مهندسي آنزيم پراكسيداز ترب كوهي براي توليد سامانه زيست‌حسگر هيبريدي آنزيم–نانوزايم به‌منظور شناسايي مواد پلي و پرفلوروالكيلي(PFAS)

Enzymes, as biocatalysts, play crucial roles in many industrial, environmental, an‎d biochemical processes; however, limitations such as low stability an‎d sensitivity to environmental conditions restrict their optimal use. Enzyme engineering is an effective strategy to improve their stability, activity, an‎d applicability. One of the key approaches in enzyme engineering is immobilization onto various carriers, which enhances thermal an‎d chemical stability, enables recovery an‎d reuse, an‎d improves catalytic performance. In this thesis, horseradish peroxidase (HRP) was engineered through immobilization onto biomimetic carriers. These carriers included Fe3O4 an‎d MFe2O4 (M=Ni, Zn, Cd) nanozymes with peroxidase-like activity. Nanozymes, as biomimetic materials, mimic the behavior of enzymes an‎d, compared to natural enzymes, possess higher stability, lower cost, an‎d the ability to function under broader environmental conditions. The goal was to develop a hybrid enzyme–nanozyme system in which HRP could maintain its peroxidase activity while the nanozyme, in addition to acting as a carrier, would also contribute as a catalytic component, thereby creating a synergistic effect between the two. The results demonstrated that, after immobilizing HRP onto different nanozymes an‎d comparing the peroxidase activity of the hybrid systems with that of each individual component, the Fe3O4 nanozyme coated with sodium citrate (Fe3O4-Cit@HRP) exhibited the strongest synergistic effect. Kinetic analysis showed that the Km value for the TMB substrate in the hybrid system was about 15 times lower than that of free HRP, indicating a higher substrate affinity. Moreover, theν max for H2O2 was more than fivefold higher than that of the free enzyme, reflecting a significant enhancement in catalytic reaction rate in the presence of the nanozyme. The optimal temperature an‎d pH conditions for the hybrid system were also determined. Subsequently, the hybrid system was applied as a turn-off biosensor for detecting per- an‎d polyfluoroalkyl substances (PFAS). PFAS are a family of persistent an‎d harmful pollutants, whose chemical resistance an‎d hazardous impacts on human health an‎d the environment make their detection an‎d removal a major challenge. In this system, in the absence of PFAS, both components catalyzed the oxidation of TMB by H₂O₂, producing ox-TMB, whose blue color served as an indicator of peroxidase activity. The presence of PFAS inhibited the oxidation of TMB, an‎d the decrease in blue color intensity, correlated with pollutant concentration, was used for PFAS detection. The limit of detection (LOD) of the system was calculated as 200 nM. Finally, its performance was eva‎luated in real samples, including water, milk, an‎d human serum, with recovery rates of 83.5–106.9%. Overall, the findings indicate that the Fe3O4-Cit@HRP hybrid system is an effective strategy for designing sensitive an‎d stable biosensors. By offering improved performance an‎d a synergistic effect between the enzyme an‎d the nanozyme, this hybrid system enables accurate PFAS detection an‎d holds promise as a practical an‎d reliable tool for environmental pollutant monitoring.

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