Practical and Rapid Membrane-Based Biosensor for Phenol Using Copper/Calcium-Enzyme Hybrid Nanoflowers

Phenol, a pollutant frequently found in chemical industries effluents, is highly toxic even in low concentrations. This study reports a green, simple, and rapid method for qualitative phenol biosensing using horseradish peroxidase (HRP) hybrid nanoflowers made with copper (Cu²⁺-hNF) or calcium (Ca²⁺-hNF) ions. The enzyme was immobilized through protein-inorganic self-assembly into hybrid structures and subsequently supported onto a polyvinylidene fluoride (PVDF) membrane. SEM, EDS, FTIR, and XRD techniques sustained the effective enzyme encapsulation into hybrid structures. The protein concentration in the structures was 0.25 mg.mL^-1 for both ions. The best temperature and pH were 60 °C and 7.4, respectively, for both hybrids and the free enzyme, suggesting that the immobilization did not affect the optimal conditions of the free HRP. Thermal stability from 25 to 70 °C and pH stability from 4.0 to 9.0 of the hybrids were also determined. Finally, using copper and calcium hybrids, both biosensors produced onto a PVDF membrane could detect phenol in concentrations ranging from 0.72 to 24.00 µmol.mL^-1 in 1 min. In contrast, control biosensors produced with free enzyme have not presented a visible color change in the same conditions. The findings suggest a promising application of the developed biosensors in functional phenol detection.