Development of a paper-based analytical device incorporating NADH-sensitive polymer dots for rapid fluorometric quantification of branched-chain amino acids in blood

Branched-chain amino acids (BCAAs) are essential amino acids that play a crucial role in regulating animal physiology and metabolism. The lack of sensitive, rapid, and portable methods for measuring BCAA concentrations in blood samples hinders precise and dynamic monitoring of nutritional status during swine production. In this work, a paper-based analytical device (PAD) was developed for quantifying BCAAs in blood samples. The device utilized a redox reaction between BCAAs and NAD+ catalyzed by leucine dehydrogenase, producing NADH that could be detected by NADH-responsive polymer dots with high selectivity, sensitivity, and accuracy. A separation membrane was incorporated in the PAD for spontaneous blood-plasma separation. The fluorescent images were captured by using a smartphone. It required only 20 μL of blood volume, and the entire assay could be completed in less than 5 minutes. The quantitation range spanned from 35 μM to 1000 μM, covering the normal BCAA concentration range in porcine bloods. The BCAA concentration measured with the PAD assay was in good agreement with those obtained with an ELISA kit. In sum, this PAD-based BCAA assay offers a point-of-care testing approach for monitoring BCAA levels in pig production, especially with a low-protein diet.