Self-Calibrating Ratiometric Colorimetric Microfluidic Paper-Based Analytical Device for Visual Quantification of Hypoxanthine

Existing self-calibrating strategies improve quantitative paper-based colorimetry by correcting imaging variations, yet their analytical reliability remains limited because heterogeneous sensing interfaces introduce errors that cannot be compensated by internal-reference calibration alone. Herein, we present a self-calibrating ratiometric microfluidic paper-based analytical devices (μPADs) based on a dual error-control strategy that integrates interface homogenization with internal-reference correction for portable hypoxanthine (Hx) quantification, an important early freshness indicator of aquatic products. A sol−gel/chitosan (Sol−Gel/CS) hybrid network uniformly immobilizes MnO2 nanoflowers (NFs), suppressing coffee-ring formation while improving reagent stability, catalytic activity, and color uniformity. Xanthine oxidase (XOD), incorporated only into the test zone, catalyzes Hx oxidation to generate H2O2 and uric acid, which decompose MnO2 NFs and weaken their oxidase-like activity toward 3,3′,5,5′-tetramethylbenzidine, producing an Hx-dependent color decrease. Meanwhile, the XOD-free reference zone provides a stable internal-reference signal. Ratiometric analysis (IT/IR) effectively compensates for imaging variations across different smartphones, enabling reliable visual quantification. The μPAD achieved a detection limit of 3.42 μM and agreed well with triple-quadrupole liquid chromatography−tandem mass spectrometry. Freshness monitoring of crucian carp, shrimp, and scallop was successfully demonstrated through both extract-based and direct-contact assays. This dual error-control strategy establishes a general framework for robust quantitative paper-based colorimetric sensing.