL 6R molecules caused an increase in Ra
value and it was measured as 48.1 nm over 5 x 5 µm2 area (Fig.
3F). The changes on the ITO surface illustrated that the
modification of the electrode surface was obtained through the
covalent binding. After BSA blockage step, a smooth surface was
observed on the ITO surface (Fig. 3G). The Ra value was measured
20.5 nm over 5 x 5 µm2 area (Fig. 3H).
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Fig. 2. Impedance (A) and voltammetric (B) characterization of the
build-up process of proposed immunosensor. (Inset: Equivalent
electrical circuit)
SEM and AFM characterizations of modified electrode surfaces
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Fig. 3. SEM and AFM micrographs of the immunosensor construction stages.
The specific interaction between IL 6R and IL 6 antigen changed
the electrode surface morphology and a protein layer was coated
on the ITO surface (Fig. 3I). After this interaction, the Ra was
measured as 38.8 nm over 5 x 5 µm2 (Fig. 3J). In conclusion, it was
concluded that there was a perfect correlation between the
results obtained with electrochemical and morphological
techniques.
coated on the ITO electrode could affect the analytical
performance of the immunosensor and therefore, it was
controlled by using different concentrations of polymer during
immunosensor fabrication. Three different PPyr-NHS polymer
concentrations (0.12 mg/mL, 0.24 mg/mL and 0.36 mg/mL) were
tried, and the obtained biosensor signals were illustrated in Fig.
4A. The low polymer concentration led to low immunosensor
response and caused a decrease in detection efficiency. Higher
polymer concentration increased the immunosensor signal and
0.24 mg/mL was chosen as an optimum polymer concentration.
The concentration of biorecognition elements is another
important parameter. The usage of low receptor concentration
caused a low immunosensor response and the high receptor
concentration increased the cost of the biosensor. As displayed in
Fig. 4B, the impedimetric signal was increased correspondingly
Optimization of experimental conditions
To achieve optimal biosensing performance, polymer
concentration, biorecognition element concentration and
incubation period, and target antigen incubation period are key
parameters.
The optimization of polymer concentration was conducted to
obtain excellent biosensor response. The amount of polymer
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with increasing with IL 6R concentration up to 15 ng/mL. The
responses of immunosensors were similar when the IL 6R
concentrations were 3 ng/mL and 15 ng/mL. Therefore, 3 ng/mL
IL 6R level was selected for the next experiments (Fig. 4B). The IL
6R incubation time was another considerable parameter affected
the immunosensor response. It could be seen that 45 min was
enough for IL 6R incubation and 45-min incubation in IL 6R
solution provided maximum immunosensor response. Because of
this, 45 min was utilized for the next experiments (Fig. 4C). Lastly,
View Article Online
the antigen incubation time was optimized . As seen Fig. 4D, the
DOI: 10.1039/D0NJ03183F
impedimetric response increased continuously with the
incubation time. The impedimetric responses were similar after
45 min and 60 min incubation in IL 6 antigen solution and 45 min
was adequate for IL 6 antigen incubation to form specific
immunocomplex. Thus, 45 min was selected as the suitable
incubating period and it was adopted in the following work.
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Fig. 4. The obtained EIS response after optimization experiments.
Analytical performance of the designed IL 6 immunosensor
∆Rct(kohm)₌ 0.235 [IL 6] + 0.246 (R2 =0.999) with a limit of detection
of 10.2 fg/mL at a signal to noise ratio of 3 (the standard deviation
of the blank solution, n₌5). The quantification limit and sensitivity of
the immunosensor were calculated as 34.1 fg/mL and 1 pg mL-1
kohm cm-2, respectively. The analytical characteristics of the
designed immunosensor was also compared with some previous
detection strategies and the results are given in table 1A.
Apparently, the developed polymer based immunosensor had a low
detection limit owing to conductive property of PPyr-NHS polymer.
Furthermore, the polymer synthesis procedure and immunosensor
fabrication process were easier than other biosensors.
Under optimum conditions, the analytical performance of the
developed immunosensor was analyzed by varying IL 6 antigen
concentrations. As illustrated in Fig. 5A, the impedimetric response
increased with the increase of IL 6 antigen levels, indicating that the
increasing amount of IL 6 antigens were specifically immobilized on
the ITO surface and produced remarkable electrochemical signals. In
addition, the increase of IL 6 antigen concentration led to decrease
in peak currents because of non-conductive protein layer formation
(Fig. 5B). The impedance analysis results were fitted to a Randles
equivalent circuit. The changes in the Rct of electrodes that
incubated in different concentration of IL6 were calculated by the
following equation;
Single frequency impedance (SFI) is an analytical tool, which
measures kinetic interaction between antibody and antigen at a
fixed frequency. The fixed frequency is selected from Bode plot (Fig.
SI- 6). In this study, 31 Hz frequency was utilized for SFI experiment.
Therefore, the change in impedance during the specific
immunoreaction was followed at 31 Hz as a function of time and
phase angle in an electrochemical cell containing IL 6 antigen
solution. As observed in Fig. 5D, the impedance was increased due
to specific immunoreaction between receptor and antigen.
Rct = Rct(IL 6) − Rct(BSA)
Rct(IL6) and Rct(BSA) values corresponded to Rct values after
interaction between IL6R and IL6 antigen, and after BSA
immobilization of ITO electrode, respectively. The impedimetric
signals of the suggested immunosensor were linearly proportional to
the IL 6 antigen level within the concentration range from 0.03
pg/mL to 22.5 pg/mL (Fig. 5C). The regression equation was
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Fig. 5. (A and B) EIS and CV responses of the immunosensor at different IL 6 concentrations (0.03, 0.15, 0.3, 1.5, 3, 7.5, 15, 22.5 pg/mL), (C)
calibration curve obtained after EIS measurements, (D) Single frequency impedance result.
Repeatability, reproducibility, selectivity, regeneration and
stability of the immunosensor
In order to investigate the repeatability of the biosensing system,
20 different modified ITO electrodes were used to measure the IL
6 antigen at the same concentration (3 pg/mL). The relative
standard deviation (RSD) was calculated as 3.2%, indicating
acceptable repeatability of the immunosensor (Fig. 6A).
Furthermore, the reproducibility of the biosensor was also
examined. 10 different immunosensors were prepared at the
Besides, the regeneration of the immunosensor was also
significant in practical analyses. The biosensing interface could be
easily regenerated by immersion the ITO electrode into acidic
solution (HCl, 10 mM) at room temperature for 5 min, followed
by being rinsed with ultrapure water. After 8 regeneration steps,
75.96% of the initial signal was observed (Fig. 6C).
In addition, the developed immunosensor had an excellent
storage stability. After long-term storage (4 oC in dry form) of the
immunosensor, as much as 80.91% (eight weeks) of initial
impedimetric response was preserved, displayed the suggested
immunosensor could be utilized for IL 6 antigen determination
with acceptable stability (Fig. 6D).
same conditions and they were used for IL
6 antigen
measurement. The RSD was calculated as 1.03%, indicating the
good reproducibility (Fig. 6B).
Another important property of a good immunosensor is high
selectivity45. The selectivity of the designed immunosensor was
based on the recognition of IL 6 antigen by IL 6R. To examine the
selectivity of the proposed immunosensor, a comparison study
was performed by using interferent biomarkers. One of the IL 6R
immobilized ITO electrode was incubated in PBS solutions
containing interferent biomarkers (IL 1α, TNF α, p53 and NSE
antigens with 300 pg/mL concentration), another IL 6R
immobilized ITO electrode was incubated in PBS solutions
containing interferent biomarkers (300 pg/mL) and IL 6 antigens
(3 pg/mL). As expected, the impedimetric response of the target
IL 6 antigen captured ITO electrode was higher than the
impedimetric response of interferent biomarkers, which
illustrated the higher selectivity of the developed immunosensor.
Practical application of the IL 6 immunosensor in human sera
To evaluate the applicability of the designed immunosensor,
human sera were analyzed after 10-fold dilution by using the
proposed immunosensor. To test accuracy of the immunosensor,
standard addition method was utilized, and IL 6 antigens were
added in diluted human sera. The analysis results are given in
table 1B. It could be seen that the recoveries were between
100.17% and 100.99, which illustrated that the suggested
electrochemical immunosensor had promising successful in real
human sera analyses.
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Fig. 6. Results of repeatability (A), Reproducibility (B), Regeneration (C) and Storage stability (D).
15.6-103
3-1000
5.00
3.00
4.69
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ELISA
ELISA
ELISA
-
Table 1. Comparison with other works using different techniques
for IL 6 detection (A), human sera results of the immunosensor.
(A) Modification
Electrode Linear
Detection Ref.
limit
-
7.81-500
Type
Range
(pg/mL)
(pg/mL)
Found by
biosensor
(pg/mL)
0.59
Found by biosensor
after IL 6 addition
(0.5 pg/mL)
AuNPs/single walled
carbon nanotubes
(SWCNTs)
(B)
Recovery
(%)
Si/SiO2
substrate
10-5 – 10-1
0.00001
1.00
46
18
17
SD*
SD*
1
1
2
2
3
3
4
4
5
5
1.09
100.18
100.83
97.95
104.0
101.57
95.0
AuNPs-polydopamine
ITO
4-800
0.04 %
0.02 %
0.02%
0.02 %
0.02 %
0.04 %
0.54
0.65
0.61
0.71
0.74
0.68
0.63
0.78
0.74
1.06
1.12
1.16
1.23
1.17
1.12
1.20
1.24
1.29
Graphene oxide
nanosheets/polyaniline
/CdSe quantum dot
GCE
0.5-104
0.17
3-aminopropyltriethoxy
silane (APTES)
47
Dip-probe
-
0.12
p-aminobenzoic acid/p-
aminothiophenol/AuNPs
GCE
5-105
1.60
0.02
94.78
105.80
97.16
104.64
19
48
0.04 %
0.03%
0.05 %
0.04 %
AuNPs
Gold
0.02-20
Graphite
screen
printed
electrode
(SPE)
14
Polypyrrole-AuNPs
1-15x106
0.33
SD* : Standard deviation.
Conclusion
11-mercaptoundecanic
acid/6-mercapto-1-
hexanol
49
Gold chip
-
1.30
An ultrasensitive label-free electrochemical immunosensor based
on PPyr-NHS modified ITO electrode was developed for
determination of IL 6 antigen. PPyr-NHS polymer as an interface
material improved the electron transfer efficiency and provided
attachment points for IL6R. Furthermore, PPyr-NHS polymer was
employed as an electrode matrix material for the first time. The
basic principle of this analytical tool was based on the covalently
immobilization of IL 6R on PPyr-NHS modified ITO electrode.
During the immobilization, this tool did not require a crosslinking
agent. The designed immunosensor offered excellent advantages
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Polydopamine
Europium nanoparticles
APTES
Glass
LFA
2-2000
2-500
2.00
0.37
0.10
0.50
0.01
1.00
Optic fiber
0.4-400
0.5-30
SWCNTs
Graphite SPE
This
work
54
0.03-22.5
10.24–400
PPyr-NHS Polymer
ITO
-
ELISA
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L. Wu and X. Qu, Chemical Society Reviews, 2015, 44, 2963-
like long storage-stability, good selectivity and sensitivity because
of to the covalently attachment of IL 6R on the sensing surface,
and the usage of PPyr-NHS polymer increased surface area. The
proposed immunosensor could sensitively detect IL 6 antigen in a
range from 0.03 pg/mL and 22.5 pg/mL with a detection limit of
10.2 fg/mL. Additionally, it illustrated good repeatability and
excellent reproducibility for IL 6 antigen detection. The developed
biosensor could be regenerated by immersion in acidic solution,
and the regenerated surface could be reused at least 8 times.
Moreover, the developed immunosensor had low cost than
traditional ELISA kit. In conclusion, the designed immunosensor
will not only be proper for determination of IL 6 as shown above
but also for other cancer biomarkers.
View Article Online
2997.
DOI: 10.1039/D0NJ03183F
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https://www.thermofisher.com/elisa/product/IL-6-Human-
ELISA-Kit/EH2IL6
https://www.mybiosource.com/human-elisa-kits/il-6/355306
https://www.raybiotech.com/human-il6-elisa-kit/
www.elabscience.com/phuman_il_6(interleukin_6)_elisa_kit-
17965. html
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Acknowledgements
This research was supported The Scientific and Technological
Research Council of by Tekirdağ Namık Kemal University, NKU-
BAP (Grant number: NKUBAP.00.ÖNAP.19.209).
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