# Immobilization of Detonation Nanodiamonds on Macroscopic Surfaces
## 3 Results
Figure 3 illustrates the attachment strength of the NDs to the surface, using the direct determination of the lateral force to move the indentation tip and ND agglomerates, respectively. The measurements in Figure 3 describe the movement of the nanoindentation tip on the surface, and show the lateral force as a function of lateral displacement. Figure 3a demonstrates the measurements for the bare Si as a reference for the subsequent coatings, whereby Figure S5 displays the SEM images of the corresponding ND-coated Si samples. The lateral force increases following the direction of the tip. For both ND-coated samples, sharp peaks (black arrows Figure 3b,c) in lateral force were detected, with greater variations for the covalently immobilized ND surface than for the dip-coated surface. Intense force jumps were observed during the lateral displacement of the tip, moving along the sample of amide-bonded NDs, where bigger ND agglomerates exert higher forces on the indenter tip. In the resistance of the agglomerates opposing the moving tip, clear differences are observable. Amide-bonded NDs exhibit 60% higher lateral forces compared to the dip-coated NDs. The ND attachment of dip-coated samples are based on physi \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Uncoated** & **Dip Coated** & **EPD-Treated** & **Amide-Bonded NDs** \\ \hline Excess surface area (\(\mu\)m\({}^{2}\)) & 1.0 & 11.7 & 5.5 & 19.6 \\ Ra (nm) & 7.3 & 17.7 & 19.6 & 49.7 \\ RMS (nm) & 10.6 & 24.4 & 25.1 & 66.3 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Excess surface area, mean deviation (Ra), and the root mean square (RMS) of surface roughness parameters for the uncoated, dip-coated, EPD-treated, and amide-bonded NDs on Ti. Projected area = 400 \(\mu\)m\({}^{2}\). Figure 3: Representative nanoindentation tests of (**a**) uncoated, (**b**) dip-coated, and (**c**) amide-bonded NDs to Si. Graphs show the lateral force as a function of the lateral displacement of the indentation tip. van der Waals force, whereas amide-bonded (chemisorbed) ND agglomerates possess strong amide compounds to the substrate. Every peak in the lateral force is likely to be associated to a single ND agglomerate resisting the movement of the tip. The sharp peaks in lateral force appear as the tip either skips or completely detaches from the same agglomerate. Nanoindentation measurements provide a first means of comparison for the binding strength of differently immobilized ND agglomerates. Comparing the absolute variations of lateral force, the covalently immobilized ND agglomerates pose a higher resistance to the tip movement, confirming that the interaction of NDs with the substrate is stronger than on the dip-coated surface. Apart from the above-shown immobilization strategies, an alternative covalent immobilization strategy--a DNA-mediated immobilization of NDs to previously ssDNA loaded substrates--will be discussed. We propose biomolecules as linkers of NDs to macroscopic surfaces. Therefore, a facile ssDNA grafting to NDs is required for further hybridization of the immobilized counterparts. Previous attempts to graft functional DNA to NDs have utilized various complex steps. [PERSON] et al. [42] used NHS-mediated amide bridges to bind NDs to peptide nuclei acids. Further approaches have focused on chopper-free click reactions with N\({}_{3}\)-functionalised NDs [43], DNA-biotin-avidin-biotin-ND origami [44], and DNA physisorption to NDs [45]. As an initial step of the proposed model, a facile conjugation of NDs to ssDNA as a platform for further immobilization on macroscopic surfaces is required. Figure 4 shows the results of the conjugation of NDs, using covalent grafting of thiol-terminated ssDNA onto carboxylated NDs through thioester formation (see inset in Figure 4a). Figure 4: (**a**) Optical bright-field microscopy image and (**b**) correlated fluorescent microscopy image of fluorescently labelled ssDNA grafted onto NDs.