Comparison of protein quantification methods for protein encapsulation with ZIF-8 metal-organic frameworks

The use of metal-organic frameworks (MOFs) as delivery systems for biologically functional macromolecules has been explored widely in recent years due to their ability to protect their payload from a wide range of harsh conditions. Given the wide usage and diversity of potential applications, optimising the encapsulation efficiency by MOFs for different biological is of particular importance. Here, several protein quantitation methods and report were compared on the accuracy, practicality, limitations, and sensitivity of these methods to assess the encapsulation efficiency of zeolitic imidazolate frameworks (ZIF)-8 MOFs for two common biologicals commonly used in nanomedicine, bovine serum albumin (BSA), and the enzyme catalase (CAT). Using these methods, ZIF-8 encapsulation of BSA and CAT was confirmed to enrich for high molecular weight and glycosylated protein forms. However, contrary to most reports, a high degree of variance was observed across all methods assessed, with fluorometric quantitation providing the most consistent results with the lowest background and greatest dynamic range. While bicinchoninic acid (BCA) assay has showed greater detection range than the Bradford (Coomassie) assay, BCA and Bradford assays were found to be susceptible to background from the organic "MOF" linker 2-methylimidazole, reducing their overall sensitivity. Finally, while very sensitive and useful for assessing protein quality SDS-PAGE is also susceptible to confounding artifacts and background. Given the increasing use of enzyme delivery using MOFs, and the diversity of potential uses in biomedicine, identifying a rapid and efficient method of assessing biomolecule encapsulation is key to their wider acceptance.