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.

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.

Towards Electrochemical Sensor Based on Molecularly Imprinted Polypyrrole for the Detection of Bacteria-Listeria monocytogenes.

Detecting bacteria-Listeria monocytogenes-is an essential healthcare and food industry issue. The objective of the current study was to apply platinum (Pt) and screen-printed carbon (SPCE) electrodes modified by molecularly imprinted polymer (MIP) in the design of an electrochemical sensor for the detection of Listeria monocytogenes. A sequence of potential pulses was used to perform the electrochemical deposition of the non-imprinted polypyrrole (NIP-Ppy) layer and Listeria monocytogenes-imprinted polypyrrole (MIP-Ppy) layer over SPCE and Pt electrodes. The bacteria were removed by incubating Ppy-modified electrodes in different extraction solutions (sulphuric acid, acetic acid, L-lysine, and trypsin) to determine the most efficient solution for extraction and to obtain a more sensitive and repeatable design of the sensor. The performance of MIP-Ppy- and NIP-Ppy-modified electrodes was evaluated by pulsed amperometric detection (PAD). According to the results of this research, it can be assumed that the most effective MIP-Ppy/SPCE sensor can be designed by removing bacteria with the proteolytic enzyme trypsin. The LOD and LOQ of the MIP-Ppy/SPCE were 70 CFU/mL and 210 CFU/mL, respectively, with a linear range from 300 to 6700 CFU/mL.

Electrochemical Immunosensor for the Determination of Antibodies against Prostate-Specific Antigen Based on ZnO Nanostructures.

In this study, ZnO nanostructures with different types of morphologies and particle sizes were evaluated and applied for the development of an immunosensor. The first material was composed of spherical, polydisperse nanostructures with a particle size in the range of 10-160 nm. The second was made up of more compact rod-like spherical nanostructures with the diameter of these rods in the range of 50-400 nm, and approximately 98% of the particles were in the range of 20-70 nm. The last sample of ZnO was made up of rod-shaped particles with a diameter of 10-80 nm. These ZnO nanostructures were mixed with Nafion solution and drop-casted onto screen-printed carbon electrodes (SPCE), followed by a further immobilization of the prostate-specific antigen (PSA). The affinity interaction of PSA with monoclonal antibodies against PSA (anti-PSA) was evaluated using the differential pulse voltammetry technique. The limit of detection and limit of quantification of anti-PSA were determined as 1.35 nM and 4.08 nM for compact rod-shaped spherical ZnO nanostructures, and 2.36 nM and 7.15 nM for rod-shaped ZnO nanostructures, respectively.

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.