Electrostatically Sealed Paper-Based Microfluidic Device for Environmentally Robust Nitrite Determination by Griess Colorimetry

Microfluidic paper-based analytical devices (µPADs) are promising for point-of-care testing, yet most operate in an open-format that is susceptible to solvent evaporation and reagent contamination, causing poor signal stability under changing environments. Here, we present an equipment-free sealing strategy to construct closed µPADs by electrostatically adsorbing two polymer films onto open chips, forming an enclosed microenvironment. Using the Griess colorimetric reaction for nitrite, we benchmarked the closed-format against an open counterpart. At a standard condition, the closed µPAD produced a higher grayscale signal, reached a stable peak within 10 min, and remained stable for >60 min. It also demonstrated superior environmental robustness, yielding consistently smaller relative standard deviations (RSDs) across varying humidity levels and temperatures. With the optimized readout time, nitrite exhibited linear calibration over 2.0–10 mg/L (R2 = 0.998), with a detection limit of 0.75 mg/L. In synthetic urine A, spike–recovery tests at 2.0, 5.0, and 9.0 mg/L gave acceptable recoveries and precision across temperature–humidity conditions (RSD < 10%). Additional verification in synthetic urine B at 5.0 mg/L under an uncontrolled environment further confirmed the practicality of this sealed µPAD for environment-tolerant quantitative nitrite analysis.