Green Synthesis of Fe₃O₄Nanozyme with Peroxidase-like Activity for Colorimetric Glucose Detection: A Comprehensive Undergraduate Laboratory Experiment

Nanomaterial-based enzyme mimics, known as nanozymes, are extensively applied in chemical sensing and biomedical fields due to their low cost, high stability, and tunable catalytic activity. However, conventional synthesis methods employing hazardous chemicals limit their promotion in undergraduate laboratory training. Herein, an eco-friendly and simple green approach is proposed that utilizes plant extracts as natural reducing agents for nanozyme synthesis. Iron oxide nanozymes (Fe₃O₄ NZs) were synthesized using lemon peel extract, and their structures and properties were analyzed via SEM, XRD, XPS, and FT-IR. The peroxidase-like activity of Fe₃O₄ NZs was evaluated using a chromogenic substrate in the presence of H₂O₂. Additionally, their effectiveness in developing nanozyme-glucose oxidase (GOx) cascade colorimetric sensors for glucose detection was also demonstrated. These experiments have been successfully conducted at various undergraduate levels and have received positive feedback. Experimental assessments indicated that students’ understanding of green chemistry, catalytic mechanisms, and interdisciplinary applications of nanozymes had improved. Moreover, the flexible design of the experiment allows instructors to modify it based on students’ backgrounds, available equipment, and teaching objectives, making it applicable to multiple fields such as chemistry, materials science, and biology. This experiment provides a generalizable case for integrating cutting-edge technology into undergraduate education.