Renewable biofuel production from plant biomass is impeded by the inherent toughness of cell walls, which limits the efficacy of enzymatic hydrolysis, and by conventional imaging techniques that fail to capture dynamic changes during pretreatment. This study introduces a novel approach employing quantum-enabled red fluorescent nanodiamonds (rFNDs) to address these limitations. The rFNDs are linked with cellulases, forming rFND-Cellulase complexes that specifically target exposed regions of cellulose. Among various nanodiamond sizes tested, 300 nm particles demonstrated an optimal balance between resolution and signal intensity, yielding superior imaging results on the QND microscope. The exceptional photostability of the nanodiamonds, combined with their unique capability for fluorescence modulation under applied microwave fields, permits background-free, real-time visualization even in the presence of lignin interference. Additionally, dynamic light scattering (DLS) is utilized to monitor the size distribution and aggregation of the complexes, ensuring stability, while high-performance liquid chromatography (HPLC) quantifies glucose production during enzymatic hydrolysis. The cellulose region before and after pretreatment will be imaged after being bound by rFND-Cellulase to demonstrate the pretreatment mechanism. This integrated approach provides comprehensive insights into enzyme–substrate interactions and guides the optimization of pretreatment conditions to enhance the efficiency of biofuel production.
