Design and functionalization of a µpad for the enzymatic determination of nitrate in urine

In this work, the design of a microfluidic paper-based analytical device (µPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination µPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed µPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14–1.0 mM. The designed µPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at −20◦C. The accuracy of the nitrate µPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.