Co-g-C₃N₄ Nanozyme-Based Colorimetric Biosensor for miR-320a Detection in Thyroid-Associated Ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) is a complex autoimmune inflammatory disease. There is an urgent clinical need for highly sensitive and minimally invasive biomarkers to enable early diagnosis and dynamic disease monitoring. Among them, miR-320a has been confirmed to be closely associated with oxidative stress regulation, fibroblast activation, and fibrosis progression. However, current detection methods, especially those based on real-time quantitative polymerase chain reaction (RT-qPCR), still suffer from cumbersome procedures, high instrumentation costs, and limited adaptability for rapid point-of-care diagnostics. To address this, we developed a nanozyme-assisted colorimetric biosensing platform integrating target-triggered probe hybridization with Co-g-C₃N₄, which exhibits enhanced peroxidase-like activity and stability. The method involves Co-g-C₃N₄ synthesis, probe-nanozyme interface construction, catalytic optimization, and colorimetric detection via TMB oxidation, validated in spiked serum and with smartphone readout. The platform achieves sensitive miR-320a detection down to picomole levels with excellent linearity and specificity. In spiked human serum analysis, it reliably quantifies endogenous miR-320a with strong recovery rates (94.6-108.9%). However, we acknowledge that direct clinical validation using confirmed TAO patient samples is required for full diagnostic implementation, and this represents a limitation of the current study. Generally speaking, this work provides a portable, cost-effective, amplification-free colorimetric nanozyme platform for miRNA detection, requiring only basic laboratory equipment, and holding significant promise for point-of-care precision diagnostics in autoimmune and fibrotic diseases.