Immobilization of horseradish peroxidase on UiO-66-NH2 for colorimetric and fluorometric sensing of nitrite†

Abstract

Immobilized enzymes play a crucial role in analytical sensing due to their exceptional stability and considerable commercial importance. In this study, a stable Zr-based metal–organic framework (UiO-66-NH₂) was prepared as an immobilization platform for horseradish peroxidase (HRP) through covalent binding. HRP@UiO-66-NH₂ retained 75% of its activity after 10 cycles. Subsequently, a colorimetric/fluorometric dual-mode sensing strategy using HRP@UiO-66-NH₂ was developed for nitrite detection. HRP@UiO-66-NH₂ facilitated the transformation of the non-colored compound 3,3′,5,5′-tetramethylbenzidine (TMB) into its blue oxidized form. When nitrite is added, oxidized TMB (ox-TMB) specifically engaged with nitrite (NO₂⁻) to generate diazotized TMB, leading to a color shift from blue to yellow. Concurrently, NO₂⁻ reacted with the amino groups of HRP@UiO-66-NH₂, forming diazonium compounds and suppressing the fluorescence of HRP@UiO-66-NH₂. The limits of detection were 0.21 μM and 0.19 μM for the colorimetric and fluorometric strategies, respectively. Furthermore, a portable kit for detecting nitrite was created by integrating gelatin with HRP@UiO-66-NH₂. The kit could visually identify nitrite from 10–400 μM in colorimetric mode and 0–400 μM in fluorometric mode. This method provides an innovative approach for nitrite sensing, paving the way for new research into multifunctional immobilized enzymes and their potential uses in biochemical sensing applications.