Sensitive sandwich-type electrochemical SARS-CoV‑2 nucleocapsid protein immunosensor

Abstract

A sensitive and fast sandwich-type electrochemical SARS-CoV‑2 (COVID-19) nucleocapsid protein immunosensor was prepared based on bismuth tungstate/bismuth sulfide composite (Bi₂WO₆/Bi₂S₃) as electrode platform and graphitic carbon nitride sheet decorated with gold nanoparticles (Au NPs) and tungsten trioxide sphere composite (g-C₃N₄/Au/WO₃) as signal amplification. The electrostatic interactions between capture antibody and Bi₂WO₆/Bi₂S₃ led to immobilization of the capture nucleocapsid antibody. The detection antibody was then conjugated to g-C₃N₄/Au/WO₃ via the affinity of amino-gold. After physicochemically characterization via transmission electron microscopy (TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) analysis were implemented to evaluate the electrochemical performance of the prepared immunosensor. The detection of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP) in a small saliva sample (100.0 µL) took just 30 min and yielded a detection limit (LOD) of 3.00 fg mL⁻¹, making it an effective tool for point-of-care COVID-19 testing.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00604-021-05092-6.

Introduction

The recent coronavirus disease (COVID-19), which the World Health Organization (WHO) has proclaimed as a pandemic, has a significant impact on not just health but also the economy [1–3]. Therefore, the WHO urged the international community to conduct widespread diagnostic testing in an effort to fight against the virus spread and reduce the number of cases that go undetected [4]. The tools for diagnostic are critical in determining early treatment and isolation decisions for affected people, preventing or halting the spread of infectious diseases. As a consequence, developing a highly-sensitive, precise, and prompt diagnostics tools can be vital in deciding what is best about whether or not to isolate affected individuals, thereby controlling the spread rate of this fatal virus [4–6]. The “gold standard” technique in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that leads COVID-19 can be regarded as the reverse-transcription polymerase chain reaction (RT-PCR) [7]. Although PCR-based tests seem to be specifically tailored for SARS-CoV-2, many variables, including sample variances and viral RNA persistence in the nasal cavity/throat, impact their accuracy, resulting in false-negative/false-positive test findings [8–10]. Furthermore, radiological imaging techniques like computed tomography (CT) are considered to be one of the most effective ways to detect the COVID-19. However, owing to some constraints such as being available only at health facilities, difficulties in accessibility, and high cost, it is unlikely to be utilized for swift and large-scale testing [11, 12]. Therefore, the development of time-saving, extremely sensitive, and precise, cost-effective COVID-19 electroanalytical sensing technologies is essential and would have a substantial impact on the control of the pandemic [13].

There has recently been a drive to fabricate COVID-19 serological assays that identify immunological or viral proteins in the blood sample of infected people. Since proteins are homogenously distributed in the blood, serological specimens are more persistent than viral RNA and have fewer variations than nasopharyngeal or oropharyngeal viral RNA specimens, reducing the risk of false-negative test findings [14]. In a few publications, it has been established that lateral flow immunoassay (LFA) methods incorporating colorimetry/fluorescence approaches for targeting the IgG, IgM, or IgA immunoglobulins generated as a reaction to SARS-CoV-2 during the early stages of infection may accurately diagnose viral RNA [8, 15–21]. Moreover, the serological assays will aid in assessing a population’s immunological response to spontaneous infection as well as vaccination [7]. Despite the fact that these devices are user-friendly and swift, they generally have low sensitivity and need at least two antibodies to detect. Therefore, electrochemical monitoring of SARS-CoV-2 antibodies has emerged as a viable approach to solve these limitations [7]. This technique has a high ability to distinguish tiny differences on the electrode surface from the recognition case, thereby allowing label-free detection without any need for a single antibody. Electrochemical immunosensors have significant features that are appropriate for today’s needs, such as simplicity of use, cost and time effectiveness, portability, swiftness, and high sensitivity [13]. Previously, various electrochemical biosensors were employed to detect viral antigens from a variety of fatal viruses, including Hepatitis, Dengue, Rabies, and Zika [22–24]. Moreover, due to the current COVID-19 pandemic, biosensor research aimed at identifying and quantifying SARS-CoV-2 has emerged. However, interfacial contamination caused by biomolecule adsorption is one of the most serious challenges with electrochemical sensing technologies, limiting their practical applicability [25]. Therefore, the fabrication of an effictice immunosensor surface modified with suitable nanomaterials, which restricts non-specific adsorption of molecules without interacting with particular analyte detection, is one plausible option to this challenge.

Semiconductor nanocrystals were used for various applications such as energy [26], supercapacitor electrode material [27, 28], and catalysis [29]. Especially, bismuth-based semiconductors having specific morphology show desirable sensor catalytic performance [30, 31]. Among these semiconductors, bismuth sulfide (Bi₂S₃) belonging a tunable band gap (from 1.2 to 1.8 eV) demonstrates good stability, non-toxicity, and conductivity, providing superior electronic properties for sensor/biosensor applications. Nonetheless, owing to the easy photogenerated carrier recombination, its practical applications are limited. In order to eliminate this carrier recombination problem, the several methods such as the element dopping and the preparation of heterostructures can be utilized for improvement of catalytic activity. The layered Bi₂WO₆ has received a great deal of interest thanks to its stability and superior catalytic properties [32]. Its layered structure composes of the octahedral (WO₄)²⁻ sheets and bismuth oxide (Bi₂O₂)²⁺ layers, indicating easy charge transfer. This structure was formed by an intergrowth of WO₄²⁻ ions between (Bi₂O₂)²⁺ layers [33]. Due to internal electric field, the recombination of charge carriers is reduced and sensor/biosensor’s catalytic activity increases. In addition, its application areas can be expanded by dopping treatment [34] and the heterojunction construction [35]. In these methods, the coupling with two different semiconductor into heterojunctions is effective approach [36]. Thus, we prepared Bi₂WO₆/Bi₂S₃ as electrode platform in this study.

The preparation of g-C₃N₄ can be carried out by thermal pyrolysis of nitrogen-rich precursors such as melamine and urea [37]. Due to its excellent stability, low toxicity and conductivity, it has been utilized in electrochemical applications [38]. g-C₃N₄ with band gap of 2.7 eV and WO₃ with band gap of 2.8 eV can be made up of a Z-scheme heterojunction [39], providing the prevention of the electron–hole recombination. In addition, tungsten trioxide (WO₃) has superior electronic and chemical properties and biocompatibility [40]. A mediator such as AuNPs is generally needed for facilitating the electron transfer in Z-scheme heterojunction. Due to AuNPs’ electrical conductivity and large specific surface area, the immobilization of antibodies also becomes easier [41]. Hence, g-C₃N₄/Au/WO₃ as signal amplification can separate electron–hole, providing the sensitivity of sensor performance.

Herein, in the light of all aforementioned points in mind, it was aimed to fabricate a novel electrochemical SARS-CoV‑2 nucleocapsid protein immunosensor based on Bi₂WO₆/Bi₂S₃ and g-C₃N₄/Au/WO₃ for COVID-19 detection for the first time in literature. The fabricated immunosensor provides a number of benefits, including ease of use, speed, and selectivity. Moreover, a precise LOD of 3.00 fg mL⁻¹ was acquired with high selectivity and no interference in saliva samples. Hence, the fabricated electrochemical SARS-CoV‑2 nucleocapsid protein immunosensor presents a new perspective in the point-of-care COVID-19 testing.

Experimental section

Materials

SARS-CoV-2 NP, capture human monoclonal anti-SARS-CoV-2 nucleocapsid antibody (c-SARS-CoV-2-Ab₁), detection monoclonal anti-SARS-CoV-2 nucleocapsid antibody (d-SARS-CoV-2-Ab₂), MERS-CoV nucleocapsid protein (MERS-CoV NP), coronavirus nucleoprotein (SARS-CoV NP), influenza A antigen (H1N1), Bi(NO₃)₃·5H₂O, Na₂S·9H₂O, carbamide, Na₂WO₄·2H₂O, melamine, sodium borohydride (NaBH₄), and chloroauric acid (HAuCl₄) were supplied from Sigma-Aldrich. Moreover, during the experiments, 0.1 mol.L⁻¹ phosphate-buffered saline (PBS) solution with a pH value of 7.0 was employed as a supporting electrolyte and diluting buffer solution.

Apparatus for evaluation of nanomaterials

Surface morphological characteristics were explored by using a SEM (ZEISS EVO 50) SEM and a TEM (JEOL 2100). X-ray spectrum of nanostructrues was collected by a Rigaku X-ray diffractometer with Cu-K radiation (λ = 0.150 nm). The PHI 5000 Versa Probe spectrometer was used to perform the XPS survey. UV–Vis and Raman measurements were performed by Mettler Toledo and LabRam HR Raman Spectrometer, respectively. Electrochemical characterization techniques such as cyclic voltammetry, differential pulse voltammetry, and electrochemical empdance spectroscopy were also conducted via the Gamry Reference 600 workstation (Gamry, USA).

Preparation of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃

For preparation of Bi₂S₃ nanorods, the addition of Bi(NO₃)₃·5H₂O (1.90 g) into ethanol solution (30.0 mL) was firstly performed and stirred during 30 min. Then, Na₂S·9H₂O aqueous solution (0.1 g mL⁻¹) was prepared, stirred during 45 min and slowly added into Bi(NO₃)₃·5H₂O solution, providing the black suspension. After that, carbamide solution (0.1 g mL⁻¹) was transferred into the above solution and the suspension was subjected to 200 °C for 20 h in a Teflon steel autoclave. Bi₂S₃ nanorods were filtered, washed, and dried at 25.0 °C.

For preparation of Bi₂WO₆, the mixture of Na₂WO₄·2H₂O (3.0 mmol, 10.0 mL) and Bi(NO₃)₃·5H₂O (3.0 mmol, 10.0 mL) was prepared and strongly stirred during 20 min. Then, the mixture was subjected to 200 °C for 15 h in a Teflon steel autoclave. The resulting product as Bi₂WO₆ was filtered, washed, and dried at 60.0 °C.

To prepare Bi₂WO₆/Bi₂S₃ composite, Bi₂S₃ nanorods (10.0 mmol) and Bi₂WO₆ (10.0 mmol) were mixed at 1:1, v/v for 40 min. After that, Bi₂WO₆/Bi₂S₃ composite having heterojunction was collected, filtered, and dried at 40 °C.

Bi₂WO₆/Bi₂S₃ modified GCE (Bi₂WO₆/Bi₂S₃/GCE) as electrochemical sensor platform with c-SARS-CoV-2-Ab₁ and SARS-CoV-2 NP immobilizations

The glassy carbon electrode (GCE) was prepared as follows to be utilized in the further steps [42]: firstly, 0.1 µm and 0.05 µm alumina slurries were transferred on cleaning pads, respectively. Then, the GCE was polished with these alumina slurries for 20 min. Subsequently, the electrodes were rinsed with isopropyl alcohol and acetonitrile, respectively to remove the alumina remains at 25 °C. The electrode modifications with Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃ suspensions (10.0 μL, 0.1 mg mL⁻¹) were performed by dropping these suspensions on the clean GCEs. After 20 min, the solvent removal was carried out by infrared heat lamp, providing Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃ modified GCEs (Bi₂WO₆/GCE, Bi₂S₃/GCE, and Bi₂WO₆/Bi₂S₃/GCE). The c-SARS-CoV-2-Ab₁ immobilization on Bi₂WO₆/Bi₂S₃/GCE was performed by dropping of 20 μL c-SARS-CoV-2-Ab₁ dispersion (with a concentration of 20.0 μg.mL⁻¹) on Bi₂WO₆/Bi₂S₃/GCE via strong electrostatic interactions between amino group of c-SARS-CoV-2-Ab₁ and bismuth oxide (Bi₂O₂)²⁺. The electrodes were maintained at 37.0 °C for 15 min (c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE). Then, BSA (3.0% w/v) was incubated on c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE at 37.0 °C over 15 min to eliminate the non-specific interactions (BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE). Each of SARS-CoV-2 NP with different concentrations was incubated to BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE for 15 min at 37.0 °C via specific protein-antibody interactions (SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE). Lastly, the whole electrode including SARS-CoV-2 NP and c-SARS-CoV-2-Ab₁ was interacted with 0.1 M PBS (pH 7.0) to take away non-interacting SARS-CoV-2 nucleocapsid proteins.

Preparation of g-C₃N₄, g-C₃N₄/WO₃, and g-C₃N₄/Au/WO₃

Firstly, g-C₃N₄ preparation was performed [43]. For this aim, the calcination of melamine (20.0 g) was conducted at 500 °C over 90 min. After the cooling treatment at 25 °C, the obtained yellow product was transferred into the combustion boat and the calcination treatment was performed at 480 °C for 120 min and after cooling, the white g-C₃N₄ was obtained.

For g-C₃N₄/WO₃ preparation [44], the mixture of g-C₃N₄ (0.25 g), Na₂WO₄⋅2H₂O (1.00 g), and ethanol (150.0 mL) was prepared under strong stirring. Afterward, the concentrated HCl (10.0 mL) was gently added into this mixture and the dispersion was calcined at 420 °C for 120 min, providing g-C₃N₄/WO₃ composite.

For g-C₃N₄/Au/WO₃ preparation, the mixture of g-C₃N₄/WO₃ (150.0 mg), HAuCl₄ (50.0 μL, 20.0 mM), and ultra-pure water (50.0 mL) was prepared. After that, NaBH₄ (2.0 mL, 20.0 mM) was added into this dispersion under strong stirring and g-C₃N₄/Au/WO₃ was dried at 25 °C.

g-C₃N₄/Au/WO₃ signal amplification with d-SARS-CoV-2-Ab₂ conjugation

d-SARS-CoV-2-Ab₂ conjugation was performed by addition of detection antibody (20.0 μL, 20.0 μg mL⁻¹) into g-C₃N₄/Au/WO₃ (20.0 μL, 20.0 mg mL⁻¹) signal amplification via strong amino-gold affinity [45, 46]. After the vigorous stirring at 37.0 °C for 30 min, d-SARS-CoV-2-Ab₂/g-C₃N₄/Au/WO₃ was centrifugated at 5000 rpm for 30 min.

Electrochemical characterizations

The resulting SARS-CoV‑2 nucleocapsid protein immunosensor was constructed by antibody-nucleocapsid protein interactions between d-SARS-CoV-2-Ab₂/g-C₃N₄/Au/WO₃ and SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE. By drop-casting method, 20.0 μL d-SARS-CoV-2-Ab₂/g-C₃N₄/Au/WO₃ dispersion (with a concentration of 20.0 mg mL⁻¹) was coated on electrode surface such as SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, at a 30 min immunological response time. The final electrochemical immunosensor was tagged as g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂/SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE and this electrochemical immunosensor was stored in 0.1 M PBS (pH 7.0, 3.0 mL) without pressure fluctuations at 25 °C. The electrochemical performance of the SARS-CoV‑2 nucleocapsid protein immunosensor was monitored in 0.1 M PBS (pH 7.0, 2.0 mL) containing 1.0 mM H₂O₂ solution in the range of + 0.0/ + 0.4 V. Scheme 1 shows the preparation procedure of electrochemical SARS-CoV-2 immunosensor including the preparations of electrode platform and signal amplification.

Scheme 1.

Schematic illustration of the fabrication procedure of electrochemical SARS-CoV-2 immunosensor

Processing of samples

The saliva samples were acquired from five healthy individuals (See S. M. for a further description of sample processing).

Results and discussion

Fundamental of electrochemical SARS-CoV-2 NP immunosensor based on Bi₂WO₆/Bi₂S₃ and g-C₃N₄/Au/WO₃

Bi₂WO₆/Bi₂S₃ was employed as a sensor platform in immunosensor fabrication. Bi₂WO₆ with a layered structure comprises WO₄²⁻ ions’ intergrowth between (Bi₂O₂)²⁺ layers. Due to the layered structure, Bi6s and O2p levels create the dispersed valence band, providing the catalytic effect [47]. In addition, Bi₂WO₆ has important function as a framework to prepare Bi₂WO₆/Bi₂S₃ composite. Sodium sulfide as sulfur source can release S²⁻ ion to react with Bi₂WO₆ via ion change, providing Bi₂S₃ nanorods’ dispersion [33]. In immunosensor construction, the efficient electrostatic interactions between amino group of c-SARS-CoV-2-Ab₁ and Bi₂WO₆/Bi₂S₃ composite also provided the strong capture antibody immobilization on electrode surface.

g-C₃N₄/Au/WO₃ composite was used as a signal amplification for electrochemical SARS-CoV-2 NP immunosensor. This composite composed a Z-scheme heterojunction [39], resulting in the decrease of the electron transfer. Hence, a mediator, which facilitated electron transfer, is necessary at the interface of heterojunction. AuNPs can be a candidate intermediate owing to their large specific surface area, the ability of the charge separation promotion, and the antibody’s easy immobilization [48]. Hence, g-C₃N₄/Au/WO₃ composite both integrates substantial benefits and efficiently facilitates electron transport, thereby resulting in the development of a sensitive immunosensor.

Finally, H₂O₂ was utilized as a redox probe in this work due to its easy oxidation into O₂ and continuous monitoring. The related electrochemical reaction mechanism for H₂O₂ in potential range was also provided on Scheme 1 as H₂O₂ ↔ O₂ + 2H⁺  + 2e⁻ [49, 50].

Characterizations of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃

The morphological features of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃ composite were investigated on Fig. 1. The nanoflower and ultrathin nanosheet morphological structure of Bi₂WO₆ with about 2.5 μm diameter and 490–510 nm length were shown on Fig. 1A. Figure 1B shows pure Bi₂S₃ having a nanorod structure with width of 35–45 nm. In addition, the deposition of Bi₂S₃ nanorods with 120–140 nm lengths on Bi₂WO₆ nanoflower was shown on Fig. 1C. Finally, the elemental mapping of composite material (Fig. 1D) confirmed Bi₂WO₆/Bi₂S₃ formation in presence of Bi, W, and S. Secondly, XPS analysis was carried out to show the analysis patterns of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃ (Fig.S1AS1 ). According to Fig. S1A, Bi 4f7/2 peaks at 157.8 and 163.7 eV, Bi 4f5/2 peaks at 158.8 and 164.1 eV, and S 2p3/2 peak at 161.2 eV verified the presence of Bi³⁺ and S²⁻, respectively. In addition, the peaks at 34.9 and 37.3 eV were ascribed to W 4f7/2 and W 4f5/2, respectively. Then, XRD patterns of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃ were demonstrated on Fig. S1B. The characteristic XRD peaks belonging to Bi₂WO₆ and Bi₂S₃ were attributed to the orthorhombic phases of Bi₂S₃ and Bi₂WO₆ [51, 52]. The same XRD peaks on the patterns of Bi₂WO₆ and Bi₂S₃ were observed on XRD pattern of Bi₂WO₆/Bi₂S₃ and it is concluded that Bi₂WO₆/Bi₂S₃ was prepared with a high purity.

Fig. 1.

SEM images of A Bi₂WO₆, B Bi₂S₃, and C Bi₂WO₆/Bi₂S₃; D elemental mapping of Bi₂WO₆/Bi₂S₃ composite

According to Raman spectra (Fig. 2A), the obvious peaks such as the symmetric and asymmetric stretching peaks between W and O atoms on Bi₂WO₆ in 600–1000 cm⁻¹ region were observed. Moreover, the band observed at 308 cm⁻¹ was corresponded to O–WO– group’s bending, whereas the bands detected at 788 and 822 cm⁻¹ were ascribed to asymmetrical and symmetrical modes of O–WO groups, respectively [53, 54]. The asymmetrical bridging mode relating to the tungstate chain was confirmed by the peak at 698 cm⁻¹. On Raman spectra of Bi₂WO₆/Bi₂S₃, the novel peaks at 101 and 229 cm⁻¹ and the increased peak intensity at 254 cm⁻¹ confirmed the presence of Bi₂S₃ on composite [55]. In addition, the specific bands attributing to Bi₂WO₆ were observed on Raman spectra of Bi₂WO₆/Bi₂S₃. Figure 2B demonstrates UV–Vis spectra of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃. The absorption band at about 440 nm showed the characteristic response of Bi₂WO₆ and the extended light absorption on the spectrum of Bi₂WO₆/Bi₂S₃ confirmed the synergistic effect between Bi₂WO₆ and Bi₂S₃ [56].

Fig. 2.

Raman (A) and B UV–Vis spectra of Bi₂WO₆, Bi₂S₃, and Bi₂WO₆/Bi₂S₃

Characterizations of g-C₃N₄, g-C₃N₄/WO₃, and g-C₃N₄/Au/WO₃

The crystalline structures and the surface morphologies of g-C₃N₄, g-C₃N₄/WO₃ and g-C₃N₄/Au/WO₃ were examined by TEM, SEM, XRD, and XPS techniques. The wrinkle structure of g-C₃N₄ was shown on Fig. 3A. According to Fig. 3B, it was revealed that WO₃ spheres with a diameter of 280–320 nm were uniformly decorated on g-C₃N₄ sheet, and AuNPs (red circle) with a small diameter placing between g-C₃N₄ and WO₃ were deposited on g-C₃N₄/WO₃ sheet (Fig. 3C). Furthermore, an EDX image of g-C3N4/Au/WO3 revealed the existence of C, N, O, W, and Au elements, noting that the g-C3N4/Au/WO3 was successfully fabricated (Fig. 3D). Finally, high-resolution TEM image (Fig. 3E) of g-C₃N₄/Au/WO₃ demonstrated the lattice spacing of Au NP (pink circle) and WO₃ (red circle), indicating 0.272 and 0.212 nm which were attributed to WO₃ (022) [57] and Au (200) [58].

Fig. 3.

A SEM image of g-C₃N₄, TEM images of B and C g-C₃N₄/Au/WO₃, D EDX image of g-C₃N₄/Au/WO₃, and E high-resolution TEM image of g-C₃N₄/Au/WO₃

The chemical states and the surface functionalities of g-C₃N₄/Au/WO₃ were examined through XPS analysis (Fig. S2). According to Fig. S2A of the survey spectra, the peaks belonging to C, N, O, W, and Au elements confirmed the successful preparation of g-C₃N₄/Au/WO₃ in harmony with Fig. 3D [59]. The high-resolution C1s XRD spectra of sample deconvoluted into two main peaks at 285.1 eV and 288.1 eV corresponded to C–C bond and N–C groups (Fig. S2B) [60]. Moreover, N1s spectra (Fig. S2C) were deconvoluted into three main N configurations with the peaks detected at 398.1, 399.4, and 401.1 eV attributing to C–N–C, –N = C–, and N–H, respectively [60]. The peak at 530.1 eV attributing to lattice oxygen and the peak at 532.2 eV corresponding to the chemisorbed oxygen species in WO₃ were demonstrated on Fig. S2D [61]. Fig.S2E relating to W 4f shows that W 4f7/2 and W 4f5/2 of W⁶⁺ were located at 35.1 and 37.9 eV, respectively [62]. Finally, the peaks at 84.1 and 88.1 eV were attributed to Au 4f7/2 and 4f5/2, respectively, indicating the existence of Au in composite material (Fig. S2F) [63].

The crystal structure of g-C₃N₄/Au/WO₃ composite was investigated by XRD (Fig. S3A). (002) crystal plane of g-C₃N₄ was confirmed by the peak at 27.62°. After the preparation of g-C₃N₄/Au composite, a new XRD peak at 38.31° was corresponded to Au NPs’ (111) plane [64] and all peaks belonging to WO₃ were attributed to the monoclinic phase of WO₃ [65]. The observed peaks on XRD patterns of g-C₃N₄/WO₃ and g-C₃N₄/Au/WO₃ were similar to that of WO₃. In addition, the peak at 27.62° verified the presence of g-C₃N₄ and WO₃. Nonetheless, the specific peaks of Au NPs were not observed on XRD pattern of g-C₃N₄/Au/WO₃ composite, suggesting the low amount of Au NPs. UV–Vis spectra (Fig. S3B) were also recorded for g-C₃N₄, g-C₃N₄/WO₃, and g-C₃N₄/Au/WO₃. g-C₃N₄/Au/WO₃ composite having a wide absorption range effectively absorbs light, providing the increased catalytic response.

Evaluation of the electrochemical characteristics of sensor platform and signal amplification

Firstly, the electrochemical investigations for the prepared sensor platform were progressively performed by using CV and EIS methods in presence of 1.0 mM [Fe(CN)₆]^3−/4− as redox pair. Firstly, the anodic and cathodic signals on bare GCE were observed at Epa = 550 mV and Epc = 375 mV, respectively, (curve a of Fig. 4A). Due to the high stability and catalytic activity of Bi₂WO₆/GCE in the presence of 1.0 mM [Fe(CN)₆]^3−/4−, more visible electrochemical signals were observed in contrast to bare glassy carbon electrode (curve b of Fig. 4A) [32]. Then, there was more electrochemical catalytic effect on the signals (curve c of Fig. 4A) by using Bi₂S₃/GCE, indicated the narrow band gap (1.3 eV) and the easy charge separation property [66]. Because Bi₂WO₆/Bi₂S₃ composites improved catalytic activity and the improved electron separation and transfer, the highest electrochemical responses in comparison with Bi₂S₃/GCE were obtained on Bi₂WO₆/Bi₂S₃/GCE (curve d of Fig. 4A). Electroactive surface areas of the prepared electrode surfaces were calculated as 0.173 ± 0.002 cm² for bare GCE, 0.319 ± 0.001 cm² for Bi₂WO₆/GCE, 0.647 ± 0.003 cm² for Bi₂S₃/GCE, and 1.113 ± 0.006 cm² for Bi₂WO₆/Bi₂S₃/GCE in the presence of 1.0 mM [Fe(CN)₆]³⁻ solution by i_p = 2.69 × 10⁵ A n^3/2 D^1/2 C v^1/2, where i_p was current, C (mol cm⁻³) was [Fe(CN)₆]³⁻ concentration, v was scan rate (10–500 mV s⁻¹), and A was surface area (cm²) (n = 1, D = 7.6 × 10^–6 cm² s⁻¹ for [Fe(CN)₆]³⁻) [67]. Thus, Bi₂WO₆/Bi₂S₃ composite was chosen for future sensor platform.

Fig. 4.

A Cyclic voltammograms, B EIS reponses at (a) bare GCE, (b) Bi₂WO₆/GCE, (c) Bi₂S₃/GCE, (d) Bi₂WO₆/Bi₂S₃/GCE, (e) c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, (f) BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, (g) SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE, (h) the final immunosensor including c-SARS-CoV-2-Ab₁, SARS-CoV-2 NP, and d-SARS-CoV-2-Ab₂ (scan rate of 50 mV s⁻¹) in 1.0 mM [Fe(CN)₆]³⁻ containing 0.1 M KCl, and C DPV responses of the proposed immunosensors incubated with 0.2000 pg mL⁻¹ SARS-CoV-2 NP using g-C₃N₄/WO₃/d-SARS-CoV-2-Ab₂ (curve b) in presence of 1.0 mM H₂O₂, g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂ (curve c) in presence of 1.0 mM H₂O₂ and in absence of target analyte (curve a)

As expected, the obvious electrochemical sensor signals decreased owing to c-SARS-CoV-2-Ab₁’s blocking effect on electron transfer (curve e of Fig. 4A). After the immobilizations of BSA (curve f of Fig. 4A) and SARS-CoV-2 NP (curve g of Fig. 4A), respectively, we observed that the sensor signals gradually decreased. Thus, it is concluded that the immobilization treatments of BSA and SARS-CoV-2 NP on electrode surface were successfully carried out. Finally, when the resulting immunosensor was used (curve h of Fig. 4A), further decrease on sensor signals was observed because of more antibody-nucleocapsid protein interactions.

Secondly, EIS experiments were performed to prove CV results and according to Fig. 4B, the obtained charge transfer resistances were calculated as 75.0, 65.0, 55.0, 40.0, 85.0, 95.0, 125.0, and 140.0 Ω for bare GCE (curve a), Bi₂WO₆/GCE (curve b), Bi₂S₃/GCE (curve c), Bi₂WO₆/Bi₂S₃/GCE (curve d), c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE (curve e), BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE (curve f), SARS-CoV-2 NP/BSA/c-SARS-CoV-2-Ab₁/Bi₂WO₆/Bi₂S₃/GCE (curve g), and the final immunosensor (curve h), respectively. Hence, the preparation procedure of immunosensor was completed successfully based on CV and EIS results.

For electrochemical performance characterization (Fig. 4C) of the prepared signal amplification, several immunosensors using g-C₃N₄/WO₃/d-SARS-CoV-2-Ab₂ (curve b) and g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂ (curve c) were developed by using 0.2000 pg mL⁻¹ SARS-CoV-2 NP at the immune reaction time of 30 min and DPV signals were observed in 1.0 mM H₂O₂. As expected, the highest electrochemical performance was obtained by the immunosensor based on g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂ in comparison with g-C₃N₄/WO₃/d-SARS-CoV-2-Ab₂. Because of the suppressing of electron transfer by g-C₃N₄/WO₃ composite, a mediator such as AuNPs was needed at the interface for improving of electron transfer in Z-scheme heterojunction [59]. In addition, the stable electrochemical signals were observed by g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂ owing to the strong covalent immobilization between g-C₃N₄/Au/WO₃ and d-SARS-CoV-2-Ab₂. Curve a of Fig. 4C also demonstrated that there was no electrochemical signal in absence of target analyte. To verify the specific interaction between antibody-nucleocapsid protein, SEM image (Fig. S4) of the resulting immunosensor indicating a spherical size and agglomeration was obtained, providing a successful immune reaction.

Optimization studies for electroanalytical characterizations

The effects of the solution pH, immune reaction time, H₂O₂, and g-C₃N₄/Au/WO₃/d-SARS-CoV-2-Ab₂ solution concentration were presented in detail (Fig. S5).

Linearity range

The obtained calibration equation by using SARS-CoV-2 NP concentrations and the observed electrochemical immunosensor signals was calculated as y = 47.231x + 1.0666, with a correlation coefficient of R² = 0.9989, where y and x represented the current (µA) and SARS-CoV-2 NP concentration (pg mL⁻¹), respectively (Fig. 5). The quantification limit (LOQ) and LOD were found to be 0.01 pg mL⁻¹ and 3.00 fg mL⁻¹, respectively. Equations (1) and (2) were employed to calculate LOQ and LOD:

$$LOQ=10.0\mathrm{S}/\mathrm{m}$$ 1

$$LOD=3.3\mathrm{S}/\mathrm{m}$$ 2

Fig. 5.

Concentration effect (from 0.01 to 1.00 pg mL⁻¹ SARS-CoV-2 NP) on immunosensor signals, Inset: calibration curve for electrochemical SARS-CoV-2 NP immunosensor (potential range is + 0.0/ + 0.4 V; Parameters are frequency of 100 Hz, pulse amplitude of 25 mV, and scan increment of 5 mV)

In addition, Table 1 shows some comparison features between the developed sandwich-type electrochemical SARS-CoV-2 NP and the other new detection methods. Firstly, the sensitive SARS-CoV-2 NP detection (LOD: 3.00 fg mL⁻¹) was performed in 30 min of immunological response time. More importantly, COVID-19 detection with high selectivity can be successfully performed from saliva samples by this immunosensor. In addition, thanks to the developed electrochemical SARS-CoV-2 NP immunosensor, the time-consuming steps in immunosensor development can be eliminated in this study. The preparation steps of Bi₂WO₆/Bi₂S₃ electrode platform and g-C₃N₄/Au/WO₃ signal amplification comprised the minimal waste generation, indicating an immunosensor that is friendly to the environment and human health. As a result, the developed selective electrochemical SARS-CoV-2 NP immunosensor may offer a potential for early COVID-19 detection.

Table 1.

The comparison of electrochemical SARS-CoV‑2 NP immunosensor with the other novel techniques

Material/method	Linear range	LOD	Assay time	Ref
Microfluidic	0.0–10.0 ng mL−1	50.0 pg mL−1	2 min	[68]
Paper-based electrochemical	1.0–1000.0 ng mL−1	1.0 ng mL−1	30 min	[7]
Chemiluminescence	0.2–100.0 ng mL−1	0.1 ng mL−1	16 min	[69]
Electrochemical/Cu2O nanocube	0.25 fg mL−1–1.00 µg mL−1	0.04 fg mL−1	20 min	[70]
Ni(OH)2 NPs	0.25 fg mL−1–1.00 µg mL−1	3.00 fg mL−1	20 min	[71]
Electrochemical immunosensor	0.01–1.00 pg mL−1	3.00 fg mL−1	30 min	This study

Recovery

The recovery experiments including saliva samples obtained from five healthy individuals were carried out by the portable electrochemical SARS-CoV-2 NP immunosensor. Table S1 indicates the close values to 100.00% confirming the preparation of high selective electrochemical SARS-CoV-2 NP immunosensor. Moreover, standard addition method was applied to saliva samples obtained from five healthy individuals and y = 47.249x + 10.171, with R² = 0.9994, was obtained as calibration equation. Thus, the close slope values between direct calibration (inset of Fig. 5) and standard addition methods again verified the high selective COVID-19 detection.

The validity of the sandwich-type electrochemical immunosensor was evaluated by using colorimetric method [72]. Table S2 indicates the comparison results, showing that no significant difference was observed between the prepared immunosensor and colorimetric method (T_calculated > T_tabulated, p > 0.05).

Selectivity, stability, reproducibility, and reusability

For selectivity measurement, the several electrochemical SARS-CoV-2 NP immunosensors were prepared by using different target dispersions such as (i) MERS-CoV NP + SARS-CoV NP + H1N1, (ii) SARS-CoV‑2 NP + MERS-CoV NP, (iii) SARS-CoV‑2 NP + SARS-CoV NP, (iv) SARS-CoV‑2 NP + H1N1. Then, these electrochemical immunosensors were applied to 1.0 mM H₂O₂ solution. Figure 6A confirms that the prepared electrochemical immunosensor demonstrated the high selectivity towards SARS-CoV‑2 nucleocapsid protein.

Fig. 6.

A Immunosensor selective responses against the prepared solutions (n = 6): (i) 10.0 pg mL⁻¹ MERS-CoV NP + 10.0 pg mL⁻¹ SARS-CoV NP + 10.0 pg mL⁻¹ H1N1, (ii) 0.2000 pg mL⁻¹ SARS-CoV‑2 NP + 10.0 pg mL⁻¹ MERS-CoV NP, (iii) 0.2000 pg mL⁻¹ SARS-CoV‑2 NP + 10.0 pg mL⁻¹ SARS-CoV NP, (iv) 0.2000 pg mL⁻¹ SARS-CoV‑2 NP + 10.0 pg mL⁻¹ H1N1; B Stability test of electrochemical SARS-CoV-2 NP immunosensor including 0.2000 pg mL⁻¹ SARS-CoV-2 NP (n = 6) at 25.0 °C

The stability test results of the constructed electrochemical SARS-CoV-2 NP immunosensor at 25.0 °C for seven weeks were depicted in Fig. 6B. It was pointed out that the immunosensor signals were about 98.73% of the original electrochemical signal, indicating strong immunosensor stability.

Finally, for reproducibility, 10 different electrochemical SARS-CoV-2 NP immunosensors were developed by the protocol which is explained in the “Electrochemical characterizations” section. The relative standard deviation (RSD) of 0.61 was calculated by using the observed 10 electrochemical signals, confirming the high reliability of immunosensor production procedure.

Reusability of prepared electrochemical SARS-CoV-2 NP immunosensor was tested in 1.0 mM H₂O₂ solution. One SARS-CoV-2 NP immunosensor was utilized at least 30 times and 0.89% of RSD was obtained for current signals, confirming high reusability of prepared electrochemical SARS-CoV-2 NP immunosensor in this study.

Precision and accuracy

The studies of same day (intra-day precision) and six consecutive days (inter-day precision) were carried out in presence of three concentrations (0.3000, 0.5000, and 0.7000 pg mL⁻¹ SARS-CoV‑2 NP) in linearity range (Table S3). The values of RSD for intra-day and inter-day precision were obtained as 0.070–0.098 and 0.035–0.098, respectively. Hence, low RSD verified high precision of prepared electrochemical SARS-CoV-2 NP immunosensor. Accuracy as Bias % was also tested and low Bias % (Table S3) suggested the high accuracy of electrochemical SARS-CoV-2 NP immunosensor.

Conclusions

COVID-19 pandemic has caused important damage to society. Until now, many kits for SARS-CoV-2 sensing have been developed. For first time, sensitive electrochemical SARS-CoV‑2 nucleocapsid protein immunosensor based on Bi₂WO₆/Bi₂S₃ as electrode platform and g-C₃N₄/Au/WO₃ as signal amplification was presented in this work. This immunosensor was constructed by c-SARS-CoV-2-Ab₁ immobilization via strong electrostatic interaction and d-SARS-CoV-2-Ab₂ incubation via gold-amino affinity. Thus, the stable electrochemical signals were accomplished in terms of COVID-19 disease detection. In addition, the prepared electrochemical immunosensor had good ability in selective detection of SARS-CoV‑2 nucleocapsid protein. In spite of these advantages, the presented detection protocol was a little time consuming in immunological response time (30 min), indicating that this analysis time is significant in terms of faster diagnosis. Furthermore, the prepared immunosensor based on Bi₂WO₆/Bi₂S₃ and g-C₃N₄/Au/WO₃ was reproducible and reusable biosensor and did not include in time-consuming steps such as sensor preparation. Finally, this developed immunosensor can be easily integrated into a commercial biosensor tool and used for the determination of the other viral diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors declare no competing interests.