Camphorsulfonic acid

Camphorsulfonic acid (CSA) (≥98.0%), dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1-butanol, acetonitrile, bis(trifluoromethane)sulfonimide lithium salt, 2,2′,7,7′-Tetrakis(N,N-di-p-methoxyphenylamine)-9,9′-spirobifluorene (Spiro-OMeTAD), methylammonium iodide (MAI), and PbI₂ were acquired from Sigma-Aldrich. Titanium diisopropoxide bis(acetylacetonate) (TAA) and cobalt dopant FK209 TFSI salt were acquired from Merck.

Polyaniline (PANi, Emeraldine base, M_W = 100,000 Da), polyacrylonitrile (PAN, M_W = 100,000 Da), camphor sulfonic acid (CSA), and N,N-dimethylformamide (DMF), acetic acid, sodium hydroxide, isopropanol, monochloroacetic acid, ethanol, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), glutaraldehyde and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. Chitosan (CTS) extracted from the crab shell with a medium weight and degree of deacetylation of > 90% was bought from Bio Basic Inc. (Canada). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and trypsin/EDTA solution were purchased from Gibco (USA).

Flat sheet polyethersulfone (PES) ultrafiltration (UF) membranes with a molecular weight cut-off (MWCO) of 50 kDa were procured from RisingSun Membrane Technology Co., Ltd. (China), cellulose nitrate (CN) membranes with a 0.45 µm pore size were purchased from Microanalytix Pty Ltd. (Australia), and commercial Trisep XN45 NF membranes were provided by Sterlitech. Inc., (USA). Chemicals including piperazine (PIP, ≥ 99%), trimesoylchloride (TMC, ≥ 98%), triethylamine (TEA), camphorsulfonic acid (CSA), sucrose, h-MoS₂ powder (< 2 µm, 98%), sodium salt of HA, sodium alginate, and dialysis tubes with MWCO of 3500 Da were supplied by Sigma Aldrich (USA). n-Hexane, hydrochloric acid (32%), sodium hydroxide, calcium chloride, magnesium chloride, and sodium chloride were purchased from Merck (Australia). Deionized (DI) water was used to prepare the organic feed solutions employed in the filtration experiments.

Samples were thawed on ice and 2 ml ice cold chloroform was added to each sample to perform a methanol:water:chloroform extraction with a volume ratio of 5∶5∶1 (modified after Wu et al. [25] (link)). Next, the internal standards were added (20 nmol ribitol, 20 nmol norvaline and 2.5 nmol camphorsulfonic acid; Sigma-Aldrich) and the extraction solution was mixed and incubated for 10 min on ice. Subsequently, the samples were centrifuged for 10 min at 3,000×g at 4°C. The separated aqueous and organic phase were transferred in a new tube, mixed again and split into two samples, which were lyophilized for GC-MS analysis and for HPLC-MS analysis.
The same extraction procedure was applied to the medium samples for analyzing nucleotides in the cell supernatants. For this purpose 1.3 ml of the thawed supernatant were lyophilized. Afterwards, the samples were resuspended in 1 ml cold doubly distilled water and metabolites were extracted as described above.

All samples were thawed on ice and the cell pellets were disrupted using a Ribolyser (MagNa Lyser, Roche, Germany), at 5000 m × s for 30 seconds. Samples were transferred on 50 ml falcon tubes and the Ribolyser tubes were washed with a total of 5 ml 75% (w/v) ethanol −20°C until all cell rests are washed out and were transferred to the falcon tube. Next, the internal standards were added (20 nmol ribitol, 20 nmol norvaline and 2.5 nmol camphorsulfonic acid; Sigma-Aldrich) and the extraction solution was mixed and incubated for 10 min on ice. Afterwards, the Falcon tubes were filled with destilated water to the final volume of 35 ml and stored at – 70°C. Finally, the samples were lyophilized for GC-MS analysis.