*3.2. Polymerisation of Dopamine*

Dissolution of dopamine in the Tris buffer resulted in an immediate colour shift to light brown. As the polymerisation proceeded, the colour became darker until the solution was black, as shown in Figure 2. As can be observed, once the polymerisation took place for 96 h, a film formed between the surface of the liquid and the air, resulting in a different reflection of light. Scanning electron microscopy (SEM) micrographs of the pDA coatings are shown in Figure 3. Figure 3a shows a clear boundary between the uncoated and uniformly coated Ti6Al4V surfaces, while Figure 3b shows the bead-like structure of the polydopamine (pDA) coating. The beads display a circular shape with an average diameter of 86 ± 20 nm. It can be observed that the beads are in fact constructed of clusters of even smaller particles with an average diameter of 10 ± 1 nm. Even though the clusters are densely packed, there are visible inequalities in the shapes of the grooves in between the clusters.

**Figure 2.** A photograph of s 24-well plate showing the change in colour of the alkaline dopamine solutions over 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, shown from left to right, respectively.

**Figure 3.** SEM micrographs showing (**a**) the Ti6Al4V surface with a polydopamine (pDA) coating (left) and uncoated (right) at a magnification of 1000×, as well as (**b**) the pDA coating at 100,000× magnification.

The thickness of the pDA coating was measured by both ellipsometry and AFM, the results of which are shown in Table 4. The results for the thickness of the coating measured by both ellipsometry and AFM agreed with one another. A 24-h immersion of Ti6Al4V plates in the dopamine solution resulted in the growth of an approximately 10-nm-thick layer. The thickness of the coating steadily increased to reach around 55 nm after 72 h, after which the growth plateaued.

**Table 4.** Polydopamine coating thickness as measured by ellipsometry and Atomic ForceM.icroscopy (AFM).

