(426h) Direct Observation of Heterogeneous Distribution of Enzymes in Metal-Organic Frameworks and Its Effects in Propelling Nanomotors

Natural biomineralization processes have inspired the in situ co-precipitation synthesis of protein-metal-organic frameworks (pMOFs) with potent competitiveness in the field of catalysis, imaging, sensing, and drug delivery. Understanding of crystallization process and biomolecule behavior of these hybrid composites helps guide the design of multifunctional pMOFs with enhanced activity and stability. While efforts have been made to investigate the nucleation and growth mechanisms of pMOFs, the specific protein distribution pattern and its role in biocatalytic functions have not been fully elaborated.

Here, we reconstructed the three-dimensional protein distribution inside pMOF composites on the molecular level by super-resolution optical microscopy and clustering analysis. Via detecting, correcting, and Gaussian fitting of signals from individually excited fluorophores, the specific localization of each detection can be acquired to overcome the resolution limit of traditional fluorescence microscopy. With clustering analysis methods, protein clusters were identified inside the porous solid particles, revealing the heterogeneity underlying the apparent symmetry. Detections centralized mostly at the center of the particles, indicating the important role protein played during the nucleation of pMOF composites. Moreover, for individual particles, the co-precipitation process promoted the scattered localization of protein clusters which generated a structural and functional asymmetry.

These results inspired us to investigate the potential of pMOFs themselves as biocatalytic nanomotors that required asymmetry for active propulsion at low-Reynold's number, a ubiquitous environment for nanoparticles in many biocatalytic and biomedical applications. Catalase@ZIF-8 (CAT@ZIF-8) was synthesized as a model and exhibited both enhanced diffusion in uniform substrate solution and positive chemotaxis in microfluidic chips with a gradient concentration. We then proposed a simple model to verify numerically that clustered protein distribution generated stronger self-diffusiophoretic propulsion than complete spatial random distributions. Different proteins exhibited similar clustered behavior, and enzyme cascades can be easily immobilized as well to increase the energy utilization efficiency of fuels and amplify the propulsion power.

In summary, the combination of super-resolution optical microscopy and clustering analysis provided a potent method to reconstruct specific protein distribution in pMOF composites. Collectively, protein clusters were found to be stochastically centralized mainly at the center of particles, which originated from protein-dependent nucleation and crystallization process. Individually, co-precipitation promoted the scattered localization of protein clusters which generated a structural and functional asymmetry leading to the active propulsion of biocatalytic pMOFs. Taken together, these results offered new insights into comprehending the co-precipitation process from a fresh perspective.