Colorimetric biosensor for phenylalanine detection based on a paper using gold nanoparticles for phenylketonuria diagnosis

Abstract In this study, a colorimetric paper-based biosensor was developed for the highly sensitive and selective determination of phenylalanine. Reagents were immobilized onto the paper substrate to form the test paper. The phenylalanine dehydrogenase (PheDH) enzyme reacts specifically with phenylalanine in the presence of NAD+ to form NADH that reacts with MTT, which is catalyzed by the diaphorase enzyme to form the purple colored formazan. The increase of phenylalanine concentration is proportional to the color intensity and can be monitored by the color change of the dye solution. It was discovered that Au NPs interact with cationic dye (MTT) which causes the enhancement of color intensity. The colored reaction product could be either qualitatively assessed by the naked eye or quantified using a smartphone camera evaluated by the freeware Image J. This paper-based test is easy to operate, and can develop the results within 10 minutes. After applying the optimal conditions, the linear range for phenylalanine was found to be from 0.8 to 33 (mg/dL) with a detection limit of 0.57 (mg/dL), using the digital image method. Furthermore, analysis of phenylalanine levels in real samples was enabled as a result of the promising detection performance of the developed approach, which could be used as a screening test for PKU in newborns in situations where current approaches cannot be used.