Biomacromolecules, such as proteins, are highly dynamic 3D polymers stabilized by intramolecular weak interactions (hydrogen bond and hydrophobic interaction etc.). It leads to the thermodynamic instability of proteins when being used in the extracellular environment. In the past decade, scientists pursue to stabilize the proteins by means of the immobilization within a porous carrier. In 2015, a pioneering work by Liu and coworkers found that the bulky protein (cytochrome C, Cyt c) was possible to be encapsulated into the microporous zeolitic imidazolate framework-8 (ZIF-8; also known as MAF-4) in situ by coprecipitation process.¹ The ZIF-8-encapsulated Cyt c displayed 10-fold increased peroxidase activity compared with the free counterpart, and the stability of encased Cyt c was significantly improved owing to the protection by the ZIF-8 shell. A closer examination by Liang and Falcaro et al found that some biomolecules, including proteins, enzymes and DNA, were feasible to trigger the nucleation of a series of MOFs (including ZIF-8, HKUST-1, Eu-BDC, Tb-BDC and MIL-88A) around their surfaces, as like the natural biomineralization process.² These initial attempts of the co-crystallization of BMOFs aroused widespread interest in the chemical, material and biological communities, since the formed symbiotic crystal well inherits the inherent MOF crystallographic structure and possesses structurally-matched 3D microenvironment for biomacromolecule confinement.³

In the previous works, our group have developed several bottom-up strategies for encapsulating proteins within the porous frameworks, including ZIF-8 and the burgeoning hydrogen-bonded organic frameworks (HOFs) materials, and also optimized the bioactivity of these biohybrids for biosensing applications.^4-9 Up to now, ZIF-8 is the popular MOFs matrix for the de novo synthesis of BMOFs crystals (Figure 1), because the mild crystallization process of ZIF-8 (room temperature and aqueous phase etc.) can avoid the denaturation of the fragile proteins. The merit of this new biotechnology is noticeable, but a puzzling question comes to us, that is, the structure-activity relationship. Under different synthesis conditions, we usually obtain the biocrystal with identical crystallographic structure, as evidenced by the PXRD and morphology analysis. However, the bioactivities of these biocrystals are of great differences in most cases, and we note that such contradiction on bioactivity is also observed in the reported literatures. For example, owing to the narrow ZIF-8 aperture (ca. 3.4 Å) that limits the mass transfer, the encased proteins were observed to be partially or even completely restrained.^10,11 Inversely, in some cases, the internalized proteins tended to maintain comparable or even superior bioactivity to the free counterpart.^12,13

Seeing is believing. Directly imaging the high-resolution structure of BMOFs crystal is an unambiguous method that allows the deep understanding of its structure-activity relationship, yet, still remains unknown. In fact, our primitive attempts based on traditional high-resolution TEM have failed to target this. The technical difficulties mainly originate from the electron beams-sensitive nature of MOFs. It means that MOFs material can be instantaneously amorphous under high-energy electron beams in TEM imaging. With the assistance by Dr. Xiaomin Ma from Cryo-EM Center of Southern University of Science and Technology and Dr. Suya Liu from Shanghai Nanoport of Thermo Fisher Scientific, we learn about a recently developed Low-electron-dose TEM technique, termed integrated differential phase contrast-scanning transmission electron microscope (iDPC-STEM). The iDPC-STEM has been reported to show a great potential for the low-dose imaging of beam-sensitive MOFs, such as MIL-101 and UiO-66.¹⁴ In short, here, we pioneered to use the emergent iDPC-STEM combining with cryo-electron microscopy and X-ray absorption fine structure techniques to unveil the microstructure of biomacromolecules-ZIF-8 (BZIF-8) composites crystallized in different pathways. The atomic structure of BZIF-8 composites are directly identified for the first time (Figure 2). These atomic-level information provide new insights into the significant activity difference of MOFs biohybrids synthesized in different scenarios, and may give a reasonable explanation on the confused bioactivity observed previously.

For more information, you can read more about our work in Nature Communications following the link: https://www.nature.com/articles/s41467-022-28615-y

Reference

1. Lyu, F., Zhang, Y., Zare, R. N. Ge, J. & Liu, Z. One-Pot Synthesis of Protein-Embedded Metal−Organic Frameworks with Enhanced Biological Activities. Nano Lett. 14, 5761−5765 (2014).
2. Liang, K. et al. Biomimetic mineralization of metal-organic frameworks as protective coatings for biomacromolecules. Nat. Commun. 6, 7240 (2015).
3. Liang, W. et al. Metal−Organic Framework-Based Enzyme Biocomposites. Chem. Rev. 121, 1077-1129 (2021).
4. G. et al. A Convenient and Versatile Amino-Acid-Boosted Biomimetic Strategy for the Nondestructive Encapsulation of Biomacromolecules within Metal–Organic Frameworks. Angew. Chem. Int. Ed. 58, 1463–1467 (2019).
5. Huang, S., Kou, X., Shen, J., Chen, G. & Ouyang G. “Armor-Plating” Enzymes with Metal–Organic Frameworks (MOFs). Angew. Chem. Int. Ed. 59, 8786–8798 (2020)
6. Chen, G., Huang, S., Kou, X., Zhu, F. & Ouyang, G. Embedding functional biomacromolecules within peptide-directed metal–organic framework (MOF) nanoarchitectures enables activity enhancement. Angew. Chem. Int. Ed. 59, 13947–13954 (2020).
7. G. et al. Modulating the Biofunctionality of Metal–Organic-Framework-Encapsulated Enzymes through Controllable Embedding Patterns. Angew. Chem. Int. Ed. 59, 2867–2874 (2020)
8. Chen, G. et al. Protein-directed, hydrogen-bonded biohybrid framework. Chem 7, 2722-2742 (2021).
9. Tang, Z. et al. A Biocatalytic Cascade in an Ultrastable Mesoporous Hydrogen-Bonded Organic Framework for Point-of-Care Biosensing. Angew. Chem. Int. Ed. 60, 23608–23613 (2021).
10. Wu, X. et al. Packaging and delivering enzymes by amorphous metal-organic frameworks. Nat. Commun. 10, 5165 (2019).
11. Chen S.-Y. et al. Probing Interactions between Metal-Organic Frameworks and Freestanding Enzymes in a Hollow Structure. Nano Lett. 20, 6630-6635 (2020).
12. Chen, W.-H., Vázquez-González, Margarita., Zoabi, A., Abu-Reziq, R., & Willner, I. Biocatalytic cascades driven by enzymes encapsulated in metal–organic framework nanoparticles. Nat. Catal. 1, 689–695 (2018).
13. Hu, C. et al. Defect-induced activity enhancement of enzyme-encapsulated metal-organic frameworks revealed in microfluidic gradient mixing synthesis. Sci. Adv. 6, eaax5785 (2020).
14. Shen, B., Chen, X., Shen, K., Xiong, H. & Wei, F. Imaging the node-linker coordination in the bulk and local structures of metal-organic frameworks. Nat. Commun. 11, 2692 (2020).