Integrated aptamer-based DNAzyme motor with pattern recognition for discriminative intracellular uric acid biosensing and high-throughput GLUT9 inhibitor discovery

Current cell-based screening methods for Glucose Transporter 9 (GLUT9) inhibitors, including manual patch-clamp (PC) electrophysiology and radioactive tracer assays, are limited by low throughput and reliance on specialized instrumentation. Here, we develop a biosensing platform combining a DNAzyme motor (DM), an aptamer sensor array (ASA) and pattern recognition-based signal processing (implemented via linear discriminant analysis, LDA), termed DM-ASA-LDA, for intracellular UA detection and GLUT9 inhibitor screening. In this system, the DM is constructed by functionalizing gold nanoparticles (AuNPs) with FAM-labeled substrate strands and DNAzymes locked by aptamers (Apts) that bind uric acid (UA) and xanthine (Xan) with varying affinities. Due to the inability of a single Apt to distinguish UA from Xan, an ASA is designed using three distinct Apts, and the resulting fluorescence signals are processed using LDA. Mechanistically, following endocytosis, the DM is internalized into cells. Intracellular UA and Xan then bind to the Apts, triggering conformational changes that unlock the DNAzyme. The activated DNAzyme cleaves FAM-labeled substrates, resulting in signal conversion and amplification. After cell lysis, the FAM fluorescence is detected and processed by LDA, enabling accurate UA quantification. This quantification capability correlates strongly with PC analysis for evaluating the GLUT9 inhibitor isobavachin (r = 0.9207). Using our platform, mangiferin was identified as a potent GLUT9 inhibitor that significantly reduces UA levels in a hyperuricemic mouse model. Implemented on a 96-well plate, this platform provides high throughput screening with operational simplicity, serving as a practical tool for routine GLUT9 inhibitor evaluation in standard laboratory settings.