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.

Enzymatic determination of D-alanine with L-alanine dehydrogenase and alanine racemase

An enzymatic assay system of D-Ala, which is reported to affect the taste, was constructed using alanine racemase and L-alanine dehydrogenase. D-Ala is converted to L-Ala by alanine racemase and then deaminated by L-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the D-Ala contents of seven crustaceans were determined.