Role of Molecular Modification and Protein Folding in the Nucleation and Growth of Protein-Metal-Organic Frameworks

Metal-organic frameworks (MOFs) are a class of porous nanomaterials that have been extensively studied as enzyme immobilization substrates. During in situ immobilization, MOF nucleation is driven by biomolecules with low isoelectric points. Investigation of how biomolecules control MOF self-assembly mechanisms on the molecular level is key to designing nanomaterials with desired physical and chemical properties. Here, we demonstrate how molecular modifications of bovine serum albumin (BSA) with fluorescein isothiocyanate (FITC) can affect MOF crystal size, morphology, and encapsulation efficiency. Final crystal properties are characterized using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), fluorescent microscopy, and fluorescence spectroscopy. To probe MOF self-assembly, in situ experiments were performed using cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction (XRD). Biophysical characterization of BSA and FITC-BSA was performed using ζ potential, mass spectrometry, circular dichroism studies, fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The combined data reveal that protein folding and stability within amorphous precursors are contributing factors in the rate, extent, and mechanism of crystallization. Thus, our results suggest molecular modifications as promising methods for fine-tuning protein@MOFs' nucleation and growth.