Electrochemical Biosensor for the Determination of Specific Antibodies against SARS-CoV-2 Spike Protein.

In this article, we report the development of an electrochemical biosensor for the determination of the SARS-CoV-2 spike protein (rS). A gold disc electrode was electrochemically modified to form the nanocrystalline gold structure on the surface. Then, it was further altered by a self-assembling monolayer based on a mixture of two alkane thiols: 11-mercaptoundecanoic acid (11-MUA) and 6-mercapto-1-hexanol (6-MCOH) (SAMmix). After activating carboxyl groups using a N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide mixture, the rS protein was covalently immobilized on the top of the SAMmix. This electrode was used to design an electrochemical sensor suitable for determining antibodies against the SARS-CoV-2 rS protein (anti-rS). We assessed the association between the immobilized rS protein and the anti-rS antibody present in the blood serum of a SARS-CoV-2 infected person using three electrochemical methods: cyclic voltammetry, differential pulse voltammetry, and potential pulsed amperometry. The results demonstrated that differential pulse voltammetry and potential pulsed amperometry measurements displayed similar sensitivity. In contrast, the measurements performed by cyclic voltammetry suggest that this method is the most sensitive out of the three methods applied in this research.

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.

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.

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation.

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Investigation and Comparison of Specific Antibodies' Affinity Interaction with SARS-CoV-2 Wild-Type, B.1.1.7, and B.1.351 Spike Protein by Total Internal Reflection Ellipsometry.

SARS-CoV-2 vaccines provide strong protection against COVID-19. However, the emergence of SARS-CoV-2 variants has raised concerns about the efficacy of vaccines. In this study, we investigated the interactions of specific polyclonal human antibodies (pAb-SCoV2-S) produced after vaccination with the Vaxzevria vaccine with the spike proteins of three SARS-CoV-2 variants of concern: wild-type, B.1.1.7, and B.1.351. Highly sensitive, label-free, and real-time monitoring of these interactions was accomplished using the total internal reflection ellipsometry method. Thermodynamic parameters such as association and dissociation rate constants, the stable immune complex formation rate constant (kr), the equilibrium association and dissociation (KD) constants and steric factors (Ps) were calculated using a two-step irreversible binding mathematical model. The results obtained show that the KD values for the specific antibody interactions with all three types of spike protein are in the same nanomolar range. The KD values for B.1.1.7 and B.1.351 suggest that the antibody produced after vaccination can successfully protect the population from the alpha (B.1.1.7) and beta (B.1.351) SARS-CoV-2 mutations. The steric factors (Ps) obtained for all three types of spike proteins showed a 100-fold lower requirement for the formation of an immune complex when compared with nucleocapsid protein.

Molecularly imprinted polypyrrole based sensor for the detection of SARS-CoV-2 spike glycoprotein.

This study describes the application of a polypyrrole-based sensor for the determination of SARS-CoV-2-S spike glycoprotein. The SARS-CoV-2-S spike glycoprotein is a spike protein of the coronavirus SARS-CoV-2 that recently caused the worldwide spread of COVID-19 disease. This study is dedicated to the development of an electrochemical determination method based on the application of molecularly imprinted polymer technology. The electrochemical sensor was designed by molecular imprinting of polypyrrole (Ppy) with SARS-CoV-2-S spike glycoprotein (MIP-Ppy). The electrochemical sensors with MIP-Ppy and with polypyrrole without imprints (NIP-Ppy) layers were electrochemically deposited on a platinum electrode surface by a sequence of potential pulses. The performance of polymer layers was evaluated by pulsed amperometric detection. According to the obtained results, a sensor based on MIP-Ppy is more sensitive to the SARS-CoV-2-S spike glycoprotein than a sensor based on NIP-Ppy. Also, the results demonstrate that the MIP-Ppy layer is more selectively interacting with SARS-CoV-2-S glycoprotein than with bovine serum albumin. This proves that molecularly imprinted MIP-Ppy-based sensors can be applied for the detection of SARS-CoV-2 virus proteins.

Biosensors for the Determination of SARS-CoV-2 Virus and Diagnosis of COVID-19 Infection.

Monitoring and tracking infection is required in order to reduce the spread of the coronavirus disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, the development and deployment of quick, accurate, and sensitive diagnostic methods are necessary. The determination of the SARS-CoV-2 virus is performed by biosensing devices, which vary according to detection methods and the biomarkers which are inducing/providing an analytical signal. RNA hybridisation, antigen-antibody affinity interaction, and a variety of other biological reactions are commonly used to generate analytical signals that can be precisely detected using electrochemical, electrochemiluminescence, optical, and other methodologies and transducers. Electrochemical biosensors, in particular, correspond to the current trend of bioanalytical process acceleration and simplification. Immunosensors are based on the determination of antigen-antibody interaction, which on some occasions can be determined in a label-free mode with sufficient sensitivity.

Affinity Sensors for the Diagnosis of COVID-19.

The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was proclaimed a global pandemic in March 2020. Reducing the dissemination rate, in particular by tracking the infected people and their contacts, is the main instrument against infection spreading. Therefore, the creation and implementation of fast, reliable and responsive methods suitable for the diagnosis of COVID-19 are required. These needs can be fulfilled using affinity sensors, which differ in applied detection methods and markers that are generating analytical signals. Recently, nucleic acid hybridization, antigen-antibody interaction, and change of reactive oxygen species (ROS) level are mostly used for the generation of analytical signals, which can be accurately measured by electrochemical, optical, surface plasmon resonance, field-effect transistors, and some other methods and transducers. Electrochemical biosensors are the most consistent with the general trend towards, acceleration, and simplification of the bioanalytical process. These biosensors mostly are based on the determination of antigen-antibody interaction and are robust, sensitive, accurate, and sometimes enable label-free detection of an analyte. Along with the specification of biosensors, we also provide a brief overview of generally used testing techniques, and the description of the structure, life cycle and immune host response to SARS-CoV-2, and some deeper details of analytical signal detection principles.

Evaluation of kinetics and thermodynamics of interaction between immobilized SARS-CoV-2 nucleoprotein and specific antibodies by total internal reflection ellipsometry.

During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔGAssoc) for SCoV-rN and anti-SCoV-rN binding was determined as -34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications.