Ultra-dense vertical electrochemical chips enable single-response multiplexing

Abstract

This project aims to develop label-free electrochemical biosensors (LEBs) with the potential to facilitate rapid and cost-effective disease diagnosis in various clinical settings, including laboratories, hospitals, and point-of-care scenarios. To achieve this objective, we intend to fabricate devices that fulfill two critical criteria: (i) real-world applicability at an economically viable cost and (ii) high testing throughput. This will be accomplished through the utilization of microfabrication techniques and vertical thin-film engineering, resulting in the production of ultra-dense chips. These chips are designed to exhibit reproducibility, compatibility for large-scale manufacturing, and high resolution (enabling the generation of both macro and microelectrodes) at a reduced cost. Exploiting the distinctive design of these chips, we propose to pioneer the implementation of single-response multiplexing analyses (SERM) in LEBs. SERM analyses can be executed through a single scan of square wave voltammetry (SWV), allowing for the use of a portable single-channel potentiostat. Importantly, this system relies on spatially isolating samples in discrete regions of the working electrodes (WEs), facilitating individual modification of the WEs and minimizing interference between redox probes (utilized for generating electrochemical responses). Each sensor comprises only two electrodes, consisting of vertical thin films of gold in a three-dimensional mesh separated by a dielectric, specifically the negative photoresist SU-8. This configuration aims to produce ultra-dense chips, featuring dozens of sensors per wafer (glass), thereby substantially reducing the final cost of the sensors. The capability to conduct SERM analyses will be achieved through two primary strategies: (i) the utilization of different redox probes (multiplex method) and (ii) the pioneering use of quasi-reference electrodes (QRE) composed of different materials within the same SWV measurement (duplex method). The multiplex method enables the monitoring of up to three samples using different redox probes, namely potassium hexacyanoferrate [Fe(CN)6]3-/4-, ferrocenemethanol (FcMeOH), and hexaammineruthenium [Ru(NH3)6]3+, resulting in distinct current peaks at different potentials. As for the duplex method, it facilitates the analysis of two samples using only the [Fe(CN)6]3-/4- probe. This is achieved by employing QREs composed of materials with distinct work functions, namely gold and Ag/AgCl. Consequently, current peaks can be obtained at specific potentials associated with each QRE. These multiplexing strategies will enable the simultaneous diagnosis of multiple samples in a single SWV analysis, thereby enhancing testing throughput. As a proof of concept, bioassays will be conducted utilizing a peptide as the recognition element for COVID-19 IgG antibodies, encompassing both standard samples and authentic serum samples.