Semi-enclosed paper sensor for highly sensitive and selective detection of proline

In the current study, we have utilized semi-enclosed, leak-proof, microfluidic paper-based analytical devices (μPAD's) modified with isatin conjugated chitosan as specific colorimetric reagent for the detection of proline. Proline is one of the globally accepted stress biomarker in plants and also one of the prominent amino acid present in wine and some processed food. Quantification of proline is regularly required in agriculture field, food and wine industries. Specific interaction of isatin with proline, uniform film forming ability of chitosan which results in uniform coloration and the presence of leak-proof layer which prevent the diffusion of colorimetric reagent deeper resulted in enhancement of color signal intensity at the reaction zone were utilized. Further, the images of the μPAD's were captured using smartphone with 3D printed imaging box which houses smartphone and μPAD's. This platform utilizes smartphone flash for uniform illumination and ensures constant positioning of μPAD's to capture images. This greatly enhances the sensitivity and accuracy of our platform. Compared to previously published highly sensitive multi-layer, paper-based platform for detection of proline, current method has enhanced detection range (∼7 fold) and has comparable limit of detection of 23.75 μM. Moreover, the developed μPAD's platform has reduced optimum reaction temperature and time compared to previous work. The developed paper based platform was utilized for evaluation of proline content in young Arabidopsis plants which are subjected to water stress for 5 days. The devised paper-based methods have the potential to be applicable for the on-site evaluation of various stresses in plants.