Paper integrated aptamer-CRISPR sensing enables multiplexed small-molecule sensing in a low-cost device.

CRISPR/Cas12a-based diagnostics achieved attomolar sensitivity for nucleic acid detection, yet their extension to small-molecule targets remains constrained by the need for solution-phase aptasensor-CRISPR coupling and laboratory infrastructure incompatible with resource-limited settings. Here we report a Paper-integrated Aptamer-Cas12a Sensing platform (PACSense) in which the complete aptasensor-CRISPR/Cas12a signal transduction cascade is air-dried within a paper substrate, enabling a one-pot, amplification-free small-molecule detection workflow that is initiated by sample addition alone. Two orthogonal sensing modalities are demonstrated: first, a signal-OFF bivalent aptamer channel for adenosine 5'-triphosphate (ATP), achieving solution- and paper-phase limits of detection of 336 nM and 446 nM respectively, and second, a signal-ON complementary strand-displacement channel for cocaine, which to our knowledge represents the first coupling of the NC195 aptamer to CRISPR/Cas12a sensing, with solution- and paper-phase LODs of 0.605 µM and 16.15 µM respectively. Both channels are operated simultaneously on a single multiplexed paper chip with a cross-channel geometry that routes sample passively to four spatially distinct reaction zones, enabling independent and simultaneous detection confirmed by fluorescence imaging under a 495 nm blue LED. Air-dried chips retain 76% of initial fluorescence signal after 30 days of ambient storage without cold chain, with a 93% signal retention measured from the functional equilibration baseline. The complete assay workflow is completed within 20 minutes using a reusable sodium acetate hand warmer for incubation and a 3D-printed smartphone-compatible reader, designed to align with ASSURED criteria for point-of-care diagnostics. PACSense establishes paper-integrated aptasensor-CRISPR sensing as a viable route to multiplexed, laboratory-instrument-free small-molecule diagnostics at the point of need.