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

Electrochemical immuno-biosensors are one of the most promising approaches for accurate, rapid, and quantitative detection of protein biomarkers. The two-working electrode strip is employed for creating a self-supporting system, as a tool for self-validating the acquired results for added reliability. However, the realization of multiplex electrochemical point-of-care testing (ME-POCT) requires advancement in portable, rapid reading, easy-to-use, and low-cost multichannel potentiostat readers. The combined multiplex biosensor strips and multichannel readers allow for suppressing the possible complex matrix effect or ultra-sensitive detection of different protein biomarkers. Herein, a handheld binary-sensing (BiSense) bi-potentiostat was developed to perform electrochemical impedance spectroscopy (EIS)-based signal acquisition from a custom-designed dual-working-electrode immuno-biosensor. BiSense employs a commercially available microcontroller and out-of-shelf components, offering the cheapest yet accurate and reliable time-domain impedance analyzer. A specific electrical board design was developed and customized for impedance signal analysis of SARS-CoV-2 nucleocapsid (N)-protein biosensor in spiked samples and alpha variant clinical nasopharyngeal (NP) swab samples. BiSense showed limit-of-detection (LoD) down to 56 fg/mL for working electrode 1 (WE1) and 68 fg/mL for WE2 and reported with a dynamic detection range of 1 pg/mL to 10 ng/mL for detection of N-protein in spiked samples. The dual biosensing of N-protein in this work was used as a self-validation of the biosensor. The low-cost (∼USD$40) BiSense bi-potentiostat combined with the immuno-biosensors successfully detected COVID-19 infected patients in less than 10 min, with the BiSense reading period shorter than 1.5 min, demonstrating its potential for the realization of ME-POCTs for rapid and hand-held diagnosis of infections.

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

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.mm2 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.

Immuno-biosensor on a chip: a self-powered microfluidic-based electrochemical biosensing platform for point-of-care quantification of proteins.

The realization of true point-of-care (PoC) systems profoundly relies on integrating the bioanalytical assays into "on-chip" fluid handing platforms, with autonomous performance, reproducible functionality, and capacity in scalable production. Specifically for electrochemical immuno-biosensing, the complexity of the procedure used for ultrasensitive protein detection using screen-printed biosensors necessitates a lab-centralized practice, hindering the path towards near-patient use. This work develops a self-powered microfluidic chip that automates the entire assay of electrochemical immuno-biosensing, enabling controlled and sequential delivery of the biofluid sample and the sensing reagents to the surface of the embedded electrochemical biosensor. Without any need for active fluid handling, this novel sample-to-result testing kit offers antibody-antigen immunoreaction within 15 min followed by the subsequent automatic washing, redox probe delivery, and electrochemical signal recording. The redox molecules ([Fe(CN)6]3-/4-) are pre-soaked and dried in fiber and embedded inside the chip. The dimensions of the fluidic design and the parameters of the electrochemical bioassay are optimized to warrant a consistent and reproducible performance of the autonomous sensing device. The uniform diffusion of the dried redox into the injected solution and its controlled delivery onto the biosensor are modeled via a two-phase flow computational fluid dynamics simulation, determining the suitable time for electrochemical signal measurement from the biosensor. The microfluidic chip performs well with both water-based fluids and human plasma with the optimized sample volume to offer a proof-of-concept ultrasensitive biosensing of SARS-CoV-2 nucleocapsid proteins spiked in phosphate buffer saline within 15 min. The on-chip N-protein biosensing demonstrates a linear detection range of 10 to 1000 pg mL-1 with a limit of detection of 3.1 pg mL-1. This is the first self-powered microfluidic-integrated electrochemical immuno-biosensor that promises quantitative and ultrasensitive PoC biosensing. Once it is modified for its design and dimensions, it can be further used for autonomous detection of one or multiple proteins in diverse biofluid samples.