Impedimetric Nanobiosensor for the Detection of SARS-CoV-2 Antigens and Antibodies.

Detection of antigens and antibodies (Abs) is of great importance in determining the infection and immunity status of the population, as they are key parameters guiding the handling of pandemics. Current point-of-care (POC) devices are a convenient option for rapid screening; however, their sensitivity requires further improvement. We present an interdigitated gold nanowire-based impedance nanobiosensor to detect COVID-19-associated antigens (receptor-binding domain of S1 protein of the SARS-CoV-2 virus) and respective Abs appearing during and after infection. The electrochemical impedance spectroscopy technique was used to assess the changes in measured impedance resulting from the binding of respective analytes to the surface of the chip. After 20 min of incubation, the sensor devices demonstrate a high sensitivity of about 57 pS·sn per concentration decade and a limit of detection (LOD) of 0.99 pg/mL for anti-SARS-CoV-2 Abs and a sensitivity of around 21 pS·sn per concentration decade and an LOD of 0.14 pg/mL for the virus antigen detection. Finally, the analysis of clinical plasma samples demonstrates the applicability of the developed platform to assist clinicians and authorities in determining the infection or immunity status of the patients.

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

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.

Sensitive Detection of SARS-CoV-2 Variants Using an Electrochemical Impedance Spectroscopy Based Aptasensor.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a threat to public health and a worldwide crisis. This raised the need for quick, effective, and sensitive detection tools to prevent the rapid transmission rate of the infection. Therefore, this study aimed to develop an electrochemical impedance spectroscopy (EIS)-based aptasensor employing an interdigitated gold electrode (IDE) to detect SARS-CoV-2 Spike (S) glycoprotein and viral particles. This allowed us to sensitively detect SARS-CoV-2 S glycoprotein with a limit of detection (LOD) of 0.4 pg/mL in a buffer solution and to obtain a linear increase for concentrations between 0.2 to 0.8 pg/mL with high specificity. The proposed aptasensor also showed a good sensitivity towards the heat-inactivated SARS-CoV-2 variants in a buffer solution, where the Delta, Wuhan, and Alpha variants were captured at a viral titer of 6.45 ± 0.16 × 103 TCID50/mL, 6.20 × 104 TCID50/mL, and 5.32 ± 0.13 × 102 TCID50/mL, respectively. Furthermore, the detection of SARS-CoV-2 performed in a spiked human nasal fluid provided an LOD of 6.45 ± 0.16 × 103 TCID50/mL for the Delta variant in a 50 µL sample and a detection time of less than 25 min. Atomic force microscopy images complemented the EIS results in this study, revealing that the surface roughness of the IDE after each modification step increased, which indicates that the target was successfully captured. This label-free EIS-based aptasensor has promising potential for the rapid detection of SARS-CoV-2 in complex clinical samples.

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.

Improving immunoassay detection accuracy of anti-SARS-CoV-2 antibodies through dual modality validation.

A novel test strategy is proposed with dual-modality detection techniques for COVID-19 antibody detection. The full-length S protein of SARS-CoV-2 was chemically immobilized on a glass surface to capture anti-SARS-CoV-2 IgG in patient serum and was detected through either Electrochemical Impedance Spectroscopy (EIS) or fluorescence imaging with labeled secondary antibodies. Gold nanoparticles conjugated with protein G were used as the probe and the bound GNP-G was detected through EIS measurements. Anti-human-IgG conjugated with the fluorescent tag Alexa Fluor 488 was used as the probe for fluorescence imaging. Clinical SARS-CoV-2 IgG positive serum and negative controls were used to validate both modalities. For fluorescence-based detection, a high sensitivity was noticed with a quantification range of 0.01-0.1 A.U.C. and a LOD of 0.004 A.U.C. This study demonstrates the possibility of utilizing different measurement techniques in conjunction for improved COVID-19 serology testing.

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.

SenSARS: A Low-Cost Portable Electrochemical System for Ultra-Sensitive, Near Real-Time, Diagnostics of SARS-CoV-2 Infections.

A critical path to solving the SARS-CoV-2 pandemic, without further socioeconomic impact, is to stop its spread. For this to happen, pre- or asymptomatic individuals infected with the virus need to be detected and isolated opportunely. Unfortunately, there are no current ubiquitous (i.e., ultra-sensitive, cheap, and widely available) rapid testing tools capable of early detection of SARS-CoV-2 infections. In this article, we introduce an accurate, portable, and low-cost medical device and bio-nanosensing electrode dubbed SenSARS and its experimental validation. SenSARS' device measures the electrochemical impedance spectra of a disposable bio-modified screen-printed carbon-based working electrode (SPCE) to the changes in the concentration of SARS-CoV-2 antigen molecules ("S" spike proteins) contained within a sub-microliter fluid sample deposited on its surface. SenSARS offers real-time diagnostics and viral load tracking capabilities. Positive and negative control tests were performed in phosphate-buffered saline (PBS) at different concentrations (between 1 and 50 fg/mL) of SARS-CoV-2(S), Epstein-Barr virus (EBV) glycoprotein gp350, and Influenza H1N1 M1 recombinant viral proteins. We demonstrate that SenSARS is easy to use, with a portable and lightweight (< 200 g) instrument and disposable test electrodes (

Development of flexible electrochemical impedance spectroscopy-based biosensing platform for rapid screening of SARS-CoV-2 inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of its spike protein (S-protein) to the cell surface-expressing angiotensin-converting enzyme 2 (ACE2). Thus, inhibition of S-protein-ACE2 binding may impede SARS-CoV-2 cell entry and attenuate the progression of Coronavirus disease 2019 (COVID-19). In this study, an electrochemical impedance spectroscopy-based biosensing platform consisting of a recombinant ACE2-coated palladium nano-thin-film electrode as the core sensing element was fabricated for the screening of potential inhibitors against S-protein-ACE2 binding. The platform could detect interference of small analytes against S-protein-ACE2 binding at low analyte concentration and small volume (0.1 µg/mL and ~1 µL, estimated total analyte consumption < 4 pg) within 21 min. Thus, a few potential inhibitors of S-protein-ACE2 binding were identified. This includes (2S,3aS,6aS)-1-((S)-N-((S)-1-Carboxy-3-phenylpropyl)alanyl)tetrahydrocyclopenta[b] pyrrole-2-carboxylic acid (ramiprilat) and (2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-Carboxybutyl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid (perindoprilat) that reduced the binding affinity of S-protein to ACE2 by 72% and 67%; and SARS-CoV-2 in vitro infectivity to the ACE2-expressing human oral cavity squamous carcinoma cells (OEC-M1) by 36.4 and 20.1%, respectively, compared to the PBS control. These findings demonstrated the usefulness of the developed biosensing platform for the rapid screening of modulators for S-protein-ACE2 binding.