Electrochemical immunosensor for diagnosis of COVID-19

An immunosensor is a biosensor that detects antigen interactions using a particular antibody bound on the transducer's surface. These biosensors have high selectivity and sensitivity due to their interaction specificity. Owing to this characteristic, this type of sensor is attractive for several applications, especially in the medical area and bioanalysis. Among the types of immunosensors, electrochemical immunosensors have gained prominence due to their simplicity and portability, potentially enabling in situ detection as promising characteristic for analysis in emergency care. In this chapter, the potential of electrochemical immunosensors is presented, especially in applications related to clinical examinations and mainly in the diagnosis of SARS-CoV-2. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023. All rights reserved.

Copper-free click chemistry assisted antibodies for immunodetection of interleukin-10 in saliva

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is secreted in response to an acute phase inflammation in patients who are suffering from heart failure (HF). The aim of this work was to develop an electrochemical biosensor for determining salivary IL-10 levels. Biofunctionalization strategy was improved through the use of copper-free click chemistry for the developed sensor due to its advantages, leading to high quantitative yields of stable triazoles, rapid reaction, no cytotoxic Cu(I) catalyst requirement, and high specificity of cyclooctynes toward azides. The approach involved in binding of dibenzocyclooctyne acid (DBCO-COOH) to thiol-azide assembled gold microelectrodes, later capturing the monoclonal IL-10 antibody (IL-10 mAb), and ultimately allowing direct detection of IL-10 antigen. Fourier transform infrared spectroscopy (FTIR) and nanoplotter associated with fluorescence microscopy methods have been employed to analyze and prove the biofunctionalization of the gold microelectrodes. Moreover, the electrochemical impedance spectroscopy (EIS) technique was used for detecting IL-10 antigen. The developed immunosensor showed a semi-logarithmic linear range, from 0.1 pg/mL to 5 pg/mL with R2 = 0.9815 and a limit of quantitation (LOQ) of 0.1 pg/mL with relative standard deviation (RSD) of 10.67%. The specificity of the immunosensor was evaluated using an inflammatory cytokine, and none of it generated detectable EIS signals. Finally, the successful analysis of saliva samples from a healthy volunteer without Coronavirus (COVID-19) infection demonstrated the usefulness of the developed immunosensor.

PdPtRu trimetallic nanozymes and application to electrochemical immunosensor for sensitive SARS-COV-2 antigen detection.

Herein, a ternary PdPtRu nanodendrite as novel trimetallic nanozyme was reported, which possessed excellent peroxidase-like activity as well as electro-catalytic activity on account of the synergistic effect between the three metals. Based on the excellent electro-catalytic activity of trimetallic PdPtRu nanozyme toward the reduction of H2O2, the trimetallic nanozyme was applied to construct a brief electrochemical immunosensor for SARS-COV-2 antigen detection. Concretely, trimetallic PdPtRu nanodendrite was used to modify electrode surface, which not only generated high reduction current of H2O2 for signal amplification, but also provided massive active sites for capture antibody (Ab1) immobilization to construct immunosensor. In the presence of target SARS-COV-2 antigen, SiO2 nanosphere labeled detection antibody (Ab2) composites were introduced on the electrode surface according sandwich immuno-reaction. Due to the inhibitory effect of SiO2 nanosphere on the current signal, the current signal was decreased with the increasing target SARS-COV-2 antigen concentration. As a result, the proposed electrochemical immunosensor presented sensitive detection of SARS-COV-2 antigen with linear range from 1.0 pg/mL to 1.0 µg/mL and limit of detection down to 51.74 fg/mL. The proposed immunosensor provide a brief but sensitive antigen detection tool for rapid diagnosis of COVID-19.

Synergistically functionalized molybdenum disulfide-reduced graphene oxide nanohybrid based ultrasensitive electrochemical immunosensor for real sample analysis of COVID-19.

Herein, we have proposed a straightforward and label-free electrochemical immunosensing strategy supported on a glassy carbon electrode (GCE) modified with a biocompatible and conducting biopolymer functionalized molybdenum disulfide-reduced graphene oxide (CS-MoS2/rGO) nanohybrid to investigate the SARS-CoV-2 virus. CS-MoS2/rGO nanohybrid-based immunosensor employs recombinant SARS-CoV-2 Spike RBD protein (rSP) that specifically identifies antibodies against the SARS-CoV-2 virus via differential pulse voltammetry (DPV). The antigen-antibody interaction diminishes the current responses of the immunosensor. The obtained results indicate that the fabricated immunosensor is extraordinarily capable of highly sensitive and specific detection of the corresponding SARS-CoV-2 antibodies with a LOD of 2.38 zg mL-1 in phosphate buffer saline (PBS) samples over a broad linear range between 10 zg mL-1-100 ng mL-1. In addition, the proposed immunosensor can detect attomolar concentrations in spiked human serum samples. The performance of this immunosensor is assessed using actual serum samples from COVID-19-infected patients. The proposed immunosensor can accurately and substantially differentiate between (+) positive and (-) negative samples. As a result, the nanohybrid can provide insight into the conception of Point-of-Care Testing (POCT) platforms for cutting-edge infectious disease diagnostic methods.

ZnO-Based Electrochemical Immunosensor to Assess Vaccine-Induced Antibody-Mediated Immunity against Wild-Type and Gamma SARS-CoV-2 Strains.

The evaluation of serological responses to COVID-19 is crucial for population-level surveillance, developing new vaccines, and evaluating the efficacy of different immunization programs. Research and development of point-of-care test technologies remain essential to improving immunity assessment, especially for SARS-CoV-2 variants that partially evade vaccine-induced immune responses. In this work, an impedimetric biosensor based on the immobilization of the recombinant trimeric wild-type spike protein (S protein) on zinc oxide nanorods (ZnONRs) was employed for serological evaluation. We successfully assessed its applicability using serum samples from spike-based COVID-19 vaccines: ChAdOx1-S (Oxford-AstraZeneca) and BNT162b2 (Pfizer-BioNTech). Overall, the ZnONRs/ spike-modified electrode displayed accurate results for both vaccines, showing excellent potential as a tool for assessing and monitoring seroprevalence in the population. A refined outcome of this technology was achieved when the ZnO immunosensor was functionalized with the S protein from the P.1 linage (Gamma variant). Serological responses against samples from vaccinated individuals were acquired with excellent performance. Following studies based on traditional serological tests, the ZnONRs/spike immunosensor data reveal that ChAdOx1-S vaccinated individuals present significantly less antibody-mediated immunity against the Gamma variant than the BNT162b2 vaccine, highlighting the great potential of this point-of-care technology for evaluating vaccine-induced humoral immunity against different SARS-CoV-2 strains.

Using low-cost disposable immunosensor based on flexible PET screen-printed electrode modified with carbon black and gold nanoparticles for sensitive detection of SARS-CoV-2.

To help meet the global demand for reliable and inexpensive COVID-19 testing and environmental analysis of SARS-CoV-2, the present work reports the development and application of a highly efficient disposable electrochemical immunosensor for the detection of SARS-CoV-2 in clinical and environmental matrices. The sensor developed is composed of a screen-printed electrode (SPE) array which was constructed using conductive carbon ink printed on polyethylene terephthalate (PET) substrate made from disposable soft drink bottles. The recognition site (Spike S1 Antibody (anti-SP Ab)) was covalently immobilized on the working electrode surface, which was effectively modified with carbon black (CB) and gold nanoparticles (AuNPs). The immunosensing material was subjected to a multi-technique characterization analysis using X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) with elemental analysis via energy dispersive spectroscopy (EDS). The electrochemical characterization of the electrode surface and analytical measurements were performed using cyclic voltammetry (CV) and square-wave voltammetry (SWV). The immunosensor was easily applied for the conduct of rapid diagnoses or accurate quantitative environmental analyses by setting the incubation period to 10 min or 120 min. Under optimized conditions, the biosensor presented limits of detection (LODs) of 101 fg mL-1 and 46.2 fg mL-1 for 10 min and 120 min incubation periods, respectively; in addition, the sensor was successfully applied for SARS-CoV-2 detection and quantification in clinical and environmental samples. Considering the costs of all the raw materials required for manufacturing 200 units of the AuNP-CB/PET-SPE immunosensor, the production cost per unit is 0.29 USD.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Gold-binding peptide as a selective layer for electrochemical detection of SARS-CoV-2 antibodies.

Electrochemical immunosensors are excellent alternatives to prepare portable platforms used for rapid and inexpensive diagnostic of infectious diseases such as the recently emerged COVID-19. Incorporating synthetic peptides as selective recognition layers combined with nanomaterials such as gold nanoparticles (AuNPs) can significantly enhance the analytical performance of immunosensors. In the present study, an electrochemical immunosensor based on solid-binding peptide was built and evaluated towards SARS-CoV-2 Anti-S antibodies detection. The peptide used as recognition site has two important portions: one based on the viral receptor binding domain (RBD), capable of recognizing antibodies of the spike protein (Anti-S), and another suitable for interacting with gold nanoparticles. Gold-binding peptide (Pept/AuNP) dispersion was used directly to modify a screen-printed carbon electrode (SPE). The voltammetric behavior of the [Fe(CN)6]3-/4- probe after every construction and detection step was recorded using cyclic voltammetry by assessing the stability of the Pept/AuNP as a recognition layer onto the electrode surface. Differential pulse voltammetry was used as a detection technique, and a linear working range from 75 ng mL-1 to 15 µg mL-1 was established, with 1.059 µA dec-1 of sensitivity and R2 = 0.984. The response selectivity against SARS-CoV-2 Anti-S antibodies was investigated in presence of concomitant species. The immunosensor was used to detect SARS-CoV-2 Anti-spike protein (Anti-S) antibodies in human serum samples, successfully differentiating between negative and positive responses of samples at a 95% confidence level. Therefore, the gold-binding peptide is a promising tool to be applied as a selective layer for antibody detection.

Electrochemical paper-based antigen sensing platform using plant-derived monoclonal antibody for detecting SARS-CoV-2.

The current approaches of diagnostic platforms for detecting SARS-CoV-2 infections mostly relied on adapting the existing technology. In this work, a simple and low-cost electrochemical sensing platform for detecting SAR-CoV-2 antigen was established. The proposed sensor combined the innovative disposable paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. The cellulose nanocrystal was modified on screen-printed graphene electrode to provide the abundant COOH functional groups on electrode surface, leading to the high ability for antibody immobilization. The quantification of the presence receptor binding domain (RBD) spike protein of SARS-CoV-2 was performed using differential pulse voltammetry by monitoring the changing current of [Fe(CN)6]3-/4- redox solution. The current change of [Fe(CN)6]3-/4- before and after the presence of target RBD could be clearly distinguished, providing a linear relationship with RBD concentration in the range from 0.1 pg/mL to 500 ng/mL with the minimum limit of detection of 2.0 fg/mL. The proposed platform was successfully applied to detect RBD in nasopharyngeal swab samples with satisfactory results. Furthermore, the paper-based immunosensor was extended to quantify the RBD level in spiked saliva samples, demonstrating the broadly applicability of this system. This electrochemical paper-based immunosensor has the potential to be employed as a point-of-care testing for COVID-19 diagnosis.

An scFv-Based Impedimetric Immunosensor Using SPCE/AuNP for RBD of SARS-CoV-2 Detection

A single-chain variable fragment (scFv) is an antibody fragment composed of VH and VL linked by a hydrophilic linker that can be designed according to the shape of the target molecule and synthesized in prokaryotic or eukaryotic cells via biotechnology engineering. This study developed an electrochemical immunosensor that detects the RBD of SARS-CoV-2 using a screen-printed carbon electrode modified with gold nanoparticles, and scFv as a bioreceptor. Electrochemical impedance spectroscopy was employed to measure specific interactions of antigens with antibodies. The developed immunosensor had a limit of detection and a quantification limit of 4.86 ng mL(-1) and 16.20 ng mL(-1), respectively. The immunosensor was stable at room temperature for up to 30 days' storage. The immunosensor was assessed at biosafety level 3 using 33 nasopharyngeal swab specimens (clinical samples);the pieces of data were compared with quantitative Reverse Transcriptase-PCR. The agreement of the data, the low detection limit achieved, the rapid analysis (30 min), the miniaturization, and the portability of the instrument combined with the easiness to use has the potential to become Point of Care (POC) for diagnosing the COVID-19 disease.

Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva.

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81-15,000 U/mL, salivary IgG: 3.4-330 U/mL, and salivary IgA: 58-870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.

Biochar: An environmentally friendly platform for construction of a SARS-CoV-2 electrochemical immunosensor

Waste management is a key feature to ensure sustainable consumption and production patterns, and to combat the impacts of climate change. In this scenario, the production of biochar from different biomasses results in environmental and economic advantages. In this study, biochar was produced from sugarcane bagasse pyrolysis, to immobilize biomolecules, in order to assemble an electrochemical immunosensor to detect antibodies against SARS-CoV-2. For this, screen-printed carbon electrodes (SPCE) were modified with a dispersion of biochar and used to immobilize the receptor-binding-domain (RBD) against virus S-protein, through EDC/NHS crosslinking reaction. Under the best set of experimental conditions, negative and positive serum samples responses distinguished based on a cutoff value of 82.3 %, at a 95 % confidence level. The immunosensor showed selective behavior to antibodies against yellow fever and its performance was stable up to 7 days of storage. Therefore, biochar yielded from sugarcane bagasse is an ecofriendly material that can be used as a platform to immobilize biomolecules for construction of electrochemical biosensors.

Functionalized Titanium Dioxide Nanoparticle-Based Electrochemical Immunosensor for Detection of SARS-CoV-2 Antibody.

The advancement in biosensors can overcome the challenges faced by conventional diagnostic techniques for the detection of the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, the development of an accurate, rapid, sensitive, and selective diagnostic technique can mitigate adverse health conditions caused by SARS-CoV-2. This work proposes the development of an electrochemical immunosensor based on bio-nanocomposites for the sensitive detection of SARS-CoV-2 antibodies through the differential pulse voltammetry (DPV) electroanalytical method. The facile synthesis of chitosan-functionalized titanium dioxide nanoparticles (TiO2-CS bio-nanocomposites) is performed using the sol-gel method. Characterization of the TiO2-CS bio-nanocomposite is accomplished using UV-vis spectroscopy, Raman spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). The electrochemical performance is studied using cyclic voltammetry (CV), DPV, and electrochemical impedance spectroscopy (EIS) for its electroanalytical and biosensing capabilities. The developed immunosensing platform has a high sensitivity with a wide range of detection from 50 ag mL-1 to 1 ng mL-1. The detection limit of the SARS-CoV-2 antibody in buffer media is obtained to be 3.42 ag mL-1 and the limit of quantitation (LOQ) to be 10.38 ag mL-1. The electrochemical immunosensor has high selectivity in different interfering analytes and is stable for 10 days. The results suggest that the developed electrochemical immunosensor can be applicable for real sample analysis and further high-throughput testing.

Graphene-Binding Peptide in Fusion with SARS-CoV-2 Antigen for Electrochemical Immunosensor Construction.

The development of immunosensors to detect antibodies or antigens has stood out in the face of traditional methods for diagnosing emerging diseases such as the one caused by the SARS-CoV-2 virus. The present study reports the construction of a simplified electrochemical immunosensor using a graphene-binding peptide applied as a recognition site to detect SARS-CoV-2 antibodies. A screen-printed electrode was used for sensor preparation by adding a solution of peptide and reduced graphene oxide (rGO). The peptide-rGO suspension was characterized by scanning electron microscopy (SEM), Raman spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). The electrochemical characterization (electrochemical impedance spectroscopy-EIS, cyclic voltammetry-CV and differential pulse voltammetry-DPV) was performed on the modified electrode. The immunosensor response is based on the decrease in the faradaic signal of an electrochemical probe resulting from immunocomplex formation. Using the best set of experimental conditions, the analytic curve obtained showed a good linear regression (r2 = 0.913) and a limit of detection (LOD) of 0.77 µg mL-1 for antibody detection. The CV and EIS results proved the efficiency of device assembly. The high selectivity of the platform, which can be attributed to the peptide, was demonstrated by the decrease in the current percentage for samples with antibody against the SARS-CoV-2 S protein and the increase in the other antibodies tested. Additionally, the DPV measurements showed a clearly distinguishable response in assays against human serum samples, with sera with a response above 95% being considered negative, whereas responses below this value were considered positive. The diagnostic platform developed with specific peptides is promising and has the potential for application in the diagnosis of other infections that lead to high antibody titers.

Determination of rSpike Protein by Specific Antibodies with Screen-Printed Carbon Electrode Modified by Electrodeposited Gold Nanostructures.

In this research, we assessed the applicability of electrochemical sensing techniques for detecting specific antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins in the blood serum of patient samples following coronavirus disease 2019 (COVID-19). Herein, screen-printed carbon electrodes (SPCE) with electrodeposited gold nanostructures (AuNS) were modified with L-Cysteine for further covalent immobilization of recombinant SARS-CoV-2 spike proteins (rSpike). The affinity interactions of the rSpike protein with specific antibodies against this protein (anti-rSpike) were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. It was revealed that the SPCE electroactive surface area increased from 1.49 ± 0.02 cm2 to 1.82 ± 0.01 cm2 when AuNS were electrodeposited, and the value of the heterogeneous electron transfer rate constant (k0) changed from 6.30 × 10-5 to 14.56 × 10-5. The performance of the developed electrochemical immunosensor was evaluated by calculating the limit of detection and limit of quantification, giving values of 0.27 nM and 0.81 nM for CV and 0.14 nM and 0.42 nM for DPV. Furthermore, a specificity test was performed with a solution of antibodies against bovine serum albumin as the control aliquot, which was used to assess nonspecific binding, and this evaluation revealed that the developed rSpike-based sensor exhibits low nonspecific binding towards anti-rSpike antibodies.

Rapid Quantification of SARS-Cov-2 Spike Protein Enhanced with a Machine Learning Technique Integrated in a Smart and Portable Immunosensor.

An IoT-WiFi smart and portable electrochemical immunosensor for the quantification of SARS-CoV-2 spike protein was developed with integrated machine learning features. The immunoenzymatic sensor is based on the immobilization of monoclonal antibodies directed at the SARS-CoV-2 S1 subunit on Screen-Printed Electrodes functionalized with gold nanoparticles. The analytical protocol involves a single-step sample incubation. Immunosensor performance was validated in a viral transfer medium which is commonly used for the desorption of nasopharyngeal swabs. Remarkable specificity of the response was demonstrated by testing H1N1 Hemagglutinin from swine-origin influenza A virus and Spike Protein S1 from Middle East respiratory syndrome coronavirus. Machine learning was successfully used for data processing and analysis. Different support vector machine classifiers were evaluated, proving that algorithms affect the classifier accuracy. The test accuracy of the best classification model in terms of true positive/true negative sample classification was 97.3%. In addition, the ML algorithm can be easily integrated into cloud-based portable Wi-Fi devices. Finally, the immunosensor was successfully tested using a third generation replicating incompetent lentiviral vector pseudotyped with SARS-CoV-2 spike glycoprotein, thus proving the applicability of the immunosensor to whole virus detection.

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Highly Sensitive Electrochemical Immunosensor Platforms for Dual Detection of SARS-CoV-2 Antigen and Antibody based on Gold Nanoparticle Functionalized Graphene Oxide Nanocomposites.

In this work, we report a facile synthesis of graphene oxide-gold (GO-Au) nanocomposites by electrodeposition. The fabricated electrochemical immunosensors are utilized for the dual detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen and SARS-CoV-2 antibody. The GO-Au nanocomposites has been characterized by UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) for its biosensing properties. The linear detection range of the SARS-CoV-2 antigen immunosensor is 10.0 ag mL-1 to 50.0 ng mL-1, whereas that for the antibody immunosensor ranges from 1.0 fg mL-1 to 1.0 ng mL-1. The calculated limit of detection (LOD) of the SARS-CoV-2 antigen immunosensor is 3.99 ag mL-1, and that for SARS-CoV-2 antibody immunosensor is 1.0 fg mL-1 with high sensitivity. The validation of the immunosensor has also been carried out on patient serum and patient swab samples from COVID-19 patients. The results suggest successful utilization of the immunosensors with a very low detection limit enabling its use in clinical samples. Further work is needed for the standardization of the results and translation in screen-printed electrodes for use in portable commercial applications.

Towards an Electrochemical Immunosensor for the Detection of Antibodies against SARS-CoV-2 Spike Protein

Herein we report the electrochemical system for the detection of specific antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in blood serum patient samples after coronavirus disease 2019 (COVID-19). For this purpose, the recombinant SARS-CoV-2 spike protein (SCoV2-rS) was covalently immobilised on the surface of the gold electrode pre-modified with mixed self-assembled monolayer (SAMmix) consisting of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. The affinity interaction of SCoV2-rS with specific antibodies against this protein (anti-rS) was detected using two electrochemical methods: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The anti-rS was detected with a detection limit of 2.53 nM and 1.99 nM using CV and EIS methods, respectively. The developed electrochemical immunosensor is suitable for the confirmation of COVID-19 infection or immune response in humans after vaccination.