Cutting-edge biorecognition strategies to boost the detection performance of COVID-19 electrochemical biosensors: A review.

Electrochemical biosensors are known for their high sensitivity, selectivity, and low cost. Recently, they have gained significant attention and became particularly important as promising tools for the detection of COVID-19 biomarkers, since they offer a rapid and accurate means of diagnosis. Biorecognition strategies are a crucial component of electrochemical biosensors and determine their specificity and sensitivity based on the interaction of biological molecules, such as antibodies, enzymes, and DNA, with target analytes (e.g., viral particles, proteins and genetic material) to create a measurable signal. Different biorecognition strategies have been developed to enhance the performance of electrochemical biosensors, including direct, competitive, and sandwich binding, alongside nucleic acid hybridization mechanisms and gene editing systems. In this review article, we present the different strategies used in electrochemical biosensors to target SARS-CoV-2 and other COVID-19 biomarkers, as well as explore the advantages and disadvantages of each strategy and highlight recent progress in this field. Additionally, we discuss the challenges associated with developing electrochemical biosensors for clinical COVID-19 diagnosis and their widespread commercialization.

One-step selective layer assemble: A versatile approach for the development of a SARS-CoV-2 electrochemical immunosensor.

The appearance of new viruses and diseases has made the development of rapid and reliable diagnostic tests crucial. In light of it, we proposed a new method for assembling an electrochemical immunosensor, based on a one-step approach for selective layer formation. For this purpose, a mixture containing the immobilizing agent (polyxydroxybutyrate, PHB) and the recognition element (antibodies against SARS-CoV-2 nucleocapsid protein) was prepared and used to modify a screen-printed carbon electrode with electrodeposited graphene oxide, for the detection of SARS-CoV-2 nucleocapsid protein (N-protein). Under optimum conditions, N-protein was successfully detected in three different matrixes - saliva, serum, and nasal swab, with the lowest detectable values of 50 pg mL-1, 1.0 ng mL-1, and 50 pg mL-1, respectively. Selectivity was assessed against SARS-CoV-2 receptor-binding domain protein (RBD) and antibodies against yellow fever (YF), and no significant response was observed in presence of interferents, reinforcing the suitability of the proposed one-step approach for selective layer formation. The proposed biosensor was stable for up to 14 days, and the mixture was suitable for immunosensor preparation even after 60 days of preparation. The proposed assembly strategy reduces the cost, analysis time, and waste generation. This reduction is achieved through miniaturization, which results in the decreased use of reagents and sample volumes. Additionally, this approach enables healthcare diagnostics to be conducted in developing regions with limited resources. Therefore, the proposed one-step approach for selective layer formation is a suitable, simpler, and a reliable alternative for electrochemical immunosensing.

An electrochemical microfluidic device for non-enzymatic cholesterol determination using a lab-made disposable electrode.

Cholesterol is an important steroid and hormone precursor, and its levels in the blood are associated with risk factors for cardiovascular diseases. In this work, a non-enzymatic methodology for cholesterol determination in serum samples is described. First, a working electrode was constructed using homemade ink and a plastic substrate by a simple dunking process. Next, the dunked electrode (DWE) was modified with nickel ions (Ni-DWE) and combined with a low-cost microfluidic platform, resulting in a thread-based electroanalytical device (µTED). The arrangement of µTED consists of two coupled electrodes (one reference in the inlet reservoir and an auxiliary electrode against the outlet reservoir) and a mobile support for facile working electrode exchange. After optimization of construction parameters, the system was applied for non-enzymatic determination of cholesterol under alkaline conditions using the redox pair Ni(II)/Ni(III) as a mediator. Under the best analytical conditions, a calibration curve was constructed with a linear dynamic range (LDR) from 0.25 to 25.0 µmol L-1, and the calculated limits of detection (LOD) and quantification (LOQ) were 0.074 and 0.24 µmol L-1, respectively. No effects of possible interferents on electrochemical response were found in the presence of ascorbic acid, uric acid, dopamine, cysteine, and glucose, suggesting that the proposed device can be used for the determination of cholesterol without significant matrix effects of human plasma. Finally, cholesterol analysis was carried out using spiked plasma samples, and good recovery values were achieved.

A versatile 3D printed multi-electrode cell for determination of three COVID-19 biomarkers.

3D-printing has shown an outstanding performance for the production of versatile electrochemical devices. However, there is a lack of studies in the field of 3D-printed miniaturized settings for multiplex biosensing. In this work, we propose a fully 3D-printed micro-volume cell containing six working electrodes (WEs) that operates with 250 µL of sample. A polylactic acid/carbon black conductive filament (PLA/CB) was used to print the WEs and subsequently modified with graphene oxide (GO), to support protein binding. Cyclic voltammetry was employed to investigate the electrochemical behaviour of the novel multi-electrode cell. In the presence of K3[Fe(CN)6], PLA/CB/GO showed adequate peak resolution for subsequent label-free immunosensing. The innovative 3D-printed cell was applied for multiplex voltammetric detection of three COVID-19 biomarkers as a proof-of-concept. The multiple sensors showed a wide linear range with detection limits of 5, 1 and 1 pg mL-1 for N-protein, SRBD-protein, and anti-SRBD, respectively. The sensor performance enabled the selective sequential detection of N protein, SRBD protein, and anti-SRBD at biological levels in saliva and serum. In summary, the miniaturized six-electrode cell presents an alternative for the low-cost and fast production of customizable devices for multi-target sensing with promising application in the development of point-of-care sensors.

Biochar: An environmentally friendly platform for construction of a SARS-CoV-2 electrochemical immunosensor.

Waste management is a key feature to ensure sustainable consumption and production patterns, and to combat the impacts of climate change. In this scenario, the production of biochar from different biomasses results in environmental and economic advantages. In this study, biochar was produced from sugarcane bagasse pyrolysis, to immobilize biomolecules, in order to assemble an electrochemical immunosensor to detect antibodies against SARS-CoV-2. For this, screen-printed carbon electrodes (SPCE) were modified with a dispersion of biochar and used to immobilize the receptor-binding-domain (RBD) against virus S-protein, through EDC/NHS crosslinking reaction. Under the best set of experimental conditions, negative and positive serum samples responses distinguished based on a cutoff value of 82.3 %, at a 95 % confidence level. The immunosensor showed selective behavior to antibodies against yellow fever and its performance was stable up to 7 days of storage. Therefore, biochar yielded from sugarcane bagasse is an ecofriendly material that can be used as a platform to immobilize biomolecules for construction of electrochemical biosensors.

Novel and highly stable strategy for the development of microfluidic enzymatic assays based on the immobilization of horseradish peroxidase (HRP) into cotton threads.

The use of biological components in the development of new methods of analysis and point-of-care (POC) devices is an ever-expanding theme in analytical chemistry research, due to the immense potential for early diagnosis of diseases and monitoring of biomarkers. In the present work, the evaluation of an electrochemical microfluidic device based on the immobilization of horseradish peroxidase (HRP) enzyme into chemically treated cotton threads is described. This bioreactor was used as a channel for the build of the microfluidic device, which has allowed to use of a non-modified screen-printed electrode (SPE) as an amperometric detector. Cotton threads were treated using citric acid, and the immobilization of HRP has been performed by EDC/NHS crosslinking, connecting amine groups of the enzymes to carboxylic acids in the cellulosic structure. For the analytical evaluation, an amperometric assay for hydrogen peroxide detection was performed after the injection of H2O2 and hydroquinone (HQN) concomitantly. The enzymatic reaction consumes H2O2 leading to the formation of O-quinone, which is readily reducible at non-modified SPE. Several experimental parameters related to enzyme immobilization have been investigated and under the best set of conditions, a good analytical performance was obtained. In addition, the threads were freezer-stored and, after 12 weeks, 84% of hydrogen peroxide sensitivity was maintained, which is very reasonable for enzyme-based systems and still offers good analytical precision. Therefore, a simple and inexpensive microfluidic system was reported by crosslinking carboxylic groups to amine-containing macromolecules, suggesting a new platform for many other protein-based assays.

Microfluidic devices based on textile threads for analytical applications: state of the art and prospects.

Microfluidic devices based on textile threads have interesting advantages when compared to systems made with traditional materials, such as polymers and inorganic substrates (especially silicon and glass). One of these significant advantages is the device fabrication process, made more cheap and simple, with little or no microfabrication apparatus. This review describes the fundamentals, applications, challenges, and prospects of microfluidic devices fabricated with textile threads. A wide range of applications is discussed, integrated with several analysis methods, such as electrochemical, colorimetric, electrophoretic, chromatographic, and fluorescence. Additionally, the integration of these devices with different substrates (e.g., 3D printed components or fabrics), other devices (e.g., smartphones), and microelectronics is described. These combinations have allowed the construction of fully portable devices and consequently the development of point-of-care and wearable analytical systems.

A carbon fiber ultramicroelectrode as a simple tool to direct antioxidant estimation based on caffeic acid oxidation.

This work describes the construction and evaluation of carbon fiber ultramicroelectrodes (CF-UMEs) in the voltammetric estimation of the antioxidant capacity of wine and grape samples based on caffeic acid (HCAF) oxidation. For this, lab-made CF-UMEs were constructed using an arrangement of six carbon fibers (7 µm diameters individual) assembled in a glass capillary, and caffeic acid (HCAF) was used as a standard solution. By using the most straightforward 2-electrode cell arrangement (the CF-UME as a working electrode and Ag/AgCl as a reference/auxiliary electrode), voltammetric measurements of a 1.0 mmol L-1 HCAF solution were done in the absence of a supporting electrolyte. A sigmoidal voltammetric profile was observed in CF-UMEs caused by a more effective mass transport by radial diffusion, which leads to a rapid formation of the diffusion layer. Reproducibility studies for different 6-fiber electrodes manually constructed in different batches showed an RSD of less than 5%. For the same electrode surface, a variation of 2.7% was observed. Under optimized conditions, a linear relationship between anodic peak current and HCAF concentration from 3.0 to 500 µmol L-1 with a sensitivity of 12 µA L mol-1 was reached. The limits of detection (LOD) and quantification (LOQ) were calculated to be 0.41 and 1.26 µmol L-1, respectively. The proposed electrochemical method was applied in the estimation of the antioxidant capacity in three different wine samples as well as in green and red grapes. Concordant and satisfactory results by comparison with a proper method were obtained, which suggests that the proposed sensor can be successfully applied for direct analysis of wine and grape samples by estimation of HCAF content.

Cellulose membrane modified with polypyrrole as an extraction device for the determination of emerging contaminants in river water with gas chromatography-mass spectrometry.

In this study, a simple, efficient, and reusable device based on cellulose membranes modified with polypyrrole was developed to extract 14 emerging contaminants from aqueous matrices. For chemical polymerization, a low-cost cellulose membrane was immersed in 0.1 mol/L pyrrole and 0.5 mol/L ammonium persulfate for 40 min in an ice/water bath. The cellulose membranes modified with polypyrrole were accommodated in a polycarbonate holder suitable for solid-phase extraction disks. Solid-phase extraction parameters that affect extraction efficiency, such as sample volume, pH, flow rate, and desorption were optimized. Subsequently, determination of target compounds was performed by gas chromatography with mass spectrometry. The linear range for analytes ranged from 0.05 to 500 µg/L, with coefficients of determination above 0.990. The limits of quantification varied between 0.05 and 10 µg/L, with relative standard deviations lower than 17%. The performance of the proposed cellulose membranes modified with polypyrrole device for real samples was evaluated after extraction of emerging contaminants from a river water sample from the city of Curitiba, Brazil. Bisphenol A (6.39 µg/L), caffeine (17.83 µg/L), and paracetamol (19.28 µg/L) were found in these samples.

Microfluidic thread based electroanalytical system for green chromatographic separations.

The use of miniaturized chromatographic systems is an important strategy for reducing the consumption of supplies related to separations, allowing the development of more sustainable analytical methodologies. However, the high cost and complexity in the production of these systems combined with the operational difficulties and the need for the use of solvent and sample pretreatment are challenges to be overcome in order to make the chromatographic methods greener. Here, we report the construction and development of a low cost microfluidic system for green and solvent-free chromatographic separations with electrochemical detection integrated into cotton threads without the use of any mechanical pumping to transport the solutions. The manufacture of the proposed system was performed by simple assembly of the components, with the separation of the species based on an ion exchange mechanism and detection using gold electrodes manufactured directly on the cotton threads. A linear range of 0.025-5.0 mM was obtained for the effective separation of ascorbic acid (AA) and dopamine (DA) with detection limits of 2.89 µM (for AA) and 4.41 µM (for DA). Each analysis was performed at a low cost (less than 0.01 dollars), and with a small volume of waste generated (107.1 µL). So, the proposed system was successfully employed to determine the levels of AA and DA present in the tears of healthy volunteers without sample pretreatment, indicating the good analytical performance of the system and the possibility of performing greener chromatographic separations.

Tear glucose detection combining microfluidic thread based device, amperometric biosensor and microflow injection analysis.

The tear glucose analysis is an important alternative for the indirect, simple and less invasive monitoring of blood glucose levels. However, the high cost and complex manufacturing process of tear glucose analyzers combined with the need to exchange the sensor after each analysis in the disposable tests prevent widespread application of the tear in glucose monitoring. Here, we present the integration of a biosensor made by the electropolymerization of poly(toluidine blue O) (PTB) and glucose oxidase (GOx) with an electroanalytical microfluidic device of easy assembly based on cotton threads, low cost materials and measurements by microflow injection analysis (µFIA) through passive pumping for performing tear glucose analyses in a simple, rapid and inexpensive way. A high stability between the analyses (RSD = 2.54%) and among the different systems (RSD = 3.13%) was obtained for the determination of glucose, in addition to a wide linear range between 0.075 and 7.5mmolL-1 and a limit of detection of 22.2µmolL-1. The proposed method was efficiently employed in the determination of tear glucose in non-diabetic volunteers, obtaining a close correlation with their blood glucose levels, simplifying and reducing the costs of the analyses, making the tear glucose monitoring more accessible for the population.

Characterization and optimization of low cost microfluidic thread based electroanalytical device for micro flow injection analysis.

The micro flow injection analysis (µFIA) is a powerful technique that uses the principles of traditional flow analysis in a microfluidic device and brings a number of improvements related to the consumption of reagents and samples, speed of analysis and portability. However, the complexity and cost of manufacturing processes, difficulty in integrating micropumps and the limited performance of systems employing passive pumps are challenges that must be overcome. Here, we present the characterization and optimization of a low cost device based on cotton threads as microfluidic channel to perform µFIA based on passive pumps with good analytical performance in a simple, easy and inexpensive way. The transport of solutions is made through cotton threads by capillary force facilitated by gravity. After studying and optimizing several features related to the device, were obtained a flow rate of 2.2 ± 0.1 µL s-1, an analytical frequency of 208 injections per hour, a sample injection volume of 2.0 µL and a waste volume of approximately 40 µL per analysis. For chronoamperometric determination of naproxen, a detection limit of 0.29 µmol L-1 was reached, with a relative standard deviation (RSD) of 1.69% between injections and a RSD of 3.79% with five different devices. Thus, based on the performance presented by proposed microfluidic device, it is possible to overcome some limitations of the µFIA systems based on passive pumps and allow expansion in the use of this technique.

Low cost microfluidic device based on cotton threads for electroanalytical application.

Microfluidic devices are an interesting alternative for performing analytical assays, due to the speed of analyses, reduced sample, reagent and solvent consumption and less waste generation. However, the high manufacturing costs still prevent the massive use of these devices worldwide. Here, we present the construction of a low cost microfluidic thread-based electroanalytical device (µTED), employing extremely cheap materials and a manufacturing process free of equipment. The microfluidic channels were built with cotton threads and the estimated cost per device was only $0.39. The flow of solutions (1.12 µL s(-1)) is generated spontaneously due to the capillary forces, eliminating the use of any pumping system. To demonstrate the analytical performance of the µTED, a simultaneous determination of acetaminophen (ACT) and diclofenac (DCF) was performed by multiple pulse amperometry (MPA). A linear dynamic range (LDR) of 10 to 320 µmol L(-1) for both species, a limit of detection (LOD) and a limit of quantitation (LOQ) of 1.4 and 4.7 µmol L(-1) and 2.5 and 8.3 µmol L(-1) for ACT and DCF, respectively, as well as an analytical frequency of 45 injections per hour were reached. Thus, the proposed device has shown potential to extend the use of microfluidic analytical devices, due to its simplicity, low cost and good analytical performance.

Sensitive voltammetric determination of lead released from ceramic dishes by using of bismuth nanostructures anchored on biochar.

A simple and sensitive electroanalytical method was developed for determination of nanomolar levels of Pb(II) based on the voltammetric stripping response at a carbon paste electrode modified with biochar (a special charcoal) and bismuth nanostructures (nBi-BchCPE). The proposed methodology was based on spontaneous interactions between the highly functionalized biochar surface and Pb(II) ions followed by reduction of these ions into bismuth nanodots which promote an improvement on the stripping anodic current. The experimental procedure could be summarized in three steps: including an open circuit pre-concentration, reduction of accumulated lead ions at the electrode surface and stripping step under differential pulse voltammetric conditions (DPAdSV). SEM images revealed dimensions of bismuth nanodots ranging from 20 nm to 70 nm. The effects of main parameters related to biochar, bismuth and operational parameters were examined in detail. Under the optimal conditions, the proposed sensor has exhibited linear range from 5.0 to 1000 nmol L(-1) and detection limit of 1.41 nmol L(-1) for Pb(II). The optimized method was successfully applied for determination of Pb(II) released from overglaze-decorated ceramic dishes. Results obtained were compared with those given by inductively coupled plasma optical emission spectroscopy (ICP-OES) and they are in agreement at 99% of confidence level.

Voltammetric determination of the antioxidant capacity in wine samples using a carbon nanotube modified electrode.

A direct determination of gallic acid was achieved at a carbon paste electrode modified with carbon nanotubes under differential pulse voltammetry conditions. The values obtained for gallic acid were used to estimate the antioxidant properties of the wine sample based on gallic acid oxidation. The proposed method is based on the gallic acid oxidation process at a modified carbon paste electrode (MCPE) containing 30% (m/m) of carbon nanotubes monitored at 0.35 V versus Ag/AgCl (KCl 3 mol L(-1)). Using the optimized experimental conditions, the calibration curve for gallic acid was linear in the concentration range from 5.0 × 10(-7) to 1.5 × 10(-5) mol L(-1) with a detection limit of 3.0 × 10(-7) mol L(-1). The MCPE was successfully applied for the determination of the antioxidant capacity for red and white wine samples without interference of glucose and ascorbic acid, and the obtained results were compared with the standard spectrophotometric method.