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This work reports on fully integrated “sample‐in‐signal‐out” microfluidic paper‐based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET‐based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume‐independent and fully reagent‐free operation, including on‐device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti‐HIV1, anti‐HA, and anti‐DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user‐friendly point‐of‐care testing in low‐resource environments.

Paper‐Based Antibody Detection Devices Using Bioluminescent BRET‐Switching Sensor Proteins