Prussian Blue Nanozyme-Based Dry-Dipstick Reflectometry for Multiplex Detection of Low Molecular Weight Metabolites

Chronic diseases (CDs) and their associated hepatic and renal dysfunctions pose grim global health challenges, severely compromising quality of life while increasing morbidity and mortality rates. In China, a big bottleneck in effective CD management lies in the lack of affordable and efficient point-of-care testing (POCT) methods at the primary care level. To address this, we developed a Prussian blue (PB) nanozyme-based dry dipstick reflectometric platform for probing key metabolic markers associated with hyperglycemia, hyperuricemia, hypercholesterolemia, and hyperlipidemia. Gram-scale synthesis of PB nanozymes was successfully secured using ethylene glycol as a crowding agent and antifouling additive. The nanozyme showed superb catalytic performance, with an activity approximately 67-fold higher than that of horseradish peroxidase (HRP), while reducing the per-test cost by up to 55.9-fold compared to the HRP-based ensemble. A considerably cost-effective quadruple sensing strip was shaped up by coimmobilizing PB nanozymes with substrate-specific oxidases, chromogen, and proton donors. This enabled the simultaneous detection of glucose, uric acid, cholesterol, and triglycerides using only 50 μL of the sample. Integrated with a smartphone-mounted detector, the slides succeeded in multiplexed assay of serum-simulated samples within 6 min, demonstrating superb repeatability (overall coefficients of variation <5%). The detection limits were 0.52 mM for glucose, 0.025 mM for uric acid, 0.18 mM for cholesterol, and 0.425 mM for triglycerides, with corresponding linear ranges of 2-30, 0.1-1.0, 1-10, and 0.5-7 mM, respectively. This on-paper approach holds strong strength for expanding to other CD indicators (e.g., aspartate, alanine aminotransferase, urea, creatinine), fostering biohybrid POCT solutions for broader clinical applications.