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. 2022 Dec 7;862:160700. doi: 10.1016/j.scitotenv.2022.160700

Polyaniline-based electrochemical immunosensor for the determination of antibodies against SARS-CoV-2 spike protein

Maryia Drobysh a,b, Arunas Ramanavicius a,b,⁎, Ausra Baradoke a
PMCID: PMC9726207  PMID: 36493838

Abstract

In this work, we report an impedimetric system for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein. The sensing platform is based on recombinant Spike protein (SCoV2-rS) immobilized on the phytic acid doped polyaniline films (PANI-PA). The affinity interaction between immobilized SCoV2-rS protein and antibodies in the physiological range of concentrations was registered by electrochemical impedance spectroscopy. Analytical parameters of the sensing platform were tuned by the variation of electropolymerization times during the synthesis of PANI-PA films. The lowest limit of detection and quantification were obtained for electropolymerization time of 20 min and equalled 8.00 ± 0.20 nM and 23.93 ± 0.60 nM with an equilibrium dissociation constant of 3 nM. The presented sensing system is label-free and suitable for the direct detection of antibodies against SARS-CoV-2 in real patient serum samples after coronavirus disease 2019 and/or vaccination.

Keywords: SARS-CoV-2 Spike protein, Electrochemical impedance spectroscopy (EIS), Immunosensor, Screen-printed electrodes, Phytic acid, Polyaniline

Graphical abstract

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1. Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is enveloped virus, which has a lipid bilayer based on a membrane formed from lipids of the host cell membrane (Lu et al., 2020). The membrane comprises nucleocapsid and Spike proteins entrapped within a formed lipid bilayer (Murin et al., 2019). The Spike protein is the main target of neutralizing antibodies since it is essential for host cell recognition, attachment, and viral entry (Mittal et al., 2022). Noteworthy that it is crucial to perform testing for coronavirus disease 2019 (COVID-19) control, to constrict the spread of the infection and to minimize risk while making efforts to get social and economic functioning back to normal after the pandemic. The determination of antibodies in serum samples enables the monitoring of the stages of the disease and the evaluation of immune system status and/or recovery efficiency after COVID-19 infection or vaccinated patients (Teng et al., 2020).

Recently, COVID-19 biosensors have evolved into an effective instrument for the determination of COVID-19 related biomolecules (Drobysh et al., 2021, Drobysh et al., 2022c, Drobysh et al., 2022a; Liustrovaite et al., 2022; Plikusiene et al., 2021; Ratautaite et al., 2022; Raziq et al., 2021). Especially, immunosensors are affinity biosensors, which are based on the assessment of antibody-antigen interactions. Mostly, analytes determined by immunosensors can be both either an antigen or an antibody. The target compound is identified by establishing a stable immunocomplex between the antigen and the antibody, which generates a signal that is interpreted by the transducing system (Mollarasouli et al., 2019). Electrochemical immunosensors have advantages such as facilitated maintenance, portability, efficiency, and reliability (Drobysh et al., 2022c). Mostly, the voltage and/or current produced by immunocomplex formation are registered using electrochemical methods such as voltammetry (Do Egito et al., 2022; Raziq et al., 2021; Sadique et al., 2022), amperometry (Arévalo et al., 2022; Deepa et al., 2022), and electrochemical impedance spectroscopy (EIS) (Baradoke et al., 2020, Baradoke et al., 2019; Liustrovaite et al., 2022). The application of the EIS method in biosensors design is of great interest as it is a non-destructive approach that produces high-quality data by transforming biological events directly into electrical signals (Kongsuphol et al., 2014). Capacitance and interfacial charge-transfer resistance signals can be exploited to assess the reaction, which is taking place on the working electrode surface (Grieshaber et al., 2008).

The three-electrode cell is often used for the determination of analytical signals, which has some specific requirements that influenced the experiment, namely, electrode pre-treatment, fixed distance between electrodes and volume of electrolyte. At the same time, the screen-printed technique allows producing compact three-electrode devices on the inactive substrate, e.g., alumina ceramic (Barros Azeredo et al., 2022). Screen-printed electrodes are made up of the following components: a substrate material, a working, counter, reference electrodes, and an insulating covering. The screen-printed electrodes offer the benefits of being compact, easy to use, integrate and modify. The working area of the screen-printed electrodes is covered by conductive inks, which can be noble metals, conductive polymers, and carbon nanomaterials (Kamyshny and Magdassi, 2014). Carbon nanomaterials, such as carbon nanotubes and graphene, provide a low-cost alternative with great environmental stability, appropriate conductivity, and distinguishing features relevant to a spectrum of uses from electrochemical sensors to supercapacitors (Rouhi et al., 2011; Weiss et al., 2012). Noteworthy, that screen-printed carbon electrodes (SPCE) are widely used for the development of immunosensors for COVID-19 diagnosis (Ameku et al., 2022; Amouzadeh Tabrizi and Acedo, 2022; Wu et al., 2021).

In most cases, electrochemically driven immunosensors are created by the immobilization of a recognition element (mostly antibody or antigen) on the surface of the working electrode. The working surface covered by carbon nanomaterials is usually modified with additional compounds to increase conductivity and provide advanced support to biomolecule immobilization (Zhao et al., 2022). For instance, screen-printed carbon electrodes (SPCE) might be modified by the electrodeposition of Au-nanostructures (Baradoke et al., 2019) with subsequent formation of self-assembled monolayer (SAM) (Alonso-Lomillo et al., 2009) for the immobilization of captured biomolecules.

Whereas SAM-based immobilization techniques as a rule require redox probes for electrochemical signal gaining and/or amplification (Drobysh et al., 2022a; Liustrovaite et al., 2022), the application of conductive polymers allows performing electrochemical analysis in label-free mode (Ramanavicius et al., 2014, Ramanavicius et al., 2010; Ramanavicius and Ramanavicius, 2021). For instance, polypyrrole-based molecularly imprinted immunosensors are employed for SARS-CoV-2 detection (Ratautaite et al., 2022; Raziq et al., 2021).

Polyaniline (PANI) has been intensively researched as a conductive material with remarkable optical, electrical, and electrochemical properties (Deshmukh et al., 2017; Mu et al., 1997). Polymerization settings such as exposure time (ET), voltage, and concentration can influence PANI characteristics such as permeability to electroactive chemicals (Garjonyte and Malinauskas, 2000). Noteworthy is that doping with protic organic acids (Olinga et al., 2000), such as phytic acid (PA) (Mawad et al., 2016), promotes the enhanced solubility and conductivity of the PANI. Glutaraldehyde (GA) is a universal immobilization compound, which is often used for the covalent immobilization of biomolecules. GA can be applied for the binding PA-PANI and analyte-capturing biomolecule and/or cross-linking of biomolecules into an insoluble network (Akyilmaz et al., 2017).

Herein, we report the investigation and comparison of SPCE modified with PANI-PA films formed under the range of ET as a label-free impedimetric sensing system for the detection of antibodies against SARS-CoV-2 Spike protein in real serum samples.

2. Experimental

2.1. Reagents and other materials

Aniline (98 %, CAS# 62-53-3), phytic acid (PA) (50 % w/w H2O, CAS# 83-86-3), Na2HPO4 (≥99.0 %, CAS# 7558-79-4) and NaH2PO4 (≥99.0 %, CAS# 7558-80-7) for preparing phosphate buffer (PB) solution, glutaraldehyde solution (GA) (50 % w/w H2O, CAS# 111-30-8) were obtained from Sigma–Aldrich (Steinheim, Germany). SARS-CoV-2 recombinant Spike protein (SCoV2-rS) was developed by Baltymas (Vilnius, Lithuania) (Liustrovaite et al., 2022). Serum samples containing antibodies against SARS-CoV-2 Spike protein (anti-rS) were obtained in compliance with Lithuanian Ethics Law from a volunteer who had received a single dose of Vaxzevria vaccination and tested positive for COVID-19 two weeks later (Liustrovaite et al., 2022). Deionized water was used to prepare all aqueous solutions. 0.1 M phosphate buffer (PB), pH 7.4, was used for preparing the solutions of proteins and antibodies, as well as for electrochemical measurements.

2.2. Electropolymerization

Screen-printed carbon electrode (SPCE) was used for the design of the electrochemical system. SPCE includes a graphene working electrode with a geometrical area of 0.125 cm2, Ag/AgCl pseudo reference and graphene counter electrodes. For modifying the working electrode with PA-doped PANI film, the electrode surface was covered by 100 μL of an aqueous solution of 1 mL aniline (98 %) and 2 mL of PA (50 % w/w H2O) with the addition of 17 mL of deionized water (Baradoke et al., 2020), whereupon current of 10 μA was applied (Fig. 1 , step 1) with different ET: from 1 to 20 min. Afterwards, the PA-PANI-modified SPCE (SPCE/PA/PANI) was rinsed with a copious amount of deionized water.

Fig. 1.

Fig. 1

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Schematic representation of the experimental steps. Step 1 – electropolymerization on the top of SPCE resulting in PA/PANI film formation; step 2 – incubation with SCoV2-rS; step 3 – covalent immobilization of SCoV2-rS accompanied by GA; step 4 – anti-rS coupling.

2.3. Immobilization of SCoV2-rS and anti-rS coupling

SPCE/PA/PANI was covered with 6 μL of 10 μg/mL of SCoV2-rS (diluted in PB) and incubated for 30 min at room temperature. After complete coverage of the electrode (SPCE/PA/PANI/SCoV2-rS) (Fig. 1, step 2), the SPCE/PA/PANI/SCoV2-rS was placed in a chamber with 25 % GA solution and incubated in vapour for 15 min (Fig. 1, step 3). Further, the SPCE/PA/PANI/SCoV2-rS was kept in PB solution overnight at 4 °C.

Subsequently, eight SPCE/PA/PANI/SCoV2-rS electrodes were incubated with 6 μL of anti-rS in the concentration range from 0 to 10 μg/mL for 10 min at room temperature (SPCE/PA/PANI/SCoV2-rS/anti-rS) (Fig. 1, step 4). After each stage of incubation, the electrodes were rinsed with 1 mL of 0.1 M PB solution.

2.4. Electrochemical measurements

Electropolymerization was performed using Metrohm DropSens μStat400 potentiostat (Asturias, Spain) equipped with DropView 8400 software with an applied current of 10 μA in 1, 2, 5, 10 and 20 min. EIS measurements were performed in 100 μL of 0.1 M PB solution by the μAUTOLAB TYPE III potentiostat (Metrohm, Netherland) controlled by FRA2- EIS software from ECO-Chemie (Utrecht, Netherlands). EIS signal was registered after SPCE/PA/PANI, SPCE/PA/PANI/SCoV2-rS, and SPCE/PA/PANI/SCoV2-rS/anti-rS formation in the frequency range from 0.1 Hz and 100,000 Hz, perturbation amplitude of 0.01 V and potential 0.1 V vs. Ag/AgCl pseudo reference electrode.

3. Results and discussion

3.1. EIS characterization of SPCE/PA/PANI

With an aim to form a conductive film on the top of the SPCE, the mixture of the PA and PANI was used for electropolymerization. The process occurred with fixed current (10 μA) and electropolymerization exposure time, which was chosen as a variable and varied from 1 to 20 min. ET is one of the electropolymerization parameters that impact the permeability of the polymer for charge transfer (Wang et al., 1989). This assertion was supported by measurements performed right after electropolymerization for five SPCEs with different ET (1, 2, 5, 10, 20 min). According to a previous investigation (Baradoke et al., 2020), the standard error was calculated based on the stability of the formed polymeric film, which fluctuates in the range of 2–3 %.

Commonly, when describing processes occurring within an electrochemical system, the charge transfer resistance (Rct) parameter generated by the fitting of EIS data to the Randles equivalent circuit is used for analysis (Ionescu et al., 2010; Ramón-Azcón et al., 2008). However, this time-consuming method requires acquiring data across a full range of frequencies and to select a suitable equivalent circuit scheme, which can vary between samples thus making results incomparable. In this work, instead of Rct, we used maximal values of the imaginary part of capacitance –C″max and impedance –Z"max for non- and faradaic processes respectively gained directly from raw experimental data (Patil et al., 2015).

As shown in the Nyquist plots in Fig. 2A, −Z"max values decreased from 83.49 ± 2.34 to 48.57 ± 1.42 kΩ·cm2 with the increase of ET from 1 to 2 min and remained almost unchanged, namely, 26.79 ± 0.65, 22.12 ± 0.57, and 22.46 ± 0.57 kΩ·cm2 for 5, 10, and 20 min correspondingly. This observation suggests that starting from the 5 min of electropolymerization conducting PA/PANI film was formed. Bode format reduces the scatter of experimental data and describes the frequency-dependent behaviour of the electrochemical system in more detail than Nyquist diagrams. Furthermore, the Bode plots are better suited for the interpretation and extrapolation of low-frequency experimental data. For the higher frequency region (>1000 Hz), the Bode graph represents a plateau (Fig. 2B) with phase angles around 0° (Fig. 2C), which are the responses of the solution resistance. Average frequencies (1000–10 Hz) are characterized by the increase of phase angles up to 80°. This is a property of an electrode's capacitive behaviour that describes the dielectric characteristics of an electrically conductive surface film (Sotelo-Mazón et al., 2014). At frequencies lower than 10 Hz, electron charge transfer events, mass transfer processes, or other relaxation processes are taking place at the film-electrolyte interface or in the pores of the film. In this area, the tendency of decreasing –Z"max (Fig. 2B) from 83.49 ± 2.34 to 22.46 ± 0.57 kΩ·cm2 as well as phase angle (Fig. 2C) from 79.37 ± 1.94° to 61 ± 1.30° with the increase of ET is observed in agreement with Nyquist plots. In this frequency range, an incomplete semicircle formed on the Nyquist plots (Fig. 2A), indicating capacitive behaviour, which can be associated with a thin electrolyte layer thickness (Cheng and Chen, 2013).

Fig. 2.

Fig. 2

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Nyquist (A), Bode (B, C), and Cole-Cole (D) plots of SPCE/PA/PANI for the electrodes with ET of polymerization from 1 to 20 min. Measurements were performed in a frequency range from 100 kHz to 0.1 Hz, at 0.01 V amplitude and applied potential 0.1 V vs. Ag/AgCl pseudo reference electrode in 0.1 M PB solution, signal normalized to the area of the electrode, 0.125 cm2.

Moreover, we represented data in the Cole-Cole model for capacitance (Fig. 2D), where there is a reverse correlation, namely, there are more clear semicircle shapes and their diameters become higher with the increase of ET. As shown in Fig. 2D, for the short duration (1–2 min) of ET, the capacitive properties of the PANI-PA layer do not change much (~1.77 μF/cm2). However, for 5 min of ET, there is a rapid increase of –C″max to 2.77 ± 0.08 μF/cm2 with further increase up to 3.29 ± 0.07 μF/cm2 for ET of 20 min.

3.2. EIS characterization of SPCE/PA/PANI/SCoV2-rS/anti-rS

Following the electropolymerization, the immobilization of SCoV2-rS was performed accompanied by GA as a cross-linker on the surface of all the above electrochemically described electrodes (Baradoke et al., 2020). The immobilization was performed by the application of a SCoV2-rS solution (10 μg/mL in 0.1 PB, pH 7.4) to the working electrode surface and incubation (30 min). Further, the electrodes were treated with GA vapour for covalent binding of the SCov2-rS and SPCE/PA/PANI with the following overnight incubation in 0.1 PB, pH 7.4, to get rid of GA and SCoV2-rS remains. After this, EIS was registered for all five electrodes at the same measuring conditions. Gained data were considered as a blank (zero) concentration (Table 1 ). As shown in Fig. 3 , with the increase of ET the behaviour of the electrochemical system alters from capacitor- to resistor-like, which can be traced by appearing more clear semicircle shape in Nyquist plots (Fig. 3A-E) and the reverse tendency in the Cole-Cole graphs (Fig. 3K-O). Hence, for each electrode, a different analytical parameter was selected depending on the behaviour type. Namely, for ET of 1 and 2 min, maximal points of semicircles on the Cole-Cole plots (Fig. 3K-L) were selected and equal to 1.72 ± 0.04 μF/cm2 and 1.27 ± 0.04 μF/cm2, correspondingly. As the electrode with ET of 5 min demonstrates ‘intermediate behaviour’ with a complicated interpretation of the Nyquist and the Cole-Cole models (Fig. 3C, M), we used the maximum -Z"max value obtained from the Bode plot (Fig. 3H) and which was equal to 17.24 ± 0.40 kΩ·cm2. The maximums of semicircles in the Nyquist plots (Fig. 3D-E) were considered for ET of 10 and 20 min with corresponding -Z"max values of 11.34 ± 0.23 kΩ·cm2 and 9.59 ± 0.22 kΩ·cm2.

Table 1.

Analytical parameters were obtained from EIS values for the electrodes with different ET of electropolymerization.

C, μg/mL ET: 1 min
 ET: 2 min
 ET: 5 min
 ET: 10 min
 ET: 20 min
-C″max
 RR
 -C″max
 RR
 -Z"max
 RR
 -Z"max
 RR
 -Z"max
 RR
μF/cm2 % μF/cm2 % kΩ·cm2 % kΩ·cm2 % kΩ·cm2 %
0 1.72 ± 0.04 0 1.27 ± 0.04 0 17.24 ± 0.40 0 11.34 ± 0.23 0 9.59 ± 0.22 0
0.1 1.81 ± 0.05 12.04 ± 2.56 1.29 ± 0.03 5.50 ± 2.59 36.66 ± 0.77 19.80 ± 2.10 24.82 ± 0.66 21.17 ± 2.67 24.76 ± 0.68 20.04 ± 2.75
0.2 1.85 ± 0.05 17.30 ± 2.60 1.32 ± 0.04 11.17 ± 2.85 51.80 ± 1.39 35.24 ± 2.68 34.32 ± 0.87 36.11 ± 2.54 38.11 ± 1.09 37.68 ± 2.86
0.5 1.90 ± 0.04 22.76 ± 2.12 1.42 ± 0.04 30.27 ± 2.72 68.18 ± 1.89 51.94 ± 2.77 41.22 ± 1.06 46.95 ± 2.58 45.76 ± 0.92 47.78 ± 2.00
1.0 1.96 ± 0.05 30.53 ± 2.44 1.46 ± 0.03 38.57 ± 2.30 80.98 ± 1.94 65.00 ± 2.40 49.69 ± 1.15 60.26 ± 2.32 55.19 ± 1.51 60.23 ± 2.75
2.0 2.05 ± 0.05 42.94 ± 2.19 1.44 ± 0.03 35.36 ± 2.08 101.05 ± 2.90 85.47 ± 2.87 55.69 ± 1.65 69.69 ± 2.96 61.67 ± 1.50 68.80 ± 2.42
5.0 2.25 ± 0.06 69.05 ± 2.84 1.56 ± 0.00 58.23 ± 0.20 106.65 ± 2.47 91.17 ± 2.32 62.88 ± 1.40 80.98 ± 2.23 74.50 ± 1.97 85.74 ± 2.64
10.0 2.49 ± 0.06 100 ± 2.40 1.77 ± 0.05 100 ± 2.78 115.31 ± 3.03 100 ± 2.63 74.98 ± 2.17 100 ± 2.89 85.30 ± 2.29 100 ± 2.68
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Fig. 3.

Fig. 3

Fig. 3

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EIS measurements of SPCE/PA/PANI/SCoV2-rS/anti-rS electrodes in the range of concentrations from 0 to 10 μg/mL. Nyquist plots for different ET of electropolymerization: A-1 min; B-2 min; C-5 min; D-10 min; E-20 min. Bode plots: F-1 min; G-2 min; H-5 min; I-10 min; J-20 min. Cole-Cole plots: K-1 min; L-2 min; M-5 min; N-10 min; O-20 min. Measurements were performed in a frequency range from 100 kHz to 0.1 Hz, at 0.01 V amplitude and applied potential 0.1 V vs. Ag/AgCl pseudo reference in 0.1 M PB solution, signal normalized to the area of the electrode, 0.125 cm2.

The next step was to electrochemically register the interaction between immobilized SCoV2-rS and anti-rS in the range of concentrations from 0.1 to 10 μg/mL on the working electrode surface. The necessary concentrations were obtained by successive dilutions of a stock solution by 0.1 M PB. The stock concentration was determined by chemiluminescent microparticle based immunoassay and equalled ~5860 BAU/mL. The values were converted in μg/mL units through the ratio of 1 BAU/mL: 20 ng/mL (the molecular weight of immunoglobulin G ∼ 150 kDa) (Dietzen, 2018; Immundiagnostik AG., 2021; NIBSC, 2020). Further consistent incubations with 6 μL of anti-rS in 10 min were performed for all electrodes with different ET. After incubation with each concentration EIS signal was registered (Table 1, Fig. 3).

In Fig. 3, Nyquist, Bode and Cole-Cole plots are presented. The models show that the same as for blank concentration, for low ET (1–2 min) Nyquist plots (Fig. 3A-B) do not constitute a complete semicircle and do not contain clear resolutions for signals belonging to higher concentrations (0.5–10 μg/mL) thus delaying data interpretation. The Bode plots for ET of 1–2 min (Fig. 3F-G) do not show a clear understanding of surface changes after modification by different concentrations, namely, starting from 0.5 μg/mL –Z"max values do not change gradually. On the other hand, the Cole-Cole capacitance models (Fig. 3K-L) show complete semicircles thus making it possible to analyse data. Eventually, we can see an increase of semicircle radii, consequently the maximum of the imaginary part of capacitance, in the correlation with concentrations of anti-rS from 1.81 ± 0.05 μF/cm2 to 2.49 ± 0.06 μF/cm2 for ET 1 min and from 1.29 ± 0.03 μF/cm2 to 1.77 ± 0.05 μF/cm2 for 2 min (Table 1). However, the capacitance-related values (Table 1, ‘-C″max’ columns for ET 1 and 2 min) still show poor resolution between signals.

Nyquist graphs for the electrode electropolymerized in 5 min (Fig. 3C) still show incomplete semicircles although with noticeable improvement in signal resolutions. The Cole-Cole model for this electrode (Fig. 3M) does not demonstrate any discernible pattern with the increase of the applied anti-rS concentrations as well as it started losing semicircle shapes and the values of the imaginary part of capacitance became lower. In contrast, the Bode plot for ET of 5 min (Fig. 3H) suggests a rather clear trend of –Z"max values increase, which is combined with a relatively good resolution between signals. Hence, as an analytical parameter for this electrode, −Z"max from Bode plots were used in the range of 36.66 ± 0.77–115.31 ± 3.03 kΩ·cm2 for concentrations from 0.1 to 10 μg/mL, respectively.

For ET of 10 min Bode model (Fig. 3I) demonstrates good resolved data with a clear trend of progressive signal increase. Moreover, in the Bode plots, one can see the growth of the imaginary part of impedance starting from low frequencies with subsequent decreases around the intermediate frequency. This behaviour can be explained by relaxation processes specific to dielectric materials (Lasia, 2014). At the same time, capacitance values are very low and hardly analysed (Fig. 3N). The Nyquist plot (Fig. 3D) demonstrates rather well-shaped semicircles with a significant tendency of radii growth in agreement with concentration increase. The considered maximum values of imaginary impedance parts grew from 24.82 ± 0.23 kΩ·cm2 to 74.98 ± 2.17 kΩ·cm2 for the corresponding increase of concentrations from 0.1 to 10 μg/mL.

The electrode with maximal ET of 20 min follows and improves the trend predetermined by the electrode with ET of 10 min. Particularly, the Bode plot (Fig. 3J) possesses highly resolved data with peaks at the medium frequencies area. Noteworthy is that peak maximums increased and shifted to the side of higher frequencies. The values of the imaginary part of capacitance (Fig. 3O) increased in comparison with that determined using the previously described electrode but still are barely interpreted. Meanwhile, the Nyquist plot (Fig. 3E) demonstrates good resolved signals with nearly complete semicircles with significant stepwise increase of radii and –Z"max values from 24.76 ± 0.68 kΩ·cm2 to 85.30 ± 2.29 kΩ·cm2 .

Considering described data obtained from EIS experiments it can be concluded that electropolymerization time influences the behaviour of the sensing system from more capacitor- to a resistor-like with the increase of ET. In general, there is a growth of –C″max and –Z"max values associated with an increase in the thickness of the layer formed after the immobilization of SCoV2-rS and its subsequent interaction with anti-rS. Moreover, our experiments with high ET (10–20 min) demonstrated dielectric behaviour (Fig. 3I-J) of the formed ‘biomolecule-based layer’ related to the poor electrical conductivity of biomolecules. The type of behaviour exhibited by the electrochemical system influences the choice of the parameters, which were applied, and the characteristics that were assessed. These characteristics could be used to plot calibration curves and to determine analytical characteristics such as limit of detection (LOD) and limit of quantification (LOQ).

3.3. Analytical characterization of the electrochemical anti-rS detection systems

LOD and LOQ were calculated for the electrodes with different ET from 1 to 20 min to assess and compare the analytical properties of the impedimetric systems. For this purpose, calibration curves, normalized signal (RR%) vs. concentration, were plotted for each electrode (Fig. 4 ). The following values have been normalized: for ET 1–2 min –C″max from the Cole-Cole models (Fig. 3K-L), ET 5 min – -Z"max from the Bode plot (Fig. 3H), and ET 10–20 min – -Z"max from the Nyquist plots (Fig. 3D-E). Normalization was achieved for the maximum response, which was accepted by 100 % (Table 1). For the calculation of LOD and LOQ values, the equations LOD = 3.33·(SD/Slope) and LOQ = 10·(SD/Slope) were used, where SD is the standard deviation of intercept. The values of intercept and the slope were obtained from linear fitting of calibration curves RR% vs logarithmic concentration (Fig. 4B). Resulting values for the electrodes with different ET are shown in Table 2 .

Fig. 4.

Fig. 4

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Calibration curves for the electrodes with ET of electropolymerization from 1 to 20 min Normalized signal vs. concentration (A) and concentration in logarithmic form. The concentration range is from 0 to 10 μg/mL.

Table 2.

LOD and LOQ values in nM (Dietzen, 2018; Immundiagnostik AG., 2021; Lasia, 2014) were calculated for the electrodes with different ET of electropolymerization.

ET: 1 min ET: 2 min ET: 5 min ET: 10 min ET: 20 min
LOD, nM 14.13 ± 0.40 15.13 ± 0.33 9.13 ± 0.20 8.20 ± 0.27 8.00 ± 0.20
LOQ, nM 42.47 ± 1.07 45.40 ± 1.076 27.40 ± 0.67 24.60 ± 0.59 23.93 ± 0.60
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The calculated LOD values are comparable with other electrochemical biosensors developed for the detection of antibodies against SARS-CoV-2 (Table 3 ). However, the reported PANI-based immunosensor has such advantage as label-free detection.

Table 3.

Some of the recently reported electrochemical biosensors for the detection of antibodies against SARS-CoV-2.

Publication Biosensing element Electrochemical method LOD Redox probe
PANI-based immunosensor Spike protein EIS 8.00 nM −
(Yakoh et al., 2021) Square wave voltammetry 1 ng/mL +
(Ali et al., 2021) EIS 2.8 fM +
(Li et al., 2021) EIS 0.4 pg/mL +
(Liustrovaite et al., 2022) Cyclic voltammetry (CV), EIS 2.53 nM (CV) 1.99 nM (EIS) +
(Drobysh et al., 2022b) CV, differential pulse voltammetry (DPV) 0.27 nM (CV) 0.14 nM (DPV) +
(Braz et al., 2022) DPV 0.77 μg/mL +
(Cardoso et al., 2022) EIS 0.7 pg/mL +
(Peng et al., 2022) Chronoamperometry 10.1 ng/mL (IgG) 1.64 ng/m (IgM). +
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In order to evaluate antibody affinity quantitatively, the equilibrium dissociation constant (K d) was calculated utilizing the Langmuir fitting equation: y = B max × x / (K d + x), where y – RR% values, B max – maximum value obtained during specific binding, and x – concentration values. In the case of ET 1 and 2 min, K d values of around 22 nM were obtained, consistent with the values calculated for C-reactive protein in the previous paper (Baradoke et al., 2020). For PA/PANI films with ET 5–20 min, obtained values were around 3 nM, which indicates that these sensor systems have a higher sensitivity and binding rate.

To summarize, for the formation of PANI-PA film applicable for effective immobilization of SCoV2-rS with subsequent detection of antibodies, the most suitable ET is 20 min.

4. Conclusions

In this work, we performed the comparison of PANI-modified SPCEs with ET of the electropolymerization as a variable. The time was varying from 1 to 20 min and it can be concluded that this factor impacted the permeability of the polymer film and subsequently on such parameters as capacitance and impedance of the electrochemical system. For ET of 1 and 2 min more expressed capacitance properties, whereas for the electrodes that have undergone longer-term electropolymerization (10–20 min) impedimetric characteristics are more prevalent for the analysis. In addition, the signals obtained from impedimetric data have a better resolution and therefore a simplified interpretation. Considering the type of the system behaviour, different analytical parameters such as maximum values of the imaginary part of capacitance or impedance were chosen for the plotting of calibration curves. LOD and LOQ values were calculated for all investigated electrodes and it was revealed that the lowest LOD and LOQ values 8.00 ± 0.20 nM and 23.93 ± 0.60 nM belong to the electrode with a maximum ET of 20 min. Moreover, this sample is characterized by high antigen-antibody affinity with K d value of around 3 nM. Thus, to form a conductive film suitable for SCoV2-rS immobilization, the most appropriate time to electropolymerize the mixture of aniline and PA on the SPCE surface is 20 min. It can be concluded, that the reported label-free impedimetric system is suitable for the detection of antibodies against SARS-CoV-2 Spike protein in real patient serum samples.

CRediT authorship contribution statement

Maryia Drobysh – Methodology, Investigation, Data analysis, Writing - original draft, Interpretation of data, Data analysis.

Ausra Baradoke – Methodology, Investigation, Data analysis, Writing - original draft, Interpretation of data, Data analysis.

Arunas Ramanavicius – Interpretation of data, Data analysis, Supervision, Conceptualization, Writing - review & editing, Funding acquisition.

Declaration of competing interest

All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.

This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.

The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript.

Acknowledgements

This work has received funding from the European Social Fund (project No 09.3.3-LMT-K-712-23-0159) under grant agreement with the Research Council of Lithuania (LMTLT).

Editor: Damià Barceló

Data availability

Data will be made available on request.

References

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Data Availability Statement

Data will be made available on request.

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