Design and assembly of microfluidic paper-based analytical devices (µPADs) for the quantification of nitrite and nitrate in saliva

In the last few years, researchers, with the help of new and advanced technologies, have been working towards the development of more practical and more affordable, diagnostic and treatment devices and techniques. In this work, two different Microfluidic Paper-based Analytical Devices (μPADs) were developed for the determination of nitrite and nitrate in human saliva samples to aid in the diagnosis of some diseases and health conditions associated with these ions. To develop these nitrite and nitrate μPADs, several studies were performed to optimize the design and construction, including an interference assessment and stability studies. The final structure of developed μPAD for the nitrite determination consisted of two layers of 9.5 mm diameter filter paper disks within a plastic laminating pouch, in which the bottom layer contained 5 μL of Griess reagent. This μPAD allowed a nitrite determination in a range of 5 - 220 μM with limits of detection and quantification of 0.05 μM and 0.17 μM, respectively. The nitrate μPAD final structure consisted of three layers of filter paper, also within a plastic laminating pouch, in which the top layer contained the zinc powder and the bottom layer contained 10 μL of Griess reagent. This μPAD allowed a nitrate determination in the range 0.2 – 1.2 mM with limits of detection and quantification of 0.08 mM and 0.27 mM, respectively. Both of the μPADs were stable when stored in vacuum (the nitrite μPAD for at least 7 days and the nitrate μPAD for a maximum of 3 days) and, after the sample placement, the nitrite and nitrate μPADs could be scanned within the first 4 and 2 hours, respectively. Finally, to validate this method, nitrite μPAD measurements were compared with the ones obtained from the standard colorimetric method and there were no statistically significant differences between these two methods. So, it was possible to conclude that the developed μPADs exhibited promising properties for NOX determinations in saliva samples, especially because they are sensitive, portable, simple and affordable devices that cost less than 50 cents each.