## 3 Results and Discussion
### Single-Point Attachment by Thiol-Epoxide Click Chemistry  
As described in the previous section the preferred immobilization protocol is the single point attachment using thiol-epoxide ring-opening reactions (Figure 8) due to the proven higher binding capacities, but also due to easier handling as NHS-esters are susceptible to hydrolysis reactions. Table 4 summarizes the conducted reactions for the single-point attachment, where the impact of  
Figure 6: FT-IR spectrum of non-functionalized macroporous microclusters bearing azide on their surface (solid line) and of functionlized microclusters (dashed lines). The azide stretching band can be seen at 2100 cm\({}^{-1}\). Figure 7: Illustrative comparison between multipoint attachment and single point attachment of proteins. (**a**) amidation reaction; (**b**) thiol-epoxide ring-opening reaction. solvent is investigated on (i) the yield of the click reaction and (ii) the performance of obtained materials for antibody chromatography. For the reactions S-1 and S-2, the click reaction of propargylglycidyl ether to the base material was carried out prior to protein immobilization. On the other hand, the thiol-epoxide ring opening reaction was conducted first in reaction S-3, and the obtained molecule was clicked by CuAAC subsequently. As shown in Table 4, S-1 and S-2 reveal that DMF has a positive impact on the click yield, most probably because it swells the styrene-based polymer making the azide groups on the surface more accessible. Interestingly, the much higher yield in S-2 did not lead to higher IgG recovery, which remained substantially unchanged in the two cases. Apparently, reacting 10% (0.03 mmol\({}_{\text{epoxide}}\)/gpolymer) of the available azide groups on the surface is already sufficient to immobilize the maximum amount of the bulky protein A molecule. This is coherent with the rather low BET surface of the material that is 11.4 m\({}^{2}\)/g, which strongly supports the hypothesis that only minor quantities of protein A can be attached in any case. S-3 was carried out in an aqueous buffer without any additional organic solvent. Similar to the previously described experiment, the yield for the click step is lower than the one in pure DMF. Given the fact that the yield of S-3 is the same as for S-1, one could expect a similarly high binding capacity: instead, the observed capacity was roughly 35% smaller. However, in this case, the thiol-epoxide reaction was carried out prior to the CuAAC reaction and likely more than one propargylglycidyl ether molecule reacted per protein A molecule, thus also resulting in some degree of multipoint attachment. Despite the lower binding capacity, the material that was functionalized without organic solvent (S-3) has one crucial advantage regarding chromatographic applications. In particular, when packing the column with materials functionalized using DMF the observed increase in pressure drop with the flow rate was much higher compared to the DMF-free counterpart, as shown in Figure 9. As mentioned above, DMF swells the polymer, but protein purification is done in aqueous buffers, which in contrast results in shrinking the material. Even if the material was washed exhaustively after the click step, there is likely some DMF trapped in the polymer scaffold keeping parts of the material swollen. During the protein chromatography process, the material contracts leading to a rearrangement in the polymer three-dimensional structure, which clogs the column and consequently increases the pressure drop.