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Immunosensors based on electrical impedance spectroscopy allow for label-free, real-time detection of biologically relevant molecules and pathogens, without requiring electro-active materials. Here, we investigate the influence of bare gold nanoparticles (AuNPs), synthesized via laser ablation in solution, on the performance of an impedimetric immunosensor for detecting severe acute respiratory syndrome coronavirus (SARS-CoV-2). Graphene acetic acid (GAA) was used in the active layer for immobilizing anti-SARS-CoV-2 antibodies, owing to its high density of carboxylic groups. Immunosensors incorporating AuNPs exhibited superior performance compared to those relying solely on GAA, achieving a limit of detection (LoD) of 3 x 10 -20  g/mL to detect the Spike Receptor Binding Domain (RBD) protein of SARS-CoV-2 and of 2 PFU/mL for inactivated virus. Moreover, these immunosensors presented high selectivity against the H1N1 influenza virus. We anticipate that this platform will be versatile and applicable in the early diagnosis of various diseases and viral infections, thereby facilitating Point-of-Care testing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Osvaldo Novais de Oliveira Jr. reports financial support was provided by Coordination of Higher Education Personnel Improvement (CAPES). Osvaldo Novais de Oliveira Jr. reports financial support was provided by National Council for Scientific and Technological Development (CNPq). Osvaldo Novais de Oliveira Jr. reports financial support was provided by State of Sao Paulo Research Foundation (FAPESP). Rafael Cintra Hensel reports financial support was provided by State of Sao Paulo Research Foundation. Stefano Casalini reports financial support was provided by Nanochemistry for Energy and Health. Stefano Casalini reports financial support was provided by Nexus. Stefano Casalini reports financial support was provided by Complessità Chimica C2. Stefano Casalini reports financial support was provided by Government of Italy Commission for the Selection of Excellence University Departments. 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. Published by Elsevier B.V.)

High-throughput sensors are valuable tools for enabling massive, fast, and accurate diagnostics. To yield this type of electrochemical device in a simple and low-cost way, high-density arrays of vertical gold thin-film microelectrode-based sensors are demonstrated, leading to the rapid and serial interrogation of dozens of samples (10 μL droplets). Based on 16 working ultramicroelectrodes (UMEs) and 3 quasi-reference electrodes (QREs), a total of 48 sensors were engineered in a 3D crossbar arrangement that devised a low number of conductive lines. By exploiting this design, a compact chip (75 × 35 mm) can enable performing 16 sequential analyses without intersensor interferences by dropping one sample per UME finger. In practice, the electrical connection to the sensors was achieved by simply switching the contact among WE adjacent fingers. Importantly, a short analysis time was ensured by interrogating the UMEs with chronoamperometry or square wave voltammetry using a low-cost and hand-held one-channel potentiostat. As a proof of concept, the detection of Staphylococcus aureus in 15 samples was performed within 14 min (20 min incubation and 225 s reading). Additionally, the implementation of peptide-tethered immunosensors in these chips allowed the screening of COVID-19 from patient serum samples with 100% accuracy. Our experiments also revealed that dispensing additional droplets on the array (in certain patterns) results in the overestimation of the faradaic current signals, a phenomenon referred to as crosstalk. To address this interference, a set of analyses was conducted to design a corrective strategy that boosted the testing capacity by allowing using all on-chip sensors to address subsequent analyses (i.e., 48 samples simultaneously dispensed on the chip). This strategy only required grounding the unused rows of QRE and can be broadly adopted to develop high-throughput UME-based sensors. In practice, we could analyze 48 droplets (with [Fe(CN) 6 ] 4 - ) within ∼8 min using amperometry.

Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low-cost ultra-dense electrochemical chips, the unprecedented single-response multiplexing of typical label-free biosensors is reported. Using a cheap, handheld one-channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi-reference electrodes, Au (0 V) and Ag/AgCl ink (+0.2 V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non-spatially resolved single-response multiplex strategies. As proof of concept, peptide-tethered immunosensors are demonstrated to provide the duplex detection of COVID-19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high-throughput and multi-analyte platforms, as it can be tailored to other biosensing devices and formats., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)