Advances in electrochemiluminescence: optimizing materials and techniques for beads-based biosensor applications

Accurate antibody (Ab) detection is crucial for modern diagnostics, spanning infectious disease screening to cancer biomarker identification. Electrochemiluminescence (ECL) offers key advantages over conventional luminescence techniques, including low background noise, high signal-to-noise ratio, and broad compatibility with biological matrices. This PhD project aimed to deepen mechanistic insights into ECL and optimize ECL-based biosensors, particularly bead-based platforms. The integration of ECL microscopy (ECLM) enabled single-object-level imaging, uncovering spatial and temporal characteristics of ECL emissions, advancing real-time, high-resolution biosensing. We employed magnetic microbeads functionalized with tris(2,2'-bipyridyl)ruthenium(II) ([Ru(bpy)₃]²⁺) as the luminophore and tripropylamine (TPrA) as the coreactant. High-resolution ECLM imaging revealed spatial heterogeneity in emissions, influenced by restricted TPrA radical cation diffusion near the electrode. Emission intermittency correlated with luminophore proximity and density on the beads. Combining finite element simulation with singular spectrum analysis enabled signal denoising, isolating a coreactant-diffusion-controlled decay component and exposing previously undetected oscillatory patterns in ECL intensity. These oscillations provide mechanistic insights critical for enhancing biosensor sensitivity and performance. To further optimize ECL, we investigated alternative electrode materials, notably boron-doped diamond (BDD) and cerium oxide (CeO₂)-coated glassy carbon. Compared to platinum, BDD electrodes increased ECL intensity by 70%, doubling the signal-to-noise ratio with enhanced stability. CeO₂-coated electrodes improved electron transfer rates, essential for ultrasensitive immunoassays. Additionally, within the European Nano-ImmunoEra project, we explored screen-printed carbon electrodes (SPCEs) for point-of-care applications. Laser-treated SPCEs exhibited increased surface area and crystallinity, enhancing ECL intensity, reproducibility, and scalability. SPCEs with GST carbon paste achieved a detection limit of ~11 Abs/µm² on bead surfaces. This thesis advances ECL technology for next-generation diagnostics, establishing improvements for high-throughput, portable biosensing in clinical, environmental, and personalized health applications.