A Data-Driven Multifunctional Microfluidic Platform for in Vitro Safety Assessment of 5-Hydroxymethylfurfural

5-Hydroxymethylfurfural (5-HMF), an important Maillard reaction product formed during food processing, has received increasing attention. However, its safety and efficacy are still debated, likely due to inconsistent findings from existing evaluation models. To address these controversies, we established a multifunctional microfluidic platform integrated with a data-driven weighting strategy. This platform consisted of a concentration-gradient generator, a gut-on-a-chip module, and aptamer-based online monitoring sensors. It enabled high-throughput, sensitive analysis under physiologically relevant conditions, facilitating the systematic assessment of 5-HMF-induced cellular responses within the intestinal microenvironment. Multiparametric cytotoxicity end points were further integrated into a unified toxicity index using a statistically weighted approach based on variable loadings, enabling objective toxicity evaluation with reduced subjective bias. This platform identified a safe concentration range (0–960 μg/mL) and revealed dual antioxidant functions: preventive effects through upregulation of superoxide dismutase (SOD) and glutathione (GSH), and therapeutic potential reflected by inhibition of tumor necrosis factor-α (TNF-α) under hydrogen peroxide (H 2 O 2 )-induced stress. Nontargeted metabolomics further revealed regulation of glutathione metabolism, the tricarboxylic acid (TCA) cycle, and purine pathways, supporting its role in oxidative stress modulation. Overall, this integrated, data-driven framework held considerable potential for the evaluation of 5-HMF, paving the way for its application to a broader range of molecules within physiologically simulated systems.