Nanomaterial Based Tools for Molecular Investigation of Membrane Associated Proteins and Diverse Screening Applications
Nanomaterials exhibiting specific affinity towards target molecules constitute the foundation of several emerging technologies including nanomedicine and (bio)sensing. In this work, metal, metal oxide, semiconductor, and carbon-based nanomaterials (NMs) were functionalized with targeting ligands exhibiting high affinity towards a variety of targets including membrane-associated proteins, enzymes, antibodies, and gas molecules. The functionalization of NMs with targeting ligands was accomplished by employing a diverse range of reactive molecular platforms including bi-functional linkers, branched polymers, and polymer brushes grafted from the surfaces via surface-initiated atom transfer radical polymerization (SI-ATRP) technique.
In the first part of the work, a range of novel probes were designed and developed for potential applications in molecular imaging and protein purification. In this context, the chemical strategies historically emerged during the development of NMs for targeted drug delivery applications were exploited for the fabrication of NMs that could be employed as contrast agent in cryo-electron microscopic (cryo-EM) investigations of selected proteins. For this purpose, gold nanoparticles (AuNPs) were functionalized with bi-functional linkers bearing chemical groups that bind to the nanoparticle at one end and conjugate to the targeting-ligands at the other end. The targeting ligands employed in this work include glycine receptors (GlyR) antagonists (strychnine), glutathione, and biotin, respectively exhibiting high affinity towards GlyR, glutathione-S-transferases (GSTs), and streptavidin. Cryo-EM is still a young field, and its application landscape is limited by a number of constraints. Some of these constrains stem from the challenging sample preparation protocols. The processes used to vitrify the sample before structural determination via cryo-EM are difficult to control and they appear to cause biomolecules to come in contact with the air-water interface. In this regard, chemically functionalized graphene oxide coated TEM grids were designed that are expected to help in uniformly distributing the protein molecules onto the cryo-EM grid while holding it away from the air-water interface. The imaging of proteins using cryo-EM requires protein purification, which has posed a challenge to the state-of-the-art purification technologies in recent years. In order to simplify protein purification process, monolayer bifunctional PEG and polymer brush grafted iron oxide nanoparticles were fabricated. Ligands, which specifically bind to GlyR and GSTs, were conjugated to the monolayer and polymer brush functionalized iron oxide magnetic nanoparticles to develop ligand-specific magnetic nanobeads. The developed materials are expected to provide a simple route for purifying membrane-associated proteins and enzymes.1 In addition to surface modification of NMs with specific chemical functionalities, knowing the precise location and number of surface anchored chemical functional groups is of critical importance. Organic functional groups cannot be generally visualized with adequate contrast using electron microscopic techniques. In this context, electron dense AuNPs functionalized with the chemical functionalities complimentary to the functionalities introduced on the surface of the functionalized NMs can be used as tags revealing the precise location and density of the functional groups introduced on the surface. The inert chemical nature and higher phase contrast make AuNPs suitable for this application. Consequently, we have developed alkyne functionalized AuNPs for mapping reactive azide groups on the surface of individual 2D peptoid nanosheets (PNS) via surface confined click chemistry.
The toolbox of materials and chemical strategies developed in this work were extended to augment the multidisciplinary and integrative research efforts focused on the development of effective sensing platforms. Development of point-of-care (POC) diagnostics and onsite detection platforms hold great potential to improve healthcare and environmental monitoring in both developing and developed nations. As an applied impact of this work, natural and synthetic fibers were functionalized with different combinations of polyphenol coating and silica nanoparticles to develop surfaces for enhancing the activity of the immobilized antibody. The results presented in this context highlight the strategies for increasing the activities of immobilized antibodies (i.e., increased activity retention) without resorting to the relatively costly approaches currently being practiced.2 In addition to biosensing applications, an H2S gas sensing platform consisting of silver NPs confined on the surface of flexible polypropylene film was developed and its effectiveness to sense H2S at different concentrations was demonstrated.3
In summary, the unprecedented materials and chemical strategies developed in this work are expected to lay foundation for the development of a vast variety of novel bioimaging, biosensing, and chemical sensing technologies.
1. Azeem, I.; Çitoğlu, S.; Duran, H.; Yameen, B., Chapter 2 Design of Functionalized Magnetic Nanoparticles for Improving Stabilization, Biocompatibility and Uptake Efficiency. In Analytical Applications of Functionalized Magnetic Nanoparticles, The Royal Society of Chemistry: 2021; pp 20-53.
2. Azeem, I.; Sousa, A. M.; Yameen, B.; Lau, K. H. A., Binding enhancements of antibody functionalized natural and synthetic fibers. RSC Adv. 2021, 11 (48), 30353-30360.
3. Ashfaq, B.; Azeem, I.; Sohail, M.; Yüce, F. G. z.; Citoglu, S.; Nayab, S.; Abdullah, M.; Duran, H.; Yameen, B., Ultraviolet-Printing-Assisted Surface-Confined Growth of Silver Nanoparticles on Flexible Polymer Films for Cu2+ and H2S Sensing. ACS Applied Nano Materials 2021,4 (8), 8200-8216. (Equal contribution co-first author).
4. Nayab, S.; Trouillet, V.; Gliemann, H.; Weidler, P. G.; Azeem, I.; Tariq, S. R.; Goldmann, A. S.; Barner-Kowollik, C.; Yameen, B., Reversible Diels–Alder and Michael Addition Reactions Enable the Facile Postsynthetic Modification of Metal–Organic Frameworks. Inorg. Chem. 2021, 60 (7), 4397-4409
5. Abid, M.; Naveed, M.; Azeem, I.; Faisal, A.; Nazar, M. F.; Yameen, B., Colon specific enzyme responsive oligoester crosslinked dextran nanoparticles for controlled release of 5-fluorouracil. Int. J. Pharm. 2020, 586, 119605.
6. Sohail, M.; Ashfaq, B.; Azeem, I.; Faisal, A.; Doğan, S. Y.; Wang, J.; Duran, H.; Yameen, B., A facile and versatile route to functional poly (propylene) surfaces via UV-curable coatings. React. Funct. Polym. 2019, 144, 104366.
7. Azeem, I.; Ashfaq, B.; Sohail, M.; Yameen, B., Polymer Surface Engineering in the Food Packaging Industry. (Book Chapter under review).