Lipase–Copper(II) Hybrid Nanoflowers: Bradford Method Validation and Morphological Evaluation Under Different Synthesis Conditions

Enzyme immobilization has been widely explored to improve the stability, reusability, and catalytic performance of biocatalysts. Among the most promising approaches are hybrid organic-inorganic nanoflowers (NFs), formed by the self-assembly of enzymes and metal phosphate crystals into hierarchical flower-like structures with high surface area and catalytic potential. In this study, NFs composed of porcine pancreatic lipase and copper(II) phosphate were synthesized to evaluate the effects of temperature and chloride ions on crystal growth. Chloride (10 mM) promoted more open petals, while chloride-free conditions yielded compact NFs and inorganic spherical particles without lamellar organization. High chloride concentrations (40 mM) disrupted hierarchical organization. Refrigerated synthesis produced more organized and compact structures. The Bradford method was validated for protein quantification, showing limited selectivity in chloride/phosphate-containing matrices. Standard addition calibration minimized matrix effects, providing satisfactory linearity, precision, accuracy, LoD (0.010 mg/mL), and LoQ (0.029 mg/mL).