Photoluminescence-based biosensor for the detection of antibodies against SARS-CoV-2 virus proteins by ZnO tetrapod structure integrated within microfluidic system.

This paper reports on development of an optical biosensor for the detection of antibodies against SARS-CoV-2 virus proteins in blood serum. ZnO nanotetrapods with high surface area and stable room temperature photoluminescence (PL) were selected as transducers. Structure and optical properties of the ZnO tetrapods have been studied by XRD, SEM and Raman spectroscopy. Crystallinity, dimensions and emission peaks of the ZnO tetrapods were determined. The ZnO tetrapods were fixed on glass chip. Silanization of ZnO tetrapods surface resulted in forming of functional surface groups suitable for the immobilization of bioselective layer. Two types of recombinant proteins (rS and rN) have been used to form bioselective layer on the surface of the ZnO tetrapods. Flow through microfluidic system, integrated with optical system, has been used for the determination of antibodies against SARS-CoV-2 virus proteins present in blood samples. The SARS-CoV-2 probes, prepared in PBS solution, have been injected into the measurement chamber with a constant pumping speed. Steady-state photoluminescence spectra and photoluminescence kinetics have been studied before and after injection of the probes. The biosensor signal has been tested to anti-SARS-CoV-2 antibodies in the range of 0.001 nM-1 nM. Control measurements have been performed with blood serum of healthy person. ZnO-SARS-CoV-2-rS and ZnO-SARS-CoV-2-rN biosensors showed high stability and sensitivity to anti-SARS-CoV-2 antibodies in the range of 0.025-0.5 nM (LOD 0.01 nM) and 0.3-1 nM (LOD 0.3 nM), respectively. Gibbs free energy of interaction between ZnO/SARS-CoV-2-rS and ZnO/SARS-CoV-2-rN bioselective layers with anti-SARS-CoV-2 antibodies showed -35.5 and -21.4 kJ/mol, respectively. Average detection time of biosensor integrated within microfluidic system was 15-20 min. The detection time and pumping speed (50 µL/min) were optimized to make detection faster. The developed system and ZnO-SARS-CoV-2-rS nanostructures have good potential for detection of anti-SARS-CoV-2 antibodies from patient's probes.

Electrochemical biosensor for the evaluation of monoclonal antibodies targeting the N protein of SARS-CoV-2 virus.

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein.

Molecularly imprinted composite-based biosensor for the determination of SARS-CoV-2 nucleocapsid protein.

This article aims to present a comparative study of three polypyrrole-based molecularly imprinted polymer (MIP) systems for the detection of the recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (rN). The rN is known for its relatively low propensity to mutate compared to other SARS-CoV-2 antigens. The aforementioned systems include screen-printed carbon electrodes (SPCE) modified with gold nanostructures (MIP1), platinum nanostructures (MIP2), and the unmodified SPCE (MIP3), which was used for control. Pulsed amperometric detection (PAD) was employed as the detection technique, offering the advantage of label-free detection without the need for an additional redox probe. Calibration curves were constructed using the obtained data to evaluate the response of each system. Non-imprinted systems were also tested in parallel to evaluate the contribution of non-specific binding and assess the affinity sensor's efficiency. The analysis of calibration curves revealed that the AuNS-based MIP1 system exhibited the lowest contribution of non-specific binding and displayed a better fit with the chosen fitting model compared to the other systems. Further analysis of this system included determining the limit of detection (LOD) (51.2 ± 2.8 pg/mL), the limit of quantification (LOQ) (153.9 ± 8.3 pg/mL), and a specificity test using a recombinant receptor-binding domain of SARS-CoV-2 spike protein as a control. Based on the results, the AuNS-based MIP1 system demonstrated high specificity and sensitivity for the label-free detection of SARS-CoV-2 nucleocapsid protein. The utilization of PAD without the need for additional redox probes makes this sensing system convenient and valuable for rapid and accurate virus detection.

Electrochemical capacitance spectroscopy based determination of antibodies against SARS-CoV-2 virus spike protein.

In this study, we are reporting a novel electrochemical capacitance spectroscopy (ECS) platform designed for the sensitive and label-free detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus spike protein (anti-rS) in diluted blood serum. The determination of anti-rS is crucial for identification individuals who have been infected by SARS-CoV-2 virus and may have acquired immunity. The rS protein was immobilized on a screen-printed carbon electrode, which was incubated in diluted blood serum containing anti-rS antibodies. Label-free ECS was applied for the determination of interaction between immobilized rS and free-standing anti-rS. Here reported bioanalytical platform demonstrated high sensitivity and specificity in detecting anti-rS, achieving a limit of detection of 4.38 nM. This versatile platform could be further enhanced by applying various electrode materials and adapting this platform to detect antibodies against some other proteins. Our findings have significant implications for the development of affordable, scalable biosensing platforms capable to provide rapid and accurate public health screening and monitoring, particularly in the context of the coronavirus disease 2019 (COVID-19) pandemic.

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.

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.

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.