## 5 Innovative Approaches for Enzyme Immobilization
### Challenges and Limitations of Nanomaterial-Based Immobilization  
Nanomaterial-based enzyme immobilization enhances traditional enzymatic catalysis by mitigating mass transfer resistance, enzyme instability, and inhibition [18; 123]. This nano-biocatalyst approach integrates enzymes with functionalized nanocarriers, improving substrate specificity and catalytic efficiency. These nanomaterials offer large surface areas reusability, tunable surfaces, and options for magnetic separation [154]. However, broader adoption is limited by inconsistent material properties, high synthesis costs, and environmental concerns, i.e., toxicity and disposal, hinder wider use [155].  
A major challenge lies in the development of cost-effective nanomaterial synthesis. Large-scale production demands precise control over material properties, especially in regards to 2D nanomaterials, where low-cost methods often introduce defects [156]. For carbon nanotubes, achieving uniform chirality, conductivity, and diameter remains technically difficult [157], and only structurally pure forms deliver optimal performance, requiring advanced fabrication techniques [158].  
Additionally, nanoparticle agglomeration, driven by electrostatic forces and high surface energy, affects functionality. [PERSON] et al. [159] emphasized that managing catalysts, solvents, and impurities is critical in nanoparticle production, particularly for pharmaceuticals. Inconsistent batches highlight the need for closed or automated systems to improve safety and cut costs.  
Nanomaterials also pose significant toxicity concerns due to their small size, which facilitates penetration into human tissues and ecological systems [160]. Their toxic potential is influenced by their chemical composition, size, structure, and biological interactions. Accumulation in sensitive organs, like the lungs or brain, can lead substantial harm [161], including the generation of reactive oxygen species (ROS), oxidative stress, and cell damage [162]. For instance, silica nanoparticles may induce lipid peroxidation, membrane disruption, DNA damage, and even cell death [163].  
Although polymeric nanoparticles are widely studied for medical applications, their neurotoxicity remains poorly understood. For example, Polysorbate 80 coated with chitosan nanoparticles accumulate in rat brains, causing neuronal death, inflammation, oxidative stress, and weight loss [164]. Likewise, gold nanoparticles contaminated withlipopolysaccharide disrupt human dendritic cell interactions [165]. Silver nanoparticles (AgNPs), widely utilized in pharmaceuticals and cosmetics, also warrant rigorous neurotoxicity assessment due to variable effects based on their size, morphology, surface chemistry, and cellular interactions [166]. Even bacterial responses to AgNP exhibit notable variability [167].  
Nanomaterials pose potential risks to both human health and the environment. For instance, powdered carbon nanotubes (CNTs) are known to be hazardous [168], and toxic solvents used in metal-based catalysts can cause additional harm. Therefore, strict toxicity assessments and environmental monitoring throughout nanomaterial production are crucial [4].  
Green synthesis, using bacteria, fungi, algae, and plants, offers a more eco-friendly and cost-effective alternative [169]. However, even green nanomaterials may mimic biomolecules and pose risks at high concentrations, with their toxicity still poorly understood. For example, AgNPs may damage tissue, DNA, and blood vessels [170].  
Toxicity testing, both in vitro and in vivo, is crucial. Studies show that green-synthesized AgNPs and CuO NPs are often less toxic than their chemically synthesized counterparts, likely due to differences in size and composition [171; 172]. To ensure safe use, regulators, industry experts, and researchers must collaborate to balance nanomaterial benefits against risks to safeguarding both public health and the environment.