Non-covalent and covalent binding of proteins on ultrananocrystalline diamond films with different surface terminations

The immobilization of proteins on solid surfaces is a critical aspect of biosensor development, offering enhanced sensitivity and specificity for molecular recognition. Ultrananocrystalline diamond (UNCD) films have emerged as a promising platform for protein immobilization due to their exceptional biocompatibility, chemical stability, and tunable surface properties. In this study, we investigate the non-covalent and covalent binding of green fluorescent protein (GFP) on nanostructured UNCD surfaces employing fluorescence spectroscopy to assess the efficiency of protein immobilization. The non-covalent binding was affected by the surface terminations (hydrogen, oxygen, and fluorine) rendered by different plasma modifications. The oxygen-terminated UNCD surfaces exhibited the highest efficiency attributed to favorable wettability and electrostatic interactions, revealed by zeta potential and contact angle measurements. The covalent immobilization via linker chemistry was studied for both GFP and anti-GFP nanobodies with the variation of the GFP concentration within the range of 10 μM – 10 pM. Milk powder was applied as a blocker to minimize the non-covalent binding of the target proteins. The intensity of the fluorescence signal decreased with the GFP concentration and below 1–10 nM approached that of the buffer control samples. The individual steps of the protein immobilization were investigated by zeta potential measurements, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Finally, a fluorescent dye-labeled nanobody was constructed as a reporter complex and applied to provide fluorescence signals distinguishable from those of GFP due to the different wavelengths. Such complexes combine the high specificity of the binding proteins with the bright and stable signal of the fluorescent dyes and can be implemented for realization of biosensors.