The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1–10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.

Multiplex electrochemical biosensors have been used for eliminating the matrix effect in complex bodily fluids or enabling the detection of two or more bioanalytes, overall resulting in more sensitive assays and accurate diagnostics. Many electrochemical biosensors lack reliable and low-cost multiplexing to meet the requirements of point-of-care detection due to either limited functional biosensors for multi-electrode detection or incompatible readout systems. We developed a new dual electrochemical biosensing unit accompanied by a customized potentiostat to address the unmet need for point-of-care multi-electrode electrochemical biosensing. The two-working electrode system was developed using screen-printing of a carboxyl-rich nanomaterial containing ink, with both working electrodes offering active sites for recognition of bioanalytes. The low-cost bi-potentiostat system (∼$80) was developed and customized specifically to the bi-electrode design and used for rapid, repeatable, and accurate measurement of electrochemical impedance spectroscopy signals from the dual biosensor. This binary electrochemical data acquisition (Bi-ECDAQ) system accurately and selectively detected SARS-CoV-2 Nucleocapsid protein (N-protein) in both spiked samples and clinical nasopharyngeal swab samples of COVID-19 patients within 30 min. The two working electrodes offered the limit of detection of 116 fg/mL and 150 fg/mL, respectively, with the dynamic detection range of 1–10,000 pg/mL and the sensitivity range of 2744–2936 Ω mL/pg.mm 2 for the detection of N-protein. The potentiostat performed comparable or better than commercial Autolab potentiostats while it is significantly lower cost. The open-source Bi-ECDAQ presents a customizable and flexible approach towards addressing the need for rapid and accurate point-of-care electrochemical biosensors for the rapid detection of various diseases.

Optimized self-contained microfluidic platform allows for single-step detection of proteins, through passive delivery of the dry-stored reagents required for immunosensing. Novel autonomous performance of the platform advances the point-of-care utilization of electrochemical protein sensing.

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Future point-of-care (PoC) and wearable electrochemical biosensors explore new technology solutions to eliminate the need for multistep electrode modification and functionalization, overcome the limited reproducibility, and automate the sensing steps. In this work, a new screen-printed immuno-biosensor strip is engineered and characterized using a hybrid graphene nanosheet intermixed with the conductive poly­(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) polymers, all embedded within the base carbon matrix (GiPEC) of the screen-printing ink. This intermixed nanocomposite ink is chemically designed for self-containing the “carboxyl” functional groups as the most specific chemical moiety for protein immobilization on the electrodes. The GiPEC ink enables capturing the target antibodies on the electrode without any need for extra surface preparation. As a proof of concept, the performance of the non-functionalized ready-to-immobilize strips was assessed for the detection of glial fibrillary acidic protein (GFAP) as a known central nervous system injury blood biomarker. This immuno-biosensor exhibits the limit of detection of 281.7 fg mL–1 (3 signal-to-noise ratio) and the sensitivity of 322.6 Ω mL pg–1 mm–2 within the clinically relevant linear detection range from 1 pg mL–1 to 10 ng mL–1. To showcase its potential PoC application, the bio-ready strip is embedded inside a capillary microfluidic device and automates electrochemical quantification of GFAP spiked in phosphate-buffered saline and the human serum. This new electrochemical biosensing platform can be further adapted for the detection of various protein biomarkers with the application in realizing on-chip immunoassays.

The integration of electrochemical biosensors into fluid handling units such as paper-based, centrifugal, and capillary microfluidic devices has been explored with the purpose of developing point-of-care platforms for quantitative detection...

Current laboratory diagnostic approaches for virus detection give reliable results, but they require a lengthy procedure, trained personnel, and expensive equipment and reagents; hence, they are not a suitable choice for home monitoring purposes. This paper addresses this challenge by developing a portable impedimetric biosensing system for the identification of COVID-19 patients. This sensing system has two main parts: a throwaway two-working electrode (2-WE) strip and a novel read-out circuit, specifically designed for simultaneous signal acquisition from both working electrodes. Highly reliable electrochemical signal tracking from multiplex immunosensors provides a potential for flexible and portable multi-biomarker detection. The electrodes' surfaces were functionalized with SARS-CoV-2 Nucleocapsid Antibody enabling the selective detection of Nucleocapsid protein (N-protein) along with self-validation in the clinical nasopharyngeal swab specimens. The proposed programmable highly sensitive impedance read-out system allows for a wide dynamic detection range, which makes the sensor capable of detecting N-protein concentrations between 0.116 and 10,000 pg/mL. This lightweight and economical read-out arrangement is an ideal prospect for being mass-produced, especially during urgent pandemic situations. Also, such an impedimetric sensing platform has the potential to be redesigned for targeting not only other infectious diseases but also other critical disorders.

The widespread accessibility of commercial/clinically-viable electrochemical diagnostic systems for rapid quantification of viral proteins demands translational/preclinical investigations. Here, Covid-Sense (CoVSense) antigen testing platform; an all-in-one electrochemical nano-immunosensor for sample-to-result, self-validated, and accurate quantification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N)-proteins in clinical examinations is developed. The platform's sensing strips benefit from a highly-sensitive, nanostructured surface, created through the incorporation of carboxyl-functionalized graphene nanosheets, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) conductive polymers, enhancing the overall conductivity of the system. The nanoengineered surface chemistry allows for compatible direct assembly of bioreceptor molecules. CoVSense offers an inexpensive (<$2 kit) and fast/digital response (<10 min), measured using a customized hand-held reader (<$25), enabling data-driven outbreak management. The sensor shows 95% clinical sensitivity and 100% specificity (Ct<25), and overall sensitivity of 91% for combined symptomatic/asymptomatic cohort with wildtype SARS-CoV-2 or B.1.1.7 variant (N = 105, nasal/throat samples). The sensor correlates the N-protein levels to viral load, detecting high Ct values of ≈35, with no sample preparation steps, while outperforming the commercial rapid antigen tests. The current translational technology fills the gap in the workflow of rapid, point-of-care, and accurate diagnosis of COVID-19.