Bi-ECDAQ: An electrochemical dual-immuno-biosensor accompanied by a customized bi-potentiostat for clinical detection of SARS-CoV-2 Nucleocapsid proteins

Abstract

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.

A compact, low-cost, and binary sensing (BiSense) platform for noise-free and self-validated impedimetric detection of COVID-19 infected patients

“…1 B). It is noted that the iCPG ink composition has carboxyl functionalized groups, therefore the antibodies are immobilized on the electrode without any need for common functionalization steps, as we previously demonstrated in our previous work ( Salahandish et al, 2022a ).…”

“…S1A ). ( Salahandish et al, 2022a ) The numerical simulation was applied to three different electrode designs with different distances between the two WEs while having a similar distance of WEs to the counter and reference electrodes. The three different electrode designs with the distances between WEs set to 0.750, 0.300, and 0.125 mm (denoted DL, DM, and DS, respectively) were subject to computational modeling ( Fig.…”

“…These biosensors have been used for the early diagnosis of several diseases, such as brain injuries ( Khetani et al 2018 , 2021 ), cancer ( Akhavan et al, 2014 ; Chung et al, 2021 ; Pimalai et al, 2021 ), infectious diseases ( Idili et al, 2021 ; Wu et al, 2018 ), and other clinical and pharmaceutical applications ( Arduini et al, 2016 ; Castle et al, 2021 ; El Harrad et al, 2018 ; Monošík et al, 2012 ). For complex bodily fluids such as the blood, the matrix effect has been a critical challenge for the reliable detection of several biomarkers for which multiplex biosensors with the capability of detecting matrix effect are highly beneficial ( Gao et al, 2017 ; Liu et al, 2021 ; Salahandish et al, 2022a ; Timilsina et al, 2021 ; Zupančič et al, 2021 ). The use of multiple electrodes (Panes-Ruiz et al, 2021) in the format of electrode arrays has been also investigated as a self-validation mechanism for avoiding false-positive results, relying on the fact that the results from each electrode were comparable.…”

“…Multiplex electrochemical point-of-care testing (ME-POCT) devices are expected to have four major compartments: 1) reliable and reproducible electrodes created using inkjet or screen printing methods, 2) an accurate biosensing protocol for mediating the electrode surface to create accurate biosensors, 3) microfluidics for automating electrochemical biosensing steps, and 4) and an accurate and low-cost multichannel potentiostat with an easy-to-use interface for data interpretation and wireless data transfer. Despite enormous advances in developing multiplex screen printed electrodes and electrochemical biosensors ( Dhanapala et al, 2020 ; Khetani et al, 2019 ; Kinnamon et al, 2018 ; Noah and Ndangili, 2019 ; Salahandish et al 2019 , 2022a ) as well as the recent progress in self-powered microfluidic devices ( Haghayegh et al 2022a , 2022b ; Nyein et al, 2018 ; Salahandish et al, 2022b ), the realization of ME-POCT requires the development of new hand-held, rapid and remote reading, easy-to-use, noise-free, and cheaper multichannel potentiostat readers ( Alam et al, 2020 ; Bianchi et al, 2021 ).…”

“…To reach accurate signals from each of the electrodes, particularly when using accurate but slow electrochemical impedance spectroscopy (EIS) reading module ( Capaldo et al, 2016 ), the signals need to be recorded at each testing frequency for all working electrodes prior to reading the response in the subsequent testing frequency. In our recent bipotentiostat design (Bi-ECDAQ), we addressed this concern via a parallel signal reading of two biosensors simultaneously wherein the reader showed an improved response compared to the expensive commercial potentiostat ( Salahandish et al, 2022a ). However, further advances are required to cancel noises, improve signal acquisition and interpretation, increase the accuracy of curve fitting, reduce the cost, and enable remote control and access, all needed for the realization of ME-POCT.…”