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Rapid, point-of-care tests are critical for early diagnosis of disease and detection of biological threats. Lateral flow immunoassays (LFIAs) are well-suited for point-of-care testing due to their ease of use and straightforward readout. However, limitations in sensitivity, quantification, and integration into sample-to-result systems indicate the need for further advancements. This paper introduces a novel all-in-one LFIA that integrates a test strip with optimized geometry within a newly designed finger-prick blood collection cartridge. This innovative system offers an efficient solution for the detection and quantification of hepatitis B antigens, combining precision and user-friendliness in a single device. To enhance assay performance, the conventional test strip's response to the target protein was significantly improved by incorporating a constricted test zone and upstream mixing elements. To meet self-testing requirements, the blood collection cartridge incorporates an innovative two-step rotation mechanism that simplifies sample collection, processing, and application onto the test strip. Numerical simulations of flow dynamics and antibody-antigen interactions using COMSOL guided the optimization of the test strip geometry, achieving a substantial improvement in the limit of detection from 1.78 ± 0.08 to 0.55 ± 0.04 ng/mL compared to the classic rectangular strip geometry. The optimized design also increased analytical sensitivity from 1.4 ± 0.1 to 2.8 ± 0.1 RU.mL/ng. The system demonstrated complete functionality, from sample collection to analyte quantification. This integrated, user-friendly platform provides an advanced, sample-to-result diagnostic solution for detecting disease markers from finger-prick blood samples. Its simplicity makes it suitable for point-of-care testing, including home use by non-professionals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Razieh Salahandish reports financial support was provided by Mitacs Canada. Razieh Salahandish has patent #63/643,523 pending to Scanbo. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Early-stage disease detection, particularly in Point-Of-Care (POC) wearable formats, assumes pivotal role in advancing healthcare services and precision-medicine. Public benefits of early detection extend beyond cost-effectively promoting healthcare outcomes, to also include reducing the risk of comorbid diseases. Technological advancements enabling POC biomarker recognition empower discovery of new markers for various health conditions. Integration of POC wearables for biomarker detection with intelligent frameworks represents ground-breaking innovations enabling automation of operations, conducting advanced large-scale data analysis, generating predictive models, and facilitating remote and guided clinical decision-making. These advancements substantially alleviate socioeconomic burdens, creating a paradigm shift in diagnostics, and revolutionizing medical assessments and technology development. This review explores critical topics and recent progress in development of 1) POC systems and wearable solutions for early disease detection and physiological monitoring, as well as 2) discussing current trends in adoption of smart technologies within clinical settings and in developing biological assays, and ultimately 3) exploring utilities of POC systems and smart platforms for biomarker discovery. Additionally, the review explores technology translation from research labs to broader applications. It also addresses associated risks, biases, and challenges of widespread Artificial Intelligence (AI) integration in diagnostics systems, while systematically outlining potential prospects, current challenges, and opportunities., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., (Copyright © 2022. Published by Elsevier B.V.)

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.

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.

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 of bodily fluid markers. However, the present fluidic device designs largely lack the capacity of full assay automation, needing manual loading of one or multiple reagents or requiring external devices for liquid manipulation. Such fluidic handing platforms also require universality for detecting various biomarkers. These platforms are also largely produced using materials unsuitable for scalable manufacturing and with a high production cost. The mechanism of fluid flow also often induces noise to the embedded biosensors which adversely impacts the accuracy of biosensing. This work addresses these challenges by presenting a reliable design of a fully automated and universal capillary-driven microfluidic platform that automates several steps of label-free electrochemical biosensing assays. These steps include sample aliquoting, controlled incubation, removal of non-specific bindings, reagent mixing and delivery to sensing electrodes, and electrochemical detection. The multilayer architecture of the microfluidic device is made of polymeric and adhesive materials commercially used for the fabrication of point-of-care devices. The design and geometry of different components of the device ( e.g. , sampling unit, mixer, resistances, delay valves, interconnecting components) were optimized using a combined experimental testing and numerical fluid flow modeling to reach high reproducibility and minimize the noise-induced to the biosensor. As a proof of concept, the performance of this on-chip immunosensing platform was demonstrated for rapid and autonomous detection of glial fibrillary acidic proteins (GFAP) in phosphate-buffered saline (PBS). The microfluidic immunosensing device exhibited a linear detection range of 10-1000 pg mL -1 for the detection of GFAP within 30 min, with a limit of detection (LoD) and sensitivity of 3 pg mL -1 and 39 mL pg -1 mm -2 in PBS, respectively. Owing to its simplicity, sample-to-result performance, universality for handing different biofluids, low cost, high reproducibility, compatibility with scalable production, and short analysis time, the proposed biosensing platform can be further adapted for the detection of other biomarkers in different clinical bodily fluids for rapid diagnostic and prognostic applications.

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·mm 2 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.

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.