Sensitive Detection of SARS-CoV-2 Spike Protein based on Electrochemical Impedance Spectroscopy of Fe3O4@SiO2-Au/GCE Biosensor

Highly contagious COVID-19 disease is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which poses a serious threat to global public health. Therefore, the development of a fast and reliable method for the detection of SARS-CoV-2 is an urgent research need. The Fe3O4@SiO2-Au is enriched with a variety of functional groups, which can be used to fabricate a sensitive electrochemical biosensor by biofunctionalization with angiotensin-converting enzyme 2 (ACE2). Accordingly, we developed a novel electrochemical sensor by chemically modifying a glassy carbon electrode (GCE) with Fe3O4@SiO2-Au nanocomposites (hereafter Fe3O4@SiO2-Au/GCE) for the rapid detection of S-protein spiked SARS-CoV-2 by electrochemical impedance spectroscopy (EIS). The new electrochemical sensor has a low limit detection (viz., 4.78 pg/mL) and a wide linear dynamic range (viz., 0.1 ng/mL to 10 μg/mL) for detecting the EIS response signal of S-protein. The robust Fe3O4@SiO2-Au/GCE biosensor has high selectivity, stability, and reproducibility for the detection of S-protein with good recovery of saliva samples.

Portable microfluidic impedance biosensor for SARS-CoV-2 detection.

Pandemics as the one we are currently facing, where fast-spreading viruses present a threat to humanity, call for simple and reliable methods to perform early diagnosis, enabling detection of very low pathogen loads even before symptoms start showing in the host. So far, standard polymerase chain reaction (PCR) is the most reliable method for doing so, but it is rather slow and needs specialized reagents and trained personnel to operate it. Additionally, it is expensive and not easily accessible. Therefore, developing miniaturized and portable sensors which perform early detection of pathogens with high reliability is necessary to not only prevent the spreading of the disease but also to monitor the effectiveness of the developed vaccines and the appearance of new pathogenic variants. Thus, in this work we develop a sensitive microfluidic impedance biosensor for the direct detection of SARS-CoV-2, towards a mobile point-of-care (POC) platform. The operational parameters are optimized with the aid of design-of-experiment (DoE), for an accurate detection of the viral antigens using electrochemical impedance spectroscopy (EIS). We perform the biodetection of buffer samples spiked with fM concentration levels and validate the biosensor in a clinical context of relevance by analyzing 15 real patient samples up to a Ct value (cycle threshold) of 27. Finally, we demonstrate the versatility of the developed platform using different settings, including a small portable potentiostat, using multiple channels for self-validation, as well as with single biosensors for a smartphone-based readout. This work contributes to the rapid and reliable diagnostics of COVID-19 and can be extended to other infectious diseases, allowing the monitoring of viral load in vaccinated and unvaccinated people to anticipate a potential relapse of the disease.

Customizable Fabrication Process for Flexible Carbon-Based Electrochemical Biosensors

In recent research, 3D printing has become a powerful technique and has been applied in the last few years to carbon-based materials. A new generation of 3D-printed electrodes, more affordable and easier to obtain due to rapid prototyping techniques, has emerged. We propose a customizable fabrication process for flexible (and rigid) carbon-based biosensors, from biosensor design to printable conductive inks. The electrochemical biosensors were obtained on a 50 µm Kapton® (polyimide) substrate and transferred to a 500 µm PDMS substrate, using a 3D-extrusion-based printing method. The main features of our fabrication process consist of short-time customization implementation, fast small-to-medium batch production, ease of electrochemical spectroscopy measurements, and very good resolution for an extrusion-based printing method (100 µm). The sensors were designed for future integration into a smart wound dressing for wound monitoring and other biomedical applications. We increased their sensibility with electro-deposited gold nanoparticles. To assess the biosensors' functionality, we performed surface functionalization with specific anti-N-protein antibodies for SARS-CoV 2 virus, with promising preliminary results.

A portable, low-cost and high-throughput electrochemical impedance spectroscopy device for point-of-care biomarker detection

This paper presents a portable, fast and accurate electrochemical impedance spectroscopy (EIS) device with 8-well interdigitated electrode chips for biomarker detection. The design adopts low crest factor multisine signal synthesis at low frequencies (<1 kHz) and single-tone signals at high frequencies (>1 kHz), which significantly increases measurement speed without sacrificing accuracy. In addition, the low excitation amplitude of 10 mV preserves impedance linearity and protects the biosamples. The system achieved an average magnitude accuracy error of 0.30% in the frequency range of interest and it requires only 0.46 s to scan 28 frequency points from 10 Hz to 1 MHz. Experiments were conducted to test the capability to detect antibodies against SARS-CoV-2. Gold nanoparticles bound with protein G (GNP-G) were employed as the conjugated secondary antibody probe to detect anti-SARS-CoV-2 IgG in serum. A highly statistical significance (p = 7×10−6) could be found in the impedance data at 10 kHz. The impedance magnitude alteration caused by the GNP-G of the positive and negative groups were 27.2%±13.6% and 4.1%±1.7%, respectively. The results imply that the proposed system enables rapid COVID-19 antibody biomarker detection. Moreover, the EIS system and GNPs have the potential to be modified to detect other biomarkers. © 2022 The Author(s)

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.

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.

A portable, low-cost and high-throughput electrochemical impedance spectroscopy device for point-of-care biomarker detection

This paper presents a portable, fast and accurate electrochemical impedance spectroscopy (EIS) device with 8-well interdigitated electrode chips for biomarker detection. The design adopts low crest factor multisine signal synthesis at low frequencies (<1 kHz) and single-tone signals at high frequencies (>1 kHz), which significantly increases measurement speed without sacrificing accuracy. In addition, the low excitation amplitude of 10 mV preserves impedance linearity and protects the biosamples. The system achieved an average magnitude accuracy error of 0.30% in the frequency range of interest and it requires only 0.46 s to scan 28 frequency points from 10 Hz to 1 MHz. Experiments were conducted to test the capability to detect antibodies against SARS-CoV-2. Gold nanoparticles bound with protein G (GNP-G) were employed as the conjugated secondary antibody probe to detect anti-SARS-CoV-2 IgG in serum. A highly statistical significance (p = 7×10−6) could be found in the impedance data at 10 kHz. The impedance magnitude alteration caused by the GNP-G of the positive and negative groups were 27.2%±13.6% and 4.1%±1.7%, respectively. The results imply that the proposed system enables rapid COVID-19 antibody biomarker detection. Moreover, the EIS system and GNPs have the potential to be modified to detect other biomarkers.

Recent Developments in Electrochemical-Impedimetric Biosensors for Virus Detection.

Viruses, including influenza viruses, MERS-CoV (Middle East respiratory syndrome coronavirus), SARS-CoV (severe acute respiratory syndrome coronavirus), HAV (Hepatitis A virus), HBV (Hepatitis B virus), HCV (Hepatitis C virus), HIV (human immunodeficiency virus), EBOV (Ebola virus), ZIKV (Zika virus), and most recently SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), are responsible for many diseases that result in hundreds of thousands of deaths yearly. The ongoing outbreak of the COVID-19 disease has raised a global concern and intensified research on the detection of viruses and virus-related diseases. Novel methods for the sensitive, rapid, and on-site detection of pathogens, such as the recent SARS-CoV-2, are critical for diagnosing and treating infectious diseases before they spread and affect human health worldwide. In this sense, electrochemical impedimetric biosensors could be applied for virus detection on a large scale. This review focuses on the recent developments in electrochemical-impedimetric biosensors for the detection of viruses.

Preface for the 66th Special Feature "Novel Aspects and Approaches to Experimental Methods for ElectrochemistryӠ

The Kansai Branch of the Electrochemical Society of Japan publishes a collection of papers in Electrochemistry, which serve as a commentary to the 51st Electrochemistry Workshop. This attempt is motivated by the fact that the domestic seminars are now widely publicized through the on-demand event triggered by COVID-19. This preface consists of the significance of the publication and an introduction of the lecturers as a part of special future for "Novel Aspects and Approaches to Experimental Methods for Electrochemistry.” in this issue of Electrochemistry. © 2022 Electrochemical Society of Japan. All rights reserved.

Amplification Free Detection of SARS-CoV-2 Using Multi-Valent Binding.

We present the development of electrochemical impedance spectroscopy (EIS)-based biosensors for sensitive detection of SARS-CoV-2 RNA using multi-valent binding. By increasing the number of probe-target binding events per target molecule, multi-valent binding is a viable strategy for improving the biosensor performance. As EIS can provide sensitive and label-free measurements of nucleic acid targets during probe-target hybridization, we used multi-valent binding to build EIS biosensors for targeting SARS-CoV-2 RNA. For developing the biosensor, we explored two different approaches including probe combinations that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/µL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi-valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics.

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.

Emerging Applications of Electrochemical Impedance Spectroscopy in Tear Film Analysis.

Human tear film, with a flow rate of 1-3 µL/min, is a rich bodily fluid that transmits a variety of metabolites and hormones containing proteins, lipids and electrolytes that provide clues about ocular and systemic diseases. Analysis of disease biomarkers such as proteins, mRNA, enzymes and cytokines in the tear film, collected by noninvasive methods, can provide significant results for sustaining a predictive, preventive and personalized medicine regarding various diseases such as glaucoma, diabetic retinopathy, keratoconus, dry eye, cancer, Alzheimer's disease, Parkinson's disease and COVID-19. Electrochemical impedance spectroscopy (EIS) offers a powerful technique for analyzing these biomarkers. EIS detects electrical equivalent circuit parameters related to biorecognition of receptor-analyte interactions on the electrode surface. This method is advantageous as it performs a label-free detection and allows the detection of non-electroactive compounds that cannot be detected by direct electron transfer, such as hormones and some proteins. Here, we review the opportunities regarding the integration of EIS into tear fluid sampling approaches.

Electrochemical Sensors for New Challenges

This article focuses upon new challenges faced by today’s society which electrochemical sensors maybe able to address. Focusing primarily upon two of the major challenges faced at the time of writing;the opioid crisis caused by fentanyl and the deadly Covid-19 pandemic, the employment of electrochemical sensors is assessed to determine the contribution they could make toward tackling these problems. Although only a small scope of the electrochemical research present is covered within this article the principles discussed are directly translated to other fields where electrochemical sensors could be applied. This article to aims to highlight the uses of electrochemical sensors and discusses in detail both their advantages but also where further improvements are required to improve their applications across a range of fields.

Portable Electrochemical Biosensors Based on Microcontrollers for Detection of Viruses: A Review.

With the rise of zoonotic diseases in recent years, there is an urgent need for improved and more accessible screening and diagnostic methods to mitigate future outbreaks. The recent COVID-19 pandemic revealed an over-reliance on RT-PCR, a slow, costly and lab-based method for diagnostics. To better manage the pandemic, a high-throughput, rapid point-of-care device is needed for early detection and isolation of patients. Electrochemical biosensors offer a promising solution, as they can be used to perform on-site tests without the need for centralized labs, producing high-throughput and accurate measurements compared to rapid test kits. In this work, we detail important considerations for the use of electrochemical biosensors for the detection of respiratory viruses. Methods of enhancing signal outputs via amplification of the analyte, biorecognition of elements and modification of the transducer are also explained. The use of portable potentiostats and microfluidics chambers that create a miniature lab are also discussed in detail as an alternative to centralized laboratory settings. The state-of-the-art usage of portable potentiostats for detection of viruses is also elaborated and categorized according to detection technique: amperometry, voltammetry and electrochemical impedance spectroscopy. In terms of integration with microfluidics, RT-LAMP is identified as the preferred method for DNA amplification virus detection. RT-LAMP methods have shorter turnaround times compared to RT-PCR and do not require thermal cycling. Current applications of RT-LAMP for virus detection are also elaborated upon.

Modular Label-Free Electrochemical Biosensor Loading Nature-Inspired Peptide toward the Widespread Use of COVID-19 Antibody Tests.

Limitations of the recognition elements in terms of synthesis, cost, availability, and stability have impaired the translation of biosensors into practical use. Inspired by nature to mimic the molecular recognition of the anti-SARS-CoV-2 S protein antibody (AbS) by the S protein binding site, we synthesized the peptide sequence of Asn-Asn-Ala-Thr-Asn-COOH (abbreviated as PEP2003) to create COVID-19 screening label-free (LF) biosensors based on a carbon electrode, gold nanoparticles (AuNPs), and electrochemical impedance spectroscopy. The PEP2003 is easily obtained by chemical synthesis, and it can be adsorbed on electrodes while maintaining its ability for AbS recognition, further leading to a sensitivity 3.4-fold higher than the full-length S protein, which is in agreement with the increase in the target-to-receptor size ratio. Peptide-loaded LF devices based on noncovalent immobilization were developed by affording fast and simple analyses, along with a modular functionalization. From studies by molecular docking, the peptide-AbS binding was found to be driven by hydrogen bonds and hydrophobic interactions. Moreover, the peptide is not amenable to denaturation, thus addressing the trade-off between scalability, cost, and robustness. The biosensor preserves 95.1% of the initial signal for 20 days when stored dry at 4 °C. With the aid of two simple equations fitted by machine learning (ML), the method was able to make the COVID-19 screening of 39 biological samples into healthy and infected groups with 100.0% accuracy. By taking advantage of peptide-related merits combined with advances in surface chemistry and ML-aided accuracy, this platform is promising to bring COVID-19 biosensors into mainstream use toward straightforward, fast, and accurate analyses at the point of care, with social and economic impacts being achieved.

Impedimetric Detection Based on Label-Free Immunoassay Developed for Targeting Spike S1 Protein of SARS-CoV-2.

After the COVID-19 pandemic started all over the world, great importance was placed on the development of sensitive and selective bioanalytical assays for the rapid detection of the highly pathogenic SARS-CoV-2 virus causing COVID-19 disease. In this present work, an impedimetric immunosensor was developed and applied for rapid, reliable, sensitive and selective detection of the SARS-CoV-2 S1 protein. To detect the SARS-CoV-2 virus, targeting of the spike S1 protein was achieved herein by using S1 protein-specific capture antibody (Cab-S1) immobilized screen-printed electrode (SPE) in combination with the electrochemical impedance spectroscopy (EIS) technique. With the impedimetric immunosensor, the detection limit for S1 protein in buffer medium was found to be 0.23 ng/mL (equal to 23.92 amol in 8 µL sample) in the linear concentration range of S1 protein from 0.5 to 10 ng/mL. In the artificial saliva medium, it was found to be 0.09 ng/mL (equals to 9.36 amol in 8 µL sample) in the linear concentration range of S1 protein between 0.1 and 1 ng/mL. The selectivity of the impedimetric immunosensor toward S1 protein was tested against influenza hemagglutinin antigen (HA) in the buffer medium as well as in artificial saliva.

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.