An integrated protein detection platform based on "lock-and-cage" sensors and digital microfluidics.

Data-driven protein engineering has enabled biosensors that directly couple molecular recognition with measurable signal outputs, providing a powerful strategy for rapid protein analysis. However, their integration into automated and portable analytical systems remains limited. Here, we developed an integrated photoelectrochemical and fluorescence digital microfluidic (PF-DMF) platform for automated biomarker detection by combining a programmable "lock-and-cage" protein sensor (LucCage/LucKey) with precise digital microfluidic manipulation. The platform enabled automated detection of human tumor necrosis factor-α (hTNF-α) with a detection limit of 0.1 μM over a linear range of 0.1-10 μM, while exhibiting high specificity, good reproducibility, and minimal sample consumption. Direct analysis of hTNF-α in simulated body fluids was achieved without complex sample pretreatment. The platform was further extended with a CRISPR-based nucleic acid sensing module, enabling nucleic acid detection of Salmonella typhimurium (S. typhimurium) within the same automated workflow. In addition, a standardized reagent kit maintained stable analytical performance for at least 30 days under appropriate storage conditions. Overall, the proposed PF-DMF platform provides a versatile and scalable strategy for automated multimodal biomarker analysis, offering significant potential for portable diagnostics in healthcare and food safety.