Iron and Nitrogen Co-doped Graphene Quantum Dots-Based Nanozyme for the Detection of NADPH

Abstract Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is a redox cofactor that plays an important role in maintaining cellular redox homeostasis, driving biosynthetic pathways, and regulating antioxidant defense mechanisms. Accurate detection of NADPH is essential for investigating metabolic regulation and oxidative stress-related diseases. In this study, we leveraged the potential of nanomaterials to develop a nanozyme that would detect and quantify NADPH. Nanozymes are synthetic nanomaterials with enzyme-like catalytic activity and have attracted significant attention due to their structural tunability, stability, suitable catalytic activity, and low production cost. Among these nanozymes, graphene quantum dots (GQDs)-based nanozymes are particularly promising for biosensing applications because of their distinctive optical and electronic properties. We designed and developed iron and nitrogen co-doped graphene quantum dots (FeN4-GQDs) with catalytic activity specific toward NADPH. The FeN4-GQDs were synthesized through a hydrothermal approach using polyethylenimine (PEI) and hemin as precursors. Comprehensive structural characterization confirmed the successful incorporation of iron and nitrogen. The synthesized nanozyme exhibited strong fluorescence signals with a quantum yield of 61%, surpassing the quinine sulfate control. The nanozyme displayed uniform morphology with an average hydrodynamic diameter of 7.1 ± 1.1 nm. Notably, this work introduces an enzyme-free platform that combines strong intrinsic fluorescence with highly sensitive and selective NADPH detection. The nanozyme demonstrated a concentration-dependent and specific fluorescence quenching response toward NADPH, attributed to efficient electron transfer between NADPH and the doped graphene domains, with a limit of detection of 17.1 nM. Biocompatibility studies in mouse brain-derived microvascular endothelial cells (BMVECs) confirmed the suitability of nanozymes for use in both normal and metabolically distressed conditions, demonstrating their potential for applications in metabolic disease research.