(Nanowerk Spotlight) Metal-organic frameworks (MOFs) have emerged in recent years as an exciting new class of materials with tremendous potential in diverse fields such as gas storage, drug delivery, catalysis, and biosensing. Constructed from metal ions linked by organic molecules, MOFs contain porous networks ideal for housing guest molecules. Their highly tunable structures and biocompatibility make MOFs promising vehicles for targeted therapeutic delivery and release.
A major focus of researchers has been packing biologically active large molecules like proteins and enzymes inside MOFs. Packing these sensitive large molecules in MOFs protects them from falling apart and deterioration. This allows their use as therapies and makes them more stable for catalysis and biosensing. MOF packing also selects for protein varieties with larger molecular size and added sugar chains, boosting their effectiveness.
Despite enthusiasm about MOF-protein combinations, accurately measuring the packing efficiency has remained a key challenge. This measurement is critical for optimizing these mixed materials. But researchers have reported very different packing efficiencies for identical protein-MOF mixtures, suggesting it depends on the quantification method used. No thorough comparisons between techniques existed previously, a gap addressed in a recent Biotechnology Journal study (“Comparison of protein quantificationmethods for protein encapsulationwith ZIF-8 metal-organic frameworks”).
Previously attempted quantification methods each have disadvantages making them ill-suited for proteins encapsulated in MOFs. Colorimetric protein assays like Coomassie and bicinchoninic acid (BCA) suffer from interference by MOF components. The organic MOF linkers increase background readings while metal ions suppress color development, reducing sensitivity. Microplate fluorescence requires conjugating proteins with fluorescent tags, adding steps. Gel techniques like SDS-PAGE involve extensive handling, increasing variability.
Finding accurate encapsulation analysis methods is vital for realizing the promise of MOF platforms. Encapsulated enzymes must maintain activity after passing through biological barriers. And encapsulation efficiency directly determines the effective therapeutic dose.
“Identifying a rapid and efficient method of assessing biomolecule encapsulation is key to their wider acceptance,” the authors emphasize.
The researchers systematically evaluated several common quantification techniques using the well-studied MOF zeolitic imidazolate framework-8 (ZIF-8) and model protein bovine serum albumin (BSA). They synthesized ZIF-8 encapsulating BSA and the antioxidant enzyme catalase. The team assessed encapsulation efficiency via microplate BCA, Coomassie, and fluorescence assays, SDS-PAGE, size-exclusion chromatography, and mass spectrometry.
Results revealed striking variability across techniques, indicating encapsulation levels are highly method dependent. Fluorometric quantitation produced the most consistent encapsulation percentages with the least background. It also confirmed enrichment of high molecular weight and glycosylated proteins after MOF encapsulation. But the approach requires labeling proteins with fluorescent tags.
Of the colorimetric assays, BCA provided a wider detection range than Coomassie. However, BCA was less sensitive below 10 µg/mL due to linker interference. Coomassie showed a narrower linear range and higher background relative to fluorescence detection. Meanwhile, SDS-PAGE and size-exclusion chromatography suffered from extensive sample handling and processing artifacts.
The findings have broad relevance for assessing biomolecule encapsulation in imidazole-based MOFs. This work highlights the critical need to account for limitations of quantification techniques when designing protein-MOF platforms. Fluorometric detection provided a reasonably accurate encapsulation analysis. But fluorescent tagging may not be feasible for all proteins, especially in low quantities.
“Our results could assist the bio-MOF systems development with more accurate data, accelerating their utilization in catalysis and biomedical applications,” the researchers conclude. More efficient, reproducible encapsulation quantification methods applicable across diverse MOF-biomolecule combinations remain vital to drive the field forward.
This rigorous methodology comparison uncovered surprising variability in assessing MOF encapsulation efficiencies. The study emphasizes researchers must select quantification techniques judiciously when engineering protein-MOF composites to unlock their full potential. Accurately determining encapsulation levels will help transform promising MOF materials into transformative real-world technologies.
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