Origami Microfluidic Paper-Based Analytical Devices Integrated with Layered Double Hydroxides Nanozymes for the Colorimetric Determination of D-Amino Acids in Human Fluids

This work presents the development of NiFe-layered double hydroxides (NiFe-LDHs) as a highly effective peroxidase mimic for the rapid and sensitive colorimetric detection of D-amino acids. The nanozyme was synthesized via a simple coprecipitation method and thoroughly characterized by X-ray powder diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy. We integrated the NiFe-LDHs into an origami microfluidic paper-based analytical device (O-μPAD) featuring a laser-printed hydrophobic barrier. This platform spatially separates the enzymatic generation of H2O2 by D-amino acid oxidase (DAAO) from the subsequent nanozyme-catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine, preventing cross contamination and enabling multianalyte detection. The assay requires only a single drop of human serum or saliva, which is applied to the device housed in a custom acrylic mold. The resulting green color change, proportional to the D-amino acid concentration, is quantified using a smartphone camera within a controlled lightbox. The method demonstrated wide linear ranges for D-proline and D-alanine (0.01–5.0 mM) with low detection limits of 0.002 mM and 0.002 mM, respectively. By combining the catalytic activity of NiFe-LDHs with the portability of O-μPADs and the ubiquity of smartphone detection, this work provides a powerful and practical platform for multiplexed analysis of multiple biomarkers in point-of-care diagnostics.