Figure 2 – Fabrication of the eSF hydrogel based microfluidic chip. A) Schematic illustration of the eSF microfluidic device, including a serpentine channel. B-C) Scheme and corresponding photographs showing the main steps for fabricating the chip by photolithography and soft lithography (double replica molding): i: SU8 mold 1; ii: PDMS replica; iii-iv: PDMS mold 2; v: eSF chip). D) Photograph of the eSF chip and the corresponding SEM images of two different areas. (E) Image showing the high degree of transparency of the eSF microfluidic chip and a close up photograph of the serpentine features. Scale bars: 7.3 mm.
Next, we characterized the eSF hydrogel material. We first optimized the % of eSF using 6, 12, and 14% eSF. Rheology analysis showed that the higher the concentration of eSF, the higher the G’, or the stiffness. The 6% eSF hydrogel presented a G’ of 1963 ± 151 Pa, 12% eSF hydrogel showed a G’ value of 6585 ± 253 Pa, similar to 14% eSF hydrogel, which gave a G’ of 7172 ± 605 Pa (Figure 3 A). The reported results are in agreement with previous results (14). However, during replica molding, 6 and 12% eSF were significantly brittle, leaving eSF residues in within the channels. They were less transparent than the 14% counterpart. For this reason, 14% eSF hydrogel was selected for building the microfluidic platform.
Figure 3 B shows the ATR-FTIR spectra of silk 14% over 7days. There is an association between hydrogels network morphology and conformational changes (detected by ATR-FTIR) of the eSF proteins. When incubating the eSF hydrogel (in PBS at 37˚C), a relatively transparent morphology is observed over 7 days, starting to change at day 7 to opaque until 10 days (29). As shown in the FTIR-ATR spectra (Figure 3 B), where there is a clear shift around 1600 cm-1, indicating a protein conformation change from amorphous to β-sheet (as seen by the green line). Under these conditions, the cells encapsulated in these hydrogels enter apoptosis due to conformational change stress (19, 29). However, if cells are encapsulated on eSF hydrogels already in β-sheet, cell viability is preserved (17). This is the reason why our assays only lasted for 7 days. On the other hand, the transparency that the amorphous state of the eSF offer is a must for imaging cell dynamics and viability within the hydrogel.
We next investigated the deformation capacity of the microfluidic chip. For this, we performed tensile tests on the 14% eSF, finding a mean tensile strain of 103.96 % (Figure 3 C). This means that the hydrogel can be stretched twice its original size. Finally, regarding the Young’s modulus, the hydrogels can endure, about 11.79 kPa. Altogether, these results demonstrate that the eSF microfluidic platform is highly elastic.