Impedimetric Sensing: An Emerging Tool for Combating the COVID-19 Pandemic.

The COVID-19 pandemic revealed a pressing need for the development of sensitive and low-cost point-of-care sensors for disease diagnosis. The current standard of care for COVID-19 is quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This method is sensitive, but takes time, effort, and requires specialized equipment and reagents to be performed correctly. This make it unsuitable for widespread, rapid testing and causes poor individual and policy decision-making. Rapid antigen tests (RATs) are a widely used alternative that provide results quickly but have low sensitivity and are prone to false negatives, particularly in cases with lower viral burden. Electrochemical sensors have shown much promise in filling this technology gap, and impedance spectroscopy specifically has exciting potential in rapid screening of COVID-19. Due to the data-rich nature of impedance measurements performed at different frequencies, this method lends itself to machine-leaning (ML) algorithms for further data processing. This review summarizes the current state of impedance spectroscopy-based point-of-care sensors for the detection of the SARS-CoV-2 virus. This article also suggests future directions to address the technology's current limitations to move forward in this current pandemic and prepare for future outbreaks.

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.

Development of Aptasensor for the Diagnosis of Covid-19

Objectives: The objective is to develop a low-cost, practical, portable aptasensor platform for the diagnosis of COVID-19. Materials -Methods: Amino-terminated aptamers to be used for the design of an aptasensor were synthesized by SELEX method, and interaction of aptamers with SARS-CoV-2 S1 protein was investigated by isothermal titration calorimetry (ITC). Gold electrodes were used to design the biosensor platform. After the electrode surface was functionalized with cysteamine, the amino-terminated aptamer was conjugated to the surface via glutaraldehyde crosslinker. Then, the surface characterization and analytical parameters of the designed sensing platform were determined by adding commercial S1 proteins on the surface using differential pulse voltammetry (DPV), cyclic voltammetry (CV) and impedance spectroscopy (EIS). To evaluate the working performance of the system, S1 proteins were added to the synthetic serum samples using the standard addition method and the measurements were repeated. Result(s): Surface characterization of the platform designed with EIS and CV measurements was performed and it was found that the modification was successfully performed. In addition, DPV results and analytical parameters of the platform (calibration plot, limit of detection(LOD) , repeatability, coefficient of variation) were determined and the working performance of system was evaluated. Moreover, working performance of the biosensor in real samples and its specificity for COVID -19 were determined by experiments with synthetic serum and influenza A and B proteins. Conclusion(s): According the results, the system has potential to be used for the detection of COVID -19, and also it can be rapidly adapted in different pandemic situations that may occur in the future.

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.

Automatic Analysis of Isothermal Amplification via Impedance Time-Constant-Domain Spectroscopy: A SARS-CoV-2 Case Study

The development of sensitive and affordable testing devices for infectious diseases is essential to preserve public health, especially in pandemic scenarios. In this work, we have developed an attractive analytical method to monitor products of genetic amplification, particularly the loop-mediated isothermal amplification reaction (RT-LAMP). The method is based on electrochemical impedance measurements and the distribution of relaxation times model, to provide the so-called time-constant-domain spectroscopy (TCDS). The proposed method is tested for the SARS-CoV-2 genome, since it has been of worldwide interest due to the COVID-19 pandemic. Particularly, once the method is calibrated, its performance is demonstrated using real wastewater samples. Moreover, we propose a simple classification algorithm based on TCDS data to discriminate among positive and negative samples. Results show how a TCDS-based method provides an alternative mechanism for label-free and automated assays, exhibiting robustness and specificity for genetic detection.

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.

Characterization and application of porous PHBV-based bacterial polymers to realize novel bio-based electroanalytical (bio)sensors

The establishment of novel disruptive technologies represents a common requirement for the sustainable development as reported in the 2030 agenda established by United Nations. As demonstrated by the Covid-19 pandemic, and furtherly highlighted by the current global challenges, i.e. precision agriculture, decentralized testing, personalized medicine, the field of portable devices is growing day-by-day. Relatively to the electrochemical portable strips, globally represented by glucose strips for diabetes patients, the use of plastic-based products is still very high. In this work, two bacterial polymers have been deeply characterized and compared with the gold standard polyester that is the most used material to produce printed electrochemical strips. In particular, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV with micro-fibrillated cellulose (MFC), namely PHBV/MFC, have been produced with different porosities and have been morphologically, mechanically and electrochemically characterized. Scanning electron microscopy, contact angle, tensil tests, cyclic voltammetry, chronoamperometry, electrochemical impedance spectroscopy, stripping voltammetry and chronoamperometry have been used to evaluate and confirm the suitability of PHBV-based substrates for future sustainable application in the (bio)electroanalytical field. In particular these novel substrates have been applied towards the development of two sensing platforms, namely iron ions and organophosphate pesticides. As shown, in comparison with the gold standard polyester for sensors and biosensors development, the use of PHBV-based substrates allowed to reach similar detection limit and repeatability. In particular, iron ions were detected down to 140 and 150 ppb and dichlorvos was detect with an inhibition biosensor down to 0.4 and 0.5 ppb, respectively for PHBV and PHBV/MFC. These novel substrates may represent a starting point towards the development of sustainable platforms for decentralized applications. • PHBV-based materials are 100% bio-compatible and bio-degradable. • Cellulose merging is able to provide new functionalities. • Polyester-based substrates can be replaced by more sustainable ones. • A novel starting point to make sustainable electrochemical (bio)sensors. • Facile detection of iron ions and organophosphate as the case of study. [ FROM AUTHOR]

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.

The 66th special feature "Novel Aspects and Approaches to Experimental Methods for Electrochemistry"

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. (C) The Author(s) 2022. Published by ECSJ.

Electrochemical Gene Amplification Signal Detection of Disposable Biochips Using Electrodes.

Real-time Polymerase Chain Reaction (RT-PCR), a molecular diagnostic technology, is spotlighted as one of the quickest and fastest diagnostic methods for the actual coronavirus (SARS-CoV-2). However, the fluorescent label-based technology of the RT-PCR technique requires expensive equipment and a sample pretreatment process for analysis. Therefore, this paper proposes a biochip based on Electrochemical Impedance Spectroscopy (EIS). In this paper, it was possible to see the change according to the concentration by measuring the impedance with a chip made of two electrodes with different shapes of sample DNA.

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.