Freezone 4.5 freeze dryer

The worm samples were thawed and homogenated firstly by a sonicator (Fisher Scientific, Model 505 sonic dismembrator, Fair Lawn, NJ, USA), and then freeze-dried (FreeZone 4.5 Freeze Dryer, Labconco, Kansas City, MO, USA) for 24 hrs. The extraction of SLs from C. elegans was carried out according to a modified Bligh and Dyer's method 37 (link), 38 (link). Briefly, 30 mg of worms were added with 2 ml of methanol (MeOH) and 1 ml of chloroform (CHCl₃), vortexed for 1 min, and incubated in a water bath (TW12, Julabo, Allentown, PA) at 48°C for 24 hrs. The supernatant was collected and the solvent was evaporated by nitrogen blowing at room temperature. 75 μL of 1 M potassium hydroxide (in MeOH) was added into the residues and the mixture was sonicated for 5 min. After an incubation at 37°C for 2 hrs, 3 μL glacial acetic acid was added for neutralization followed by a phase separation generated by adding 1 mL CHCl₃ and 2 mL water. Following a centrifugation at 2,500 rpm for 10 min, the upper layer (water layer) was carefully removed and discarded; and the chloroform layer was dried under vacuum and reconstituted with CHCl₃-MeOH (2:1). The samples were centrifuged several times to further remove impurities before LC-MS analysis.

Graphene oxide synthesis followed the methodology used by Mangadlao et al. [22 (link)]. In a round flask, 3 g of graphite were mixed with 400 mL of concentrated H₂SO₄, after 10 min of stirring, 3 g of KMnO₄ were added. Subsequently, 3 g of KMnO₄ were added every 24 h for three days. The reaction was stopped at four days, for which a third of the contents of the balloon was mixed in 300 mL of a water/ice mixture and 2 mL of H₂O₂ were added. The solution was distributed in 50 mL flasks and washed in a UNIVERSAL 320R centrifuge (Hettich, Tuttlingen, Germany) for 10 min at 5000 rpm with Milli-Q water and isopropanol until reaching a neutral pH. Subsequently, it was purified by dialysis in 3.5K MWCO SnakeSkin tubes (Thermo Scientific, Waltham, MA, USA), followed by Milli-Q water washing and drying at −51 °C and 0.12 mBar pressure in a Freezone 4.5 freeze dryer (LABCONCO, Kansas City, MO, USA) for 48 h. The graphite oxide was dispersed in water for 24 h in a Branson 5800 ultrasound equipment (Branson, Madrid, Spain) to finally obtain the GO.