A nanostructured paper-based device for phenylalanine neonatal screening by LED-induced fluorescence

Abstract

In this work, a novel paper-based analytical device (PAD) coupled to LED induced fluorescence (LIF) detection (fPAD) for the rapid, selective, and sensitivequantification of phenylalanine (Phe) in neonatal samples was developed. EnzymesPhenylalanine dehydrogenase (PheDH) and diaphorase were immobilized on a papermicrozone previously modified with zinc oxide nanoparticles (ZnONPs) coated withchitosan (CH-ZnONPs). Phe was extracted from the blood spots collected samples on filter paper and was mixed with nicotinamide adenine dinucleotide (NAD+) and resazurin. Then the mixture was deposited on the reaction microzone of the fPAD where PheDH converts the Phe and NAD+ to phenylpyruvate and NADH, respectively.Finally, NADH was oxidized by diaphorase with the consequent reduction fromresazurin to resorufin. This latter was detected by LIF using an excitation wavelength of 535 nm and an emission of 580 nm in a synchronized video microscope. We compare the responses of the PADs with and without nanomaterials to demonstrate the improved analytical performance of the developed devices. For this, the PADs were modified with the same concentration of horseradish peroxidase (HRP). The fluorescent signal obtained from the PADs with nanomaterials was higher than that of the unmodified PADs.Our method exhibited within- and between-assay variation coefficients below 5.23% and 6.67%, was 0.125 μM. The proposed fPAD allowed the simple, rapid, low-cost, and sensitivedetection of Phe in neonatal blood samples.