Although DP technologies are convenient

Although 3DP technologies are convenient for users to manufacture devices automatically, the printing materials are inevitably limited by the manufacturers of 3D printers. Strategies are increasingly being developed to functionalize the raw printing materials or their printed devices with other physicochemical properties, thereby ensuring additional applications [[21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36]]. For example, Mandon et al. modified the resin and demonstrated that stereolithographic 3DP was compatible with the production of 3D biosensing components comprising enzyme [glucose oxidase (GOx) and peroxidase]-entrapped hydrogels for enzyme-based assays [31]. Compared with liquid stereolithographic resins, the thermoplastics for fused deposition modeling (FDM)–type 3DP are more difficult to functionalize because these substances incorporated into printing thermoplastics must be tolerant of a high-temperature (up to 250 °C) extrusion environment [24,[29], [30], [31],34]. Fortunately, many nanomaterials (NMs) possess intrinsic enzyme-like activities (e.g., peroxidase, catalase, oxidase, superoxide dismutase), and they can be highly stable, activity-tunable, and commercially available in large quantities [[37], [38], [39], [40]]. Most importantly, unlike natural enzymes, NMs with enzyme-like characteristics are suitable for incorporation into thermoplastics because they should maintain their enzyme-like catalytic activities after passing through the hot extrusion nozzle. Iron oxide nanoparticles (NPs) have been incorporated into polylactic hiv protease inhibitor filaments for fabrication of peroxidase-active multi-well plates, suggesting the feasibility of introducing enzyme-like NMs into FDM-type 3D printers for the manufacture of analytical devices [34]. To simplify the manufacture of low-cost testing devices was the major goal of this study. A dual-head FDM-type 3D printer and two functionalized thermoplastic filaments—acrylonitrile butadiene styrene (ABS) incorporating peroxidase-mimicking NMs and polyvinyl alcohol (PVA) infiltrated with the chromogenic substrate o-phenylenediamine (OPD)—were employed within a one-step 3DP process to manufacture multi-well plates displaying both peroxidase and chromogenic activities. Upon contacting a sample solution, these fabricated enzyme/substrate–incorporated multi-well plates could catalyze the oxidation of their released OPD in the presence of peroxidase substrate hydrogen peroxide (H2O2), allowing colorimetric visualization by the naked eye or through measurements of the sample's absorbance after loading into a conventional microplate reader. When coupling with glucose oxidase (GOx), glucose, which was oxidized to generate H2O2, could also be further analyzed. After optimizing the incorporation of the peroxidase-mimicking NMs, the infiltration and release of OPD, the immobilization of GOx, the design of the multi-well plates, and the assay conditions, the analytical applicability of this method was verified through analyses of glucose concentrations in urine, serum, and plasma samples. These 3D-printed enzyme/substrate–incorporated multi-well plates appear to be very promising systems for glucose testing and routine analyses of glucose concentrations in clinical samples. Also, it appears that suitably functionalized printing materials can allow 3DP technologies to fabricate smart diagnostic devices for rapid screening applications.
Experimental section
Results and discussion
Conclusions A multi-material FDM-type 3D printer, Fe3O4 NP–incorporated ABS filaments, and OPD-infiltrated PVA filaments have been employed for the one-step fabrication of enzyme/substrate–incorporated multi-well plates that enable the rapid determination of glucose concentrations in clinical samples. Using this novel approach, we have demonstrated that such analytical devices can be simply and automatically manufactured through 3DP coupled with printing materials satisfactorily functionalized through the pre-printing incorporation/infiltration of active substances into the raw materials. Such 3D-printed enzyme/substrate–incorporated multi-well plates should find practical applicability in rapid glucose testing; more generally, the use of functionalized materials for 3DP should substantially extend the applicability of this technology in the chemical sciences.