Xun-Hai You,Yao Liu,Yan-Yan Li,Bing Zhao,Yong Yang,Rohan Weerasooriya,Xing Chen
Sensitive detection of SARS-CoV-2 spike protein based on electrochemical impedance spectroscopy of Fe3O4@SiO2–Au/GCE biosensor
SARS-CoV-2 spike protein,Fe3O4@SiO2–Au nanocomposites,Electrochemical biosensor,Electrochemical impedance spectroscopy
Highly contagious COVID-19 disease is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which poses a serious threat to global public health. Therefore, the development of a fast and reliable method for the detection of SARS-CoV-2 is an urgent research need. The Fe3O4@SiO2–Au is enriched with a variety of functional groups, which can be used to fabricate a sensitive electrochemical biosensor by biofunctionalization with angiotensin-converting enzyme 2 (ACE2). Accordingly, we developed a novel electrochemical sensor by chemically modifying a glassy carbon electrode (GCE) with Fe3O4@SiO2–Au nanocomposites (hereafter Fe3O4@SiO2–Au/GCE) for the rapid detection of S-protein spiked SARS-CoV-2 by electrochemical impedance spectroscopy (EIS). The new electrochemical sensor has a low limit detection (viz., 4.78 pg/mL) and a wide linear dynamic range (viz., 0.1 ng/mL to 10 μg/mL) for detecting the EIS response signal of S-protein. The robust Fe3O4@SiO2–Au/GCE biosensor has high selectivity, stability, and reproducibility for the detection of S-protein with good recovery of saliva samples.
Sensitive detection of SARS-CoV-2 spike protein based on electrochemical impedance spectroscopy of Fe3O4@SiO2–Au/GCE biosensor

Highly contagious COVID-19 disease is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which poses a serious threat to global public health. Therefore, the development of a fast and reliable method for the detection of SARS-CoV-2 is an urgent research need. The Fe3O4@SiO2–Au is enriched with a variety of functional groups, which can be used to fabricate a sensitive electrochemical biosensor by biofunctionalization with angiotensin-converting enzyme 2 (ACE2). Accordingly, we developed a novel electrochemical sensor by chemically modifying a glassy carbon electrode (GCE) with Fe3O4@SiO2–Au nanocomposites (hereafter Fe3O4@SiO2–Au/GCE) for the rapid detection of S-protein spiked SARS-CoV-2 by electrochemical impedance spectroscopy (EIS). The new electrochemical sensor has a low limit detection (viz., 4.78 pg/mL) and a wide linear dynamic range (viz., 0.1 ng/mL to 10 μg/mL) for detecting the EIS response signal of S-protein. The robust Fe3O4@SiO2–Au/GCE biosensor has high selectivity, stability, and reproducibility for the detection of S-protein with good recovery of saliva samples.

Since the 21st century, three coronavirus outbreaks were reported at a global scale: severe acute respiratory syndrome (SARS) in 2002, Middle East respiratory syndrome (MERS) in 2012, and novel pneumonia caused by a coronavirus (SARS-CoV-2) in 2019 Corona Virus Disease 2019 (COVID-19) [1,2]. It is reported that the SARS-CoV-2 genome sequence is 77% and 50% homologous to SARS-CoV and MERS-CoV, respectively [3]. SARS-CoV-2 is more widespread compared to the other respiratory syndromes by spreading over two hundred countries causing 600 million infections and about 6 million deaths. Therefore, the development of a rapid and sensitive method for the detection of SARS-CoV-2 is urgently needed. Currently, virus detection methods rely on conventional laboratory techniques, including nucleic acid detection and serological testing [[4], [5], [6]]. Among nucleic acid assay routes, the reverse transcription polymerase chain reaction (RT-PCR) is the core method for SARS-CoV-2 detection. However, RT-PCR-based methods require skilled personnel and specialized equipment [7]. Serological assays viz., including antigen and antibody assays, are based on antigen-antibody-specific binding assays [8,9]. There is a voluminous literature on the benefits of serological assays for SARS-CoV-2 detection, where antigen assays can only be used adjunct to detect SARS-CoV-2 [10]. Although these methods consume less time than RT-PCR measurements, they still have limitations due to arduous sample preparations and low sensitivity. Further the production of antibody assays requires SARS-CoV-2 infected patients for a period of 5–7 days. Previously, the development of electrochemical biosensors for medical diagnostic applications [[11], [12], [13]], including diabetes, Alzheimer's, and other diseases, have shown that they can be adapted as a viral detection tool with high sensitivity, high specificity, low cost, and fast response time (Table S1). Notably, with the miniaturization and smart automation of electrochemical devices, these biosensors are also suited for clinical diagnosis and rapid detection of SARS-CoV-2 [[14], [15], [16]]. One of the key aspects of constructing electrochemical biosensors is the development of stable materials with desired conductivity and selectivity to an analyte [17,18]. There is a considerable literature on the development of different nanomaterials in electrochemical biosensors [[19], [20], [21], [22]], including gold, carbon, metal oxide nanomaterials, etc. Fe3O4 nanoparticles attract attention in electrochemical sensor development particularly due to their biocompatibility, simple preparation, magnetic properties, high sorption capacity, and environmentally benign nature [23]. However, iron-derived substrates readily agglomerate and undergo rapid oxidation which limits their efficient use in sensor developments [24,25]. To overcome these limitations and enhance stability, Fe3O4 nanoparticles are suitably compounded with carbon-derived substrates, metal oxides, and other metals, or polymers, etc. [23]. Presently, we developed a sensor by modifying glassy carbon electrode (GCE) with Fe3O4@SiO2–Au nanocomposites to detect S-protein for SARS-CoV-2 diagnosis by electrochemical impedance spectroscopy (EIS). To facilitate electron transfer, minimize agglomeration, and retard undesired oxidation, the Fe3O4 nanoparticles were first coated with a thin SiO2 layer and then doped with Au nanoparticles. To improve selectivity and sensitivity of SARS-CoV-2 detection, angiotensin-converting enzyme 2 (ACE2) was introduced to functionalize Fe3O4@SiO2–Au composite to enhance S-protein binding ability [[26], [27], [28], [29]]. This enables the rapid diagnosis of SARS-CoV-2 virus, which may open a new direction in COVID-19 research.

The SARS-CoV-2 S-protein and Fc-tag-tagged human ACE2 were obtained from Sino Biological (PR China). Analytical grade, ferric chloride hexahydrate (FeCl3·6H2O), ethylene glycol (C2H6O2), trisodium citrate dihydrate (C6H5Na3O7·2H2O), sodium acetate anhydrous (CH3COONa), tetraethyl orthosilicate (TEOS), ammonia, 3-aminopropyl-triethoxysilane (APTES), chloroauric acid (HAuCl4·4H2O), ethanol, potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4·12H2O), glutaraldehyde (GA), glucose, ascorbic acid, norfloxacin, uric acid, tenofovir, favipiravir, histidine, oxytetracycline were purchased from Sinopharm Chemical Reagent Co., Ltd (PR China) and used as received. Human IgG and bovine serum albumin (BSA) were purchased from Dingguo Changsheng Biotechnology Limited Company (PR China). The real saliva samples were collected from the Hefei University of Technology Hospital. Ultra-high pure water (conductivity 0.0548 μS/cm) was used in laboratory preparations. High-resolution scanning electron microscopy (HRSEM) images of the samples were obtained by Regulus 8230 at an operating voltage of 15 kV (Hitachi Ltd., Japan). Transmission electron microscopy (TEM) micrographs were recorded on JEM-1400FLASH (JEOL, Japan). X-ray diffraction (XRD) patterns of the samples were recorded by Xpert PRO MPD (Nalytical, Netherlands). Magnetic measurement was carried out using an MPMS 3 vibrating sample magnetometer (Quantum Design, USA). The fourier-transform infrared spectroscopy (FTIR) technique was carried out using a Nicolet IS50 iN10 instrument (Thermo Nicolet, USA). Zeta potential was carried out using a Zetasizer Nano ZS-90 (Spectris, China). X-ray photoelectron spectroscopy (XPS) technique was used with an EscaLab 250Xi instrument (Thermo, USA).

The Fe3O4 nanoparticles were synthesized as described in Liu [30] with the following modifications. 2.025 g FeCl3•6H2O was dissolved in 60 mL ethylene glycol with stirring for 30 min, then 0.88 g C6H5Na3O7•2H2O was added and heated to 60 °C with stirring continued for 30 min. Finally, 9.84 g CH3COONa were added to provide alkaline conditions enabling complete dissolution of the substrate. The dark yellow solution thus received was transferred to PTFE lined stainless-steel sealed container and autoclaved at 180 °C for 8 h. After cooling to room temperature, the substrate (Fe3O4) was magnetically separated and washed with ethanol and deionized water for three times each, then vacuum dried at 60 °C.

Using the Stöber improvement method [31], 0.05 g Fe3O4 nanoparticles were dissolved in a mixture of ethanol (80 mL) and deionized water (16 mL), sonicated for 20 min. Subsequently, to this mixture ammonia solution (2 mL, 28 wt%) was added followed by the slow addition of TEOS (1 mL) and was kept stirring at room temperature for 6 h. The resultant substrate was magnetically recovered and washed with ethanol and deionized for three times each, then vacuum dried at 60 °C to yield Fe3O4@SiO2 nanocomposites.

Au nanoparticles were obtained by reducing HAuCl4•4H2O with sodium citrate [32]. Briefly, HAuCl4•4H2O (100 mL, 1 wt %) aqueous solution was heated at 100 °C and 10 mL 38.8 mM C6H5Na3O7•2H2O was added into the stirred solution when it started boiling. Finally, the dark brown colored Au nanoparticles were obtained.

To functionalize Fe3O4@SiO2 with –NH2 groups, 0.1 g Fe3O4@SiO2 was dispersed in ethanol (47.5 mL) and deionized water (2.5 mL) mixture followed by the addition of 0.4 mL APTES into the suspension with stirring for 4 h (Fe3O4@SiO2–NH2). The Fe3O4@SiO2–NH2 was re-dissolved in 40 mL deionized water, and then a certain amount of Au nanoparticles was added with stirring for 4 h. The resultant nanoparticles were magnetically separated and washed with ethanol and deionized water for three times each, then vacuum dried at 60 °C. In addition, the Au nanoparticles loading on Fe3O4@SiO2 composite were varied between 5 mL, 15 mL and 25 mL Au nanoparticles solution (hereafter designated as Fe3O4@SiO2-Aux where x = 1,2,3). Without special instructions, Fe3O4@SiO2–Au nanocomposites synthesized from 15 mL Au nanoparticles were applied in the subsequent experiments.

The fabrication methodology of the chemically modified glassy carbon electrode (GCE) used for SARS-CoV-2 S-protein detection is shown in Scheme 1 . The GCE was polished to a mirror surface using alumina powder with decreasing particle sizes: 1.0 μm, 0.3 μm, and 0.05 μm. Then the GCEs were ultrasonically cleaned with ethanol and ultrapure water for 3 min. Afterward, 6 μL of 3 mg/mL Fe3O4@SiO2–Au suspension was added dropwise onto the electrode surface to obtain a chemically modified electrode, e.g., Fe3O4@SiO2–Au/GCE. The Fe3O4@SiO2–Au/GCE was then functionalized using glutaraldehyde (GA), ACE2, and bovine serum albumin (BSA). To modify with ACE2, the Fe3O4@SiO2–Au/GCE surface was first functionalized with GA, then the receptor protein ACE2 was attached to the electrode surface at room temperature. Subsequently, the electrode surface was incubated using BSA prepared to block the possible binding sites of GA on the electrode surface (BSA/ACE2/GA/Fe3O4@SiO2–Au/GCE).

The binding of SARS-CoV-2 S-protein onto the electrochemical biosensor was attained by adding 6 μL S-protein solution on the BSA/ACE2/GA/Fe3O4@SiO2–Au/GCE surface and incubating at 37 °C for 30 min. The cyclic voltammetry (CV) curves and electrochemical impedance spectrograms (EIS) were obtained by a three-electrode configuration, viz. chemically modified GCE, Ag/AgCl reference, and Pt counter electrodes, using 5.0 mM [Fe(CN)6]3−/4− in 0.1 M PBS with a pH 7.0 (Electrochemical station, CHI760E, China). The scan rate of the electrochemical analyzer was set at 100 mV/s in the CV experiments; EIS measurements were carried out in a frequency range of 0.1 Hz–100 kHz, with a signal amplitude (<10 mV) and open-circuit potential of 0.33 V. All experiments were performed at room temperature. The electrochemical impedance data was modeled with modified equivalent circuits using ZView software to estimate the charge transfer resistance under different experimental conditions. Finally, an electroanalytical method was developed based on EIS for S-protein detection using the newly developed chemically modified Fe3O4@SiO2–Au electrode.

Fig. 1 shows the morphology and micro-structures of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2–Au nanomaterials observed through HRSEM and TEM. Fe3O4 particulates tend to coagulate readily and citrate ligand was used to minimize coagulation [33]. As shown in Fig. 1a and d, Fe3O4 particulates are well-dispersed and spherical around the 60–70 nm size range. However, as shown in Fig. 1b, the Fe3O4@SiO2 nanocomposites are not well resolved to observe SiO2 coating around Fe3O4 (except for some tonal variations). Therefore, the SiO2 coating around the Fe3O4 forming a core-shell structure is shown in TEM analysis. The thickness of the SiO2 layer is around 5 nm (Fig. 1e). The spread of Au nanoparticles around 15 nm average size on Fe3O4@SiO2 surface is visible in both SEM and TEM images. As shown in Fig. 1c and f, the Au nanoparticles are well-spread on the Fe3O4@SiO2 surface forming a large proportion of active sites to sequestrate ACE2 receptor protein. The XRD diffractograms of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2–Au nanomaterials are shown in Fig. 2 a. The X-ray diffraction peaks of Fe3O4 at 2θ of 30.2°, 35.6°, 43.2°, 53.6°, 57.2°, and 62.7°, respectively are in agreement with spinel structure corresponding to (220), (311), (400), (422), (511), and (440) lattice planes (JCPDF:19–0629) [34,35]. In addition, the intensity of these diffraction peaks and the standard patterns are almost the same, indicating good Fe3O4 crystallinity. Further, the X-ray diffractograms of Fe3O4@SiO2 and Fe3O4 are also similar due to the amorphous nature of SiO2 coating. The XRD data of Fe3O4@SiO2–Au show the presence of Fe3O4 along (111) and (200) lattice planes of cubic Au nanoparticles corresponding to 2θ at 38.2° and 44.4°. The experimental data confirms further the successful incorporation of Au cubic nanocrystals on Fe3O4@SiO2 composites (hereafter Fe3O4@SiO2–Au). The magnetic properties of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2–Au nanomaterials are shown in Fig. 2b. The remanence and coercivity of material show their resistivity to demagnetization. Presently, all our nanomaterials observed zero remanence and coercivity values confirming their super magnetic properties. The B–H curves of Fe3O4@SiO2 and Fe3O4@SiO2–Au are almost overlapped showing that the Au addition did not appreciably alter the magnetic strength of the composite. The magnetization intensity of Fe3O4 nanoparticles decreased from 87.67 emu/g to 39.18 emu/g and 37.61 emu/g upon sequential cladding with SiO2 and Au doping, which confirms the successful synthesis of Fe3O4@SiO2–Au. The FTIR spectra of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2–Au nanomaterials are shown in Fig. 3 a. The characteristic peaks detected at 1630 cm−1 and 3430 cm−1 are attributed to the stretching vibration of –OH, while the 799 cm−1 and 1090 cm−1 are ascribed due to Si–O stretching vibrations (this band is absent in Fe3O4). The IR bands at 576 cm−1 and 1400 cm−1 are specific to the stretching of Fe–O and –COOH, respectively [36]. Interestingly, the IR intensity of Fe–O bands decreases as Fe3O4 > Fe3O4@SiO2 > Fe3O4@SiO2–Au, which may be related to the coating of SiO2 and doping of Au nanoparticles. Fig. 3b shows the zeta potential values of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2–Au suspensions measured in pH 7.0. Bare Fe3O4 nanoparticles show a −4.11 mV zeta potential. After incorporating SiO2 onto Fe3O4 nanoparticles, the surface becomes negatively charged due to the abundance of –OH offsets Fe–O charging. The negative zeta potential values show little agglomeration of Fe3O4@SiO2 nanocomposites. When APTES is used to functionalize Fe3O4@SiO2 sites a surface charge reversal occurred confirming the grafting of positively charged amino groups to the terminus of the substrates (viz., Fe3O4@SiO2–NH2). The positively charged Fe3O4@SiO2–NH2 sites adhere to Au nanoparticles readily again reversing the surface charge [37]. According to IR and zeta potential data, the –OH, –COOH, and –NH2 groups abut from the Fe3O4@SiO2–Au surface favor intimate interactions with receptor protein ACE2. Fig. 4a shows the XPS survey spectra Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2–Au nanomaterials, the presence of Fe, Si, Au, and associate valence states are confirmed. As shown in Fig. 4b, in all samples the deconvolved peaks at 710.2 eV (Fe 2p1/2) and 723.6 eV (Fe 2p2/3) with a satellite confirming the presence of Fe2+. Similarly, the peaks at 711.1 eV and 724.67 eV and the satellite show Fe3+ [38,39]. Moreover, the signatures of Fe 2p peaks do not vary which verifies the presence of Fe2+ and Fe3+ states [38,39]. The positions of the prominent Fe 2p peaks of the three substrates magnetic did not shift, which verifies that all Fe2+ and Fe3+ in all samples. As the XPS analysis was within 5 nm depth of the sample surface, the fluctuation of Fe 2p peaks during cladding and doping may be wide. Fig. S1 shows the stability tests of three nanomaterials, confirming the contribution of SiO2 coating to the stability of the nanocomposite coatings by comparing the changes in the redox peak currents of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2–Au nanomaterials. The results conclude that Fe3O4@SiO2–Au modified GCE is robust to the fabrication biosensor. Also shown in Fig. S1g and h, the oxidation peak current of the Fe3O4@SiO2–Au nanomaterials modified electrode was reduced by 9.2% from day 1 to day 14. Therefore, all electrochemical biosensor data presented in this study were obtained using the newly modified electrode. The cyclic voltametric curves (CV) obtained for 5.0 mM [Fe (CN)6]3−/4− in 0.1 M PBS at pH 7.0 using bare and chemically modified GCE sensors are shown in Fig. S2. Always the CV curves show a symmetry due to the reversible nature of Fe2+→ Fe3+ electron transfer. The highest CV current peak is observed with Fe3O4@SiO2–Au/GCE sensor due to the presence of Au nanoparticles (Fig. S2a). The current peak values decrease in order Fe3O4@SiO2–Au/GCE > Fe3O4/GCE > Fe3O4@SiO2/GCE > bare GCE showing the hindrance for electrons transfer due to the presence of SiO2. Fig. S2b shows the calibration curve to the peak current intensity with the square root of the scanning rate recorded for different nanomaterial-modified electrodes. The calculated electrochemically active surface area of bare GCE, Fe3O4/GCE, Fe3O4@SiO2/GCE, and Fe3O4@SiO2–Au/GCE obtained by Randles-Sevcik formula [40] is 0.043 cm2, 0.055 cm2, 0.051 cm2 and 0.060 cm2, respectively. The high electron transport capacity of Fe3O4 nanoparticles and Au nanoparticles can increase the electrochemically active area of the modified electrode, providing more electrochemically active sites for receptor protein ACE2 immobilization.

The as-fabricated electrochemical biosensor process was elucidated by CV and EIS in 5.0 mM [Fe(CN)6]3−/4− in 0.1 M PBS with a pH 7.0. As shown in Fig. 5 a, after the modification of GCE by the Fe3O4@SiO2–Au, the value of the redox current dramatically augmented compared to the GCE. This is due to the good electrochemical activity of Fe3O4@SiO2–Au which accelerates the electron transfer on the electrode surface. When GA and the receptor protein ACE2 are immobilized on the modified electrode surface, the redox current significantly decreased due to due to the presence of cross-linked macromolecular structures in GA and the receptor protein ACE2, which prevented electron transfer. Moreover, when the BSA was used to block the non-specific active sites, the redox peak further decreased. The lowest redox peak for the S-protein was attributed to the tight binding of the S-protein and the receptor protein ACE2, which made the exchange reaction between electrons at the electrode surface more difficult. Fig. 5b shows Nyquist plots including semicircle (a measure of electron transfer rate) and linear (a measure of charge diffusion) segments representing high and low-frequency regions [[41], [42], [43]]. The EIS measurements are in agreement with the CV data. The Fe3O4@SiO2–Au modified GCE had the smallest charge-transfer resistance (R ct) compared to the bare GCE. When it's surface was added with GA cross-linking, the R ct increased. Especially, when ACE2, BSA, and S-protein were added on the electrode surface, the R ct values increased orderly due to the non-conductive properties of these protein layers. To evaluate the electrochemical reaction kinetics of SARS-CoV-2 S-protein at the BSA/ACE2/GA/Fe3O4@SiO2–Au/GCE, different CV curves were measured in solutions containing 5.0 mM [Fe(CN)6]3−/4− in 0.1 M PBS with a pH 7.0. The redox peak currents versus the square root of the scan rate curves (Fig. S3) indicate that the electron transfer process on as-fabricated electrochemical biosensor is diffusion-controlled [44].

In order to obtain the best sensitivity of S-protein detection, the optimization experiments including the concentration of ACE2, the interaction temperature, and the interaction time of S-protein and ACE2 (Fig. 6 ). When the ACE2 is varied between 1 and 25 μg/mL, the R ct value was optimal at 20 μg/mL ACE2 and afterward, it shows a slight decline. The system temperature and time exert a significant impact on the biochemical activity of S-protein. As shown in Fig. 6b and c, optimal R ct values were obtained at 37 °C interaction temperature and 30 min interaction time. Interestingly it represents the average body temperature of humans. In subsequent studies, the following experimental conditions were used; 20 μg/mL ACE2 concentration, 37 °C interaction temperature, and 30 min interaction time.

A separate experiment was carried out to determine the performance of the Fe3O4@SiO2–Au/GCE biosensor for S-protein detection as a function of Au nanoparticles loading. In this experiment, the loading of Au nanoparticles onto Fe3O4@SiO2 was varied between 5 mL, 15 mL, 25 mL (Fig. S4). The GCE was then chemically modified using Fe3O4@SiO2-Aux where x ranged from 1, 2, 3 (Fe3O4@SiO2-Aux/GCE). The S-protein detection performance obtained by Fe3O4@SiO2-Aux/GCE sensor is shown in Fig. S5. Initially, the EIS response signal steadily increased with the Au nanoparticles loading showing an optimal value when Fe3O4@SiO2–Au2/GCE sensor is used for measurements. At suitable Au nanoparticles loading, well-dispersed particulates yield an abundance of reactive sites for S-protein binding. When the Au nanoparticles loading further increased 25 mL, the reactivity of the Fe3O4@SiO2–Au3/GCE to S-protein is somewhat hindered as a result of particulates agglomeration (Fig. S5f). Therefore, in optimizing the performance of biosensors for S-protein detection Fe3O4@SiO2–Au2/GCE (designated as Fe3O4@SiO2–Au/GCE) is used. The EIS response signals of 0.1 ng/mL S-protein solution measured with Fe3O4@SiO2–Au/GCE biosensor were also simulated using a modified Randles equivalent circuit [45]. Fig. 7 a shows the agreement between experimental observations and the modeled data. The modified Randles model was also used to interpret the Nyquist plots for a series of S-protein concentrations (0.1 ng/mL to 10 μg/mL) (Fig. 7b). The calculated R ct values show a linear dependence with the logarithmic S-protein concentration when the solution matrix conditions are matched. The relationship between S-protein concentration as a function of ΔR ct was estimated as ΔR ct = 3605 Log C + 12,121 (limit of S-protein detection 4.78 ng/mL; R 2 = 0.991) (Fig. 7c). The sensitivity and the linear dynamic range of the SARS-CoV-2 S-protein determination against our method are compared as shown in Table 1 . The sensitivity and the linear dynamic range of S-protein detection depend on the nature of the sensors, and the electrochemical method used (for comparison, data obtained by molecular spectroscopic methods were also given). In terms of sensitivity and the linear dynamic range, the Fe3O4@SiO2–Au/GCE sensor developed presently shows the highest performance for S-protein detection by the EIS method.

We examined the selectivity, repeatability, and reproductivity of Fe3O4@SiO2–Au/GCE for the detection of S-protein by the EIS method using optimal experimental conditions developed in this study. In all these experiments 0.1 ng/mL S-protein solution was used. For selectivity analysis glucose, ascorbic acid, BSA, IgG, norfloxacin, uric acid, tenofovir, favipiravir, histidine, oxytetracycline were used as potential interferants. As shown in Fig. S6a, in the presence of these interferents, the S-protein in the solution can be detected with high selectivity (RSD <4%). In evaluating the sensor reproduction, six identical Fe3O4@SiO2–Au/GCE sensors were fabricated for S-protein measurements with good reproducibility (Fig. S6b; RSD <1%). To determine the repeatability, a newly prepared Fe3O4@SiO2–Au/GCE sensor was used for two consecutive weeks for measurements of S-protein concentration. The RSD value of S-protein detection was always less than 5% (Fig. S6c).

The detection of SARS-CoV-2 S-protein in saliva using Fe3O4@SiO2–Au/GCE biosensor was also carried out by multiple standard addition method. The filtered and diluted saliva sample was spiked with S-protein at varying concentrations between 1 and 100 ng/mL, and the final analyte concentration was determined in triplicate by EIS; the results thus obtained are given in Table 2 . The spiked recovery of S-protein in the saliva is always above 97% and the relative standard deviation is below 5%. The results indicate the suitability of Fe3O4@SiO2–Au/GCE biosensor in detecting SARS-CoV-2 S-protein in saliva with high precision and accuracy.

Preventing the spread of the SARS-CoV-2 virus and its variants requires the development of a rapid and cost-effective detection method. To our knowledge for the first time, we developed an electrochemical sensor by chemically modifying a GCE with Fe3O4@SiO2–Au (Fe3O4@SiO2–Au/GCE) for rapid detection of SARS-CoV-2 S-protein with a wide dynamic range (0.1 ng/mL to 10 μg/mL) and low limit detection (4.78 pg/mL). The new electrochemical sensor shows robust behavior with excellent stability and reproducibility for S-protein detection. Moreover, the sensor could ultimately lead to corresponding determination in real samples. Once a miniaturized module of the electrochemical sensor is fabricated (currently in progress), it holds promise as a sensitive screening method to combat the SARS-CoV-2 global endemic.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.