Coelenterazine Analog Library for Specific Imaging of Serum Albumins on Conventional and Paper Microplates

Paper-based analytical devices (PADs) are promising platforms to achieve Point-of-Care Testing (POCT) due to their low cost, ease of operation, disposability, and simplicity of assaying. Inspired by the bioanalytical potential of marine luciferins as excellent optical indicators, we evaluated a panel of 30 coelenterazine (CTZ) analogs for the specific imaging of important serum proteins from various species. This panel included 20 novel CTZ analogs, recently developed by the authors and designated as the "G-series". Remarkably, several G-series CTZ analogs are exclusively bright with specific serum albumins and collectively create unique intensity patterns for these proteins. These G-series CTZ indicators produced highly quantitative luminescence signals with distinct spectral profiles across both trace and physiological albumin concentrations. This luminescence was utilized to determine kinetic constants, identify reaction sites, and establish dose-response curves for the various albumins. Finally, the optimized imaging system was transitioned to paper microplates for a rapid, on-site assay of serum albumins. HSA and BSA deposits dried on paper microplates were successfully imaged with CTZ indicators, achieving high signal-to-background ratios. This platform offers a cost-effective and portable approach to the rapid assay of serum albumins from various animal sources.