Gold Nanoparticles–Dynamic Constitutional Frameworks as Adaptive Hybrid Conjugates for Enzyme Immobilization

Dynamic constitutional frameworks (DCFs), connecting monomers via reversible covalent bonds, can initiate the assembly of gold nanoparticles (AuNps) with distinctive optoelectronic and surface chemical properties. A previous study indicated that these nanomaterials are particularly valuable for carbonic anhydrase immobilization and stabilization, with potential applications in biocatalysis and biosensing. However, further studies with different enzymes are needed to prove their universality. Therefore, this research focuses on immobilizing phosphotriesterase (PTE), an effective degrader of toxic organophosphates, on AuNp‐DCF conjugates. PTE is integrated with citrate‐ and PEG‐stabilized AuNps or imine‐based DCFs resulting in stable, homogeneous PTE‐AuNp‐DCF assemblies. The conjugates exhibit high bonding affinity and changes in the PTE's secondary structure, which did not deactivate the enzyme. The catalytic performance of immobilized PTE is evaluated by measuring the p‐nitrophenol (p‐NP) production in a similar way is sense the paraoxon during its enzymatic hydrolysis, used as a model organophosphate. PTE immobilized to PEG 2000 ‐AuNps assembled with DCF‐PEG1500 shows the highest reaction rate (13.7 × 10 −6 ± 0.82 M min −1 ), outperforming that immobilized to citrate‐stabilized AuNps (3.71 × 10 −6 ± 0.21 M min −1 ). Furthermore, these PTE‐PEG‐AuNp‐DCF conjugates at a 1/25 molar ratio display a residual reaction rate, 3.6 times higher than that of all other conjugates. Free amino groups exposed on the surface of AuNps facilitate optimal assembly with DCF‐PEG through aldehyde/amino exchange reactions, preserving PTE activity. These results highlight the potential of PTE‐DCF‐AuNp conjugates to intercept and transform small molecules like paraoxon.