Converting Commercial PCBs into Reliable Biosensors: A Study on the Impact of PCB Electrode Design and Pretreatment Techniques on DNA Deposition and Hybridization Efficiency

Biosensors are devices that detect and measure the presence of various analytes, including ions, biomolecules, cells, and microorganisms. 1 Currently, most biosensors that employ disposable electrochemical chips rely on screen-printed electrodes or electrodes fabricated through sputtering, with patterns created either by lithography or by laser ablation. 2 Printed circuit boards (PCBs) offer distinct advantages over these methods, including lower cost, scalability, and ease of integration with electronic components. 3–5 As a result, PCBs hold exciting potential for large-scale production and successful commercialization of electrochemical biosensors. 4 This study investigated the performance of commercially fabricated PCB electrodes designed for small-volume (<100uL) electrochemical biosensing. The design and geometry of the PCB three-electrode system, containing a working electrode (WE), counter electrode (CE), and reference electrode (RE), were made analogous to a screen-printed electrode. 6 Physical characterization including optical microscopy, scanning electron microscopy (SEM), surface profilometry, and X-ray photoelectron spectroscopy (XPS) revealed irregularities with the PCB electrode such as micro cracks, corrosion, and non-uniformity. To address surface contamination, we systematically evaluated multiple chemical and electrochemical pretreatment protocols, including sulfuric acid cycling, plasma cleaning, and low-temperature solvent cleaning (LTSC) methods. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) demonstrated that a low-temperature solvent clean (LTSC) achieved the best results across both electroless nickel immersion gold(ENIG) and hard gold electrodes. This protocol achieved the smallest peak-to-peak separation (ΔE ~77 mV) and the lowest charge transfer resistance (R ct ~188 Ω), indicating optimal removal of organic residues and enhanced electron transfer capabilities. To validate the biosensing capabilities of pretreated PCB electrodes, we employed a DNA hybridization assay using a methylene blue (MB)-tagged target strand as the redox reporter. 7 The PCB gold electrode is functionalized with a thiolated double-stranded capture probe CP:D1. A methylene blue reporter strand CR* displaces the D1 strand to bind with the thiolated CP. This bio-barcode assay can generate a methylene blue redox signal using square wave voltammetry. The target signal found is a 1:1 ratio of the binding of the target strand concentration, with LTSC pretreated electrodes exhibiting the highest peak intensity on hard gold PCB electrodes compared to the rest of the pretreatment methods. These results confirmed the efficacy of surface pretreatment in enhancing biomolecule immobilization and hybridization efficiency on PCB gold surfaces. In conclusion, PCB electrodes are a viable, reliable, and ultra-low-cost platform for electrochemical biosensors. In conclusion, PCB electrodes serve as a viable, reliable, and ultra-low-cost platform for electrochemical biosensors. These findings progress the understanding of creating sensitive, reproducible PCB electrodes, allowing labs and industry to use this substrate to develop low-cost integrated devices for sample-in-answer-out analysis. Future work could expand on these findings by exploring alternative materials, conducting long-term stability studies, and integrating advanced sample processing devices (digital microfluidics, optical analysis, and data transmission) to push the boundaries of PCB electrode performance in diagnostic applications. (1) Zhang, Z.; Sen, P.; Adhikari, B. R.; Li, Y.; Soleymani, L. Development of Nucleic-Acid-Based Electrochemical Biosensors for Clinical Applications. Angewandte Chemie International Edition 2022, 61 (50), e202212496. https://doi.org/10.1002/anie.202212496. (2) García-Miranda Ferrari, A.; Rowley-Neale, S. J.; Banks, C. E. Screen-Printed Electrodes: Transitioning the Laboratory in-to-the Field. Talanta Open 2021, 3 , 100032. https://doi.org/10.1016/j.talo.2021.100032. (3) Perdigones, F.; Quero, J. M. Printed Circuit Boards: The Layers’ Functions for Electronic and Biomedical Engineering. Micromachines (Basel) 2022, 13 (3). https://doi.org/10.3390/mi13030460. (4) Shamkhalichenar, H.; Bueche, C. J.; Choi, J.-W. Printed Circuit Board (PCB) Technology for Electrochemical Sensors and Sensing Platforms. Biosensors (Basel) 2020, 10 (11), 159. https://doi.org/10.3390/bios10110159. (5) Petherbridge, K.; Evans, P.; Harrison, D. The Origins and Evolution of the PCB: A Review. Circuit World 2005, 31 (1), 41–45. https://doi.org/10.1108/03056120510553211. (6) Paimard, G.; Ghasali, E.; Baeza, M. Screen-Printed Electrodes: Fabrication, Modification, and Biosensing Applications. Chemosensors 2023, 11 (2), 113. https://doi.org/10.3390/chemosensors11020113. (7) Traynor, S. M.; Wang, G. A.; Pandey, R.; Li, F.; Soleymani, L. Dynamic Bio-Barcode Assay Enables Electrochemical Detection of a Cancer Biomarker in Undiluted Human Plasma: A Sample-In-Answer-Out Approach. Angewandte Chemie - International Edition 2020, 59 (50), 22617–22622. https://doi.org/10.1002/anie.202009664.