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    Enhanced protein immobilization efficacy by nanostructuring of ultrananocrystalline diamond surface

    Year: 2023

    Journal: Diamond and Related Materials, Volume 136, 2023-06-01, page 109898

    Authors: Merker, Daniel; Bertinetti, Daniela; Merz, Rolf; Kopnarski, Michael; Herberg, Friedrich W.; Popov, Cyril

    Keywords: Functionalization; GFP; Nanostructuring; Protein immobilization; Ultrananocrystalline diamond films

    Ultrananocrystalline diamond (UNCD) films show profound biocompatibility to both cells and biomacromolecules, which can be further tailored by surface treatment with different methods. In this study the impact of nanostructuring of the UNCD surface on protein immobilization was investigated. The covalent immobilization of green fluorescent protein (GFP) was realized by photochemical grafting of ω-alkene (TFAAD) as linker on hydrogen terminated UNCD and subsequent reaction with glutaraldehyde on unstructured and structured surfaces. A structuring process involving oxygen plasma etching was developed to create nanosized structures that are robust and reliantly reproducible. After optimization of the conditions for preparation of the hard mask for structuring based on gold nanoparticles, irregularly shaped UNCD islands with deep trenches between them (“diamond wrinkles”) were prepared. The nanostructured UNCD was subjected to hydrogen plasma treatment to restore the H-termination of the as-grown surface necessary for the photochemical reaction on which the covalent GFP immobilization is based. The evaluation of the average fluorescence intensity of GFP coupled to the UNCD using a microplate reader demonstrated that both, TFAAD functionalization and nanostructuring, had a beneficial impact on their own with 3.5 and 2-fold increased fluorescence compared to the unstructured UNCD, respectively. Combining TFAAD functionalization and nanostructuring resulted in 5.5-fold higher fluorescence in comparison to the unstructured and non-functionalized UNCD surface. Finally, structured and TFAAD functionalized UNCD samples were incubated in solutions with GFP concentrations in the range of 10 μM–1 nM. Average fluorescence intensity increased with the concentration of GFP in a non-linear relation, which can be attributed to saturation in the higher concentration range.
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