Co-immobilization of dual-enzyme within hierarchical porous ZIF-8 for sensitive glucose colorimetric sensing

Metal-organic frameworks (MOFs) with high surface area and excellent stability are regarded as candidates for enzyme immobilization. However, their application is often limited by enzyme leakage or denaturation. In this work, a hierarchical porous ZIF-8 (denoted as PS-HP-ZIF-8) was prepared as an efficient immobilization platform for horseradish peroxidase (HRP) and glucose oxidase (GOx). PS-HP-ZIF-8 possesses a hierarchical porous structure, which can facilitate enzyme immobilization and prevent enzyme leakage. The resulting Dual-En@PS-HP-ZIF-8 composite exhibited high catalytic activity, exceptional pH and thermal stabilities, organic solvent tolerance, and improved affinity toward substrates. Notably, Dual-En@PS-HP-ZIF-8 maintained about 90% of initial activity at 60 °C and exhibited 14.1% activity enhancement in 10% (v/v) methanol, while also retaining over 65% activity in pH 4.0-7.0, outperforming the performance of free enzymes. Moreover, a visual colorimetric glucose sensor was established using the Dual-En@PS-HP-ZIF-8 composite. The sensor showed high sensitivity, favorable linear glucose detection range and excellent anti-interference ability, with no obvious response to common interferents including CaCl₂, NaCl, bovine serum albumin (BSA), dextran and four common coexisting sugars, enabling specific recognition of glucose. It is also shown to be applicable in glucose test strips with commercial white sponges, confirming feasibility for on-site practical biosensing.