Stopping, opening, and redirecting fluid flow in paper-based microfluidic devices using thermo-responsive gelatin...

The advancement of microfluidic paper-based analytical devices (µPADs) toward complex biochemical assays is hindered by the lack of reliable, low-cost fluid-control strategies capable of providing defined sealing and actuation times. The environmental sensitivity of passive barriers depends on their material composition and formulation, whereas many active valves require comparatively complex fabrication. This work introduces a thermo-responsive gelatin valve as a simple, inexpensive, and biocompatible approach to fluid control. Valve performance was optimized by varying gelatin concentration, cooling conditions, storage duration, and actuation temperature. A concentration of 30 mg mL^-1 provided the most suitable balance between closed-state retention and thermal responsiveness. Porometry and contact-angle measurements indicated that the gelatin-treated region reduced accessible capillary pathways and exhibited a water contact angle of approximately 99°, thereby preventing flow at room temperature. Under controlled heating at 40 °C, the optimized valve opened in approximately 20 s. The valve was integrated into a paper-based acetylcholinesterase inhibition assay for malathion. A single-shot design reduced the number of liquid-addition steps by combining sample delivery and incubation in one operation. The calculated limit of detection (LOD) was 0.24 µM, below the drinking-water guideline value of approximately 0.88 µM. A 0.5 µM malathion spike in St. Lawrence River water produced a recovery of 86.1 ± 3.2%. The device is presented as a proof-of-concept screening platform, with further matrix validation required before regulatory drinking-water analysis. These findings establish gelatin as a tunable material for timed fluid control in paper microfluidic devices.