Real-time fluorescent and microscopic uricase assay by monitoring the clearance of acridine orange-doped monosodium urate crystals under physiological conditions

Abstract

The accumulation of monosodium urate (MSU) crystals in joints plays a central role in the pathogenesis of gouty arthritis. While conventional treatments for acute gout focus on symptom relief, they do not address the root cause—crystal deposition. Recombinant uricase offers a promising long-term solution by catalyzing the conversion of poorly soluble uric acid into highly soluble allantoin, facilitating MSU crystal clearance. In this study, we developed a novel assay system using acridine orange (AO)–doped MSU crystals as a solid substrate and real-time sensing unit to monitor uricase activity under physiological conditions. The MSU-AO crystals were comprehensively characterized by spectroscopic, microscopic, and diffraction-based techniques. Uricase activity under physiological conditions was monitored by two complementary readouts: fluorescence emission from AO released during crystal dissolution, and microscopic measurement of crystal size reduction. Both outputs exhibited strong linear correlations with enzyme concentration, demonstrating the potential of this system as a platform for therapeutic uricase evaluation. Importantly, the microscopic assay required only 2 µL of sample and was successfully applied to rat serum and porcine synovial fluid, highlighting its suitability for use with limited quantities of biological specimens.