One-step three-dimensional printing of enzyme/substrate-incorporated devices for glucose testing

To substantially simplify the fabrication of analytical devices for rapid screening tests, in this study we employed multi-material fused deposition modeling-type three-dimensional printing (3DP) and two functionalized thermoplastic filaments-acrylonitrile butadiene styrene (ABS) incorporating peroxidase-mimicking iron oxide (Fe₃O₄) nanoparticles and polyvinyl alcohol (PVA) infiltrated with the chromogenic substrate o-phenylenediamine (OPD)-for the one-step manufacture of enzyme/substrate-incorporated multi-well plates. Upon contact with samples, these fabricated devices (i) released the chromogenic substrate OPD into the solution, (ii) efficiently catalyzed the oxidation of OPD mediated by the peroxidase substrate H₂O₂, (iii) enabled assays of those substances availably oxidized by their specific oxidases to generate H₂O₂, and (iv) facilitated colorimetric observation by the naked eye or through absorbance measurements after loading into a microplate reader. With glucose oxidase immobilized in each well, samples appropriately diluted could be directly loaded for derivatizing and analyzing glucose without adding any other reagents. After assay optimization, the limits of detection reached as low as 2.8 μM for H₂O₂ and 5.0 μM for glucose; the method's applicability was illustrated in terms of determining glucose concentrations in urine, serum, and plasma samples. These 3D-printed peroxidase mimic/chromogenic substrate-incorporated multi-well plates appear to be highly suitable for rapid and high-throughput screening of glucose in clinical samples. We demonstrate that adequate functionalization of raw materials for 3DP can contribute to the development of novel multifunctional devices with many potential practical applications.