Paper-based biosensor based on phenylalnine ammonia lyase hybrid nanoflowers for urinary phenylalanine measurement

The Phenylketonuria (PKU) is an inborn defect of phenylalanine (Phe) metabolism, in which Phe accumulated in the blood causing alterations at the central nervous system. Therefore, the detection of PKU is very important for the early diagnosis of PKU patients. However, existing tests for PKU are time-consuming and require high-resource laboratories. In this study, a novel paper-based biosensor based on phenylalnine ammonia lyase (PAL) hybrid nanoflowers was constructed that provides a semi-quantitative output of the concentration of Phe from urine samples. PAL@Ca₃(PO₄)₂ hybrid nanoflowers (PAL@NF) were first prepared using PAL and Ca²⁺. Synthesis conditions of the PAL@NF on the formation of the PAL@NF were optimized. The PAL@NF exhibited 90% activity recovery under optimal condition. Compared with free PAL, the PAL@NF displayed good storage stability and increased tolerance to proteolysis. After five consecutive operating cycles, the PAL@NF still retained 73% of its initial activity, indicating excellent reusability. Furthermore, the paper-based biosensor was able to detect Phe concentration in urine samples, and exhibited good linearity to the Phe concentrations in the range from 60 to 2400 μM and the response time was only about 10 min. Therefore, the paper-based biosensor can be a promising candidate as a biosensor for the detection of PKU.