An Integrated 3D-Printed Portable Device Coupling Bimetallic FeCo-MOF Nanozymes with Freeze-Dried Hydrogel Carriers for Point-of-Care Testing of Serum Acid Phosphatase.

Acid phosphatase (ACP) is a key biomarker for prostate disorders, hyperparathyroidism, and other metabolic diseases. However, current ACP assays are limited by complex protocols, bulky instrumentation, and poor stability under resource-limited conditions. Herein, we report a portable, smartphone-readable point-of-care testing (POCT) platform integrating bimetallic FeCo-MOF nanozymes entrapped in freeze-dried agarose hydrogel carriers for dual-mode (colorimetric-fluorometric) quantification of ACP in human serum. The FeCo-MOF catalyzes glucose oxidation to generate H2O2 and reactive oxygen species, converting o-phenylenediamine (OPD) to the chromogenic/fluorogenic product 2,3-diaminophenazine (DPA). ACP-mediated hydrolysis of sodium pyrophosphate (PPi) restores FeCo-MOF activity, enabling signal readout proportional to ACP concentration. Encapsulation of reagents within freeze-dried hydrogels significantly enhances substrate stability (89-100% activity retained at 4 °C for 30 days) and facilitates rapid reagent diffusion (<5 min). The 3D-printed device (∼1.3 kg) integrates a temperature control stage (±1 °C), dark chamber, dual-excitation light sources, and smartphone-based signal acquisition. Under optimized conditions (37 °C, 40 min and 75 °C, 8 min), the assay achieves linear ranges of 0.4-3.5 U/L (colorimetric) and 0.2-4.0 U/L (fluorometric), with LODs of 0.21 and 0.11 U/L, respectively. Results correlate strongly with conventional UV-vis and ratiometric fluorescence methods. Recovery in spiked serum samples ranges from 95 to 106%, with negligible interference from common serum constituents. This integrated POCT system offers a low-cost, user-friendly, and quantitatively reliable approach for ACP detection in decentralized settings.