A fully integrated Òsample-in-signal-out Ó microfluidic paper-based analytical devices are provided relying on bioluminescence resonance energy transfer (BRET) switches for target analyte recognition and colorimetric signal generation. Simultaneous colorimetric detection and quantification of multiple antibodies in a single drop of whole blood is shown. The devices make use of BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized porous layer(s) of material. The device enables sample volume independent, eliminates addition of reagents to the sample, and has on-device blood plasma separation. User operation is a single drop of a sample and the acquisition of a photograph after sample introduction, with no requirement for precise pipetting, liquid handling or analytical equipment except for a camera. Using different antibodies as targets, simultaneous detection in whole blood was achieved. This device is believed to be ideally suited for user-friendly point-of-care-testing in low- resource environments based on BRET-sensors.