Salicylic acid

All
strains and plasmids
used in this study are listed in Table S1. E. coli strain XLI-Blue was
used for plasmid construction and enrichment. Strain E. coli BW25113 F′ was used for biosensor
characterization and fluorescence assay. LB medium containing 10 g/L
NaCl, 5 g/L yeast extract, and 10 g/L tryptone medium was utilized
for the E. coli culture. Ampicillin
and kanamycin were supplemented in the medium as needed with the final
concentrations of 100 and 50 mg/mL, respectively. Different concentration
of IPTG was added into medium if needed. For the inducer preparation,
100 mg of p-coumaric acid, caffeic acid, trans-cinnamic acid, ferulic acid, 4-hydeoxybenzoic acid,
anthranilic acid, salicylic acid, and vanillic acid were dissolved
in 1 mL of methanol to make the master stock with a concentration
of 100 g/L. ferulic acid and p-coumaric acid were
purchased from MP Biomedicals. caffeic acid, anthranilic acid, and
cinnamic acid were purchased from Sigma-Aldrich. salicylic acid and
vanillic acid were purchased from Alfa Aesar.

Sodium sulfate anhydrous (Na₂SO₄, ≥99.0%) was purchased from Carlo Erba and used to prepare electrolytes with a concentration of 0.1 M. Sodium citrate dihydrate (≥99.0%), salicylic acid (99.5%), sodium nitroferricyanide de-hydrate (99.0%), ammonium chloride (NH₄Cl, 99.99%) and sodium hypochlorite solution (NaClO, 6−14%) were bought from Alfa Aesar and used to measure the ammonia in the electrolyte via the indophenol method. All the solutions were prepared with ultrapure water (MilliQ).
Nafion 117 membranes (N117), purchased from Dupont, were used for the absorption test, with a nominal thickness of 182 µm. The Zirfon Perl (ZP) separator was purchased from Agfa.

The LC–MS grade formic acid and acetonitrile (ACN) used for the mobile phases’ preparation were purchased from Fisher Scientific (Pittsburgh, PA, USA) and Scharlab, S.L. (Barcelona, Spain), respectively. The methanol (MeOH) used in the sample and reagent solution preparation was obtained from Scharlab, S.L. (Barcelona, Spain). In addition, ultra-high-purity water, obtained from tap water pre-treated by Elix reverse osmosis and a Milli-Q system from Millipore (Bedford, MA, USA), was used for mobile phase, reagent solutions, and sample preparation.
Standard reagents used to assess the proper LC–MS system operation were from different manufacturers: paracetamol, cholic acid, (±) verapamil hydrochloride, simvastatin, reserpine, and leucine enkephalin acetate salt hydrate were provided by Sigma-Aldrich (Steinheim, Germany); caffeine and salicylic acid were supplied by Alfa Aesar (Karlsruhe, Germany) and Fluka Analytical (Bucharest, Romania), respectively. Finally, sodium fluvastatin was kindly supplied by Novartis (Basel, Switzerland). A system suitability test solution (SST) was prepared with the nine compounds at a final concentration of 100 ng/mL in MeOH:H₂O 2:1 (v/v); this solvent composition was chosen as this is similar to the final solution (supernatant) of plasma samples.

CGC with a chitin content of 35.6% was extracted from the yeast Komagataella pastoris (DSM 70877) produced as described by Araújo et al. [25 (link)]. Caffeine (99%), diclofenac sodium salt (98%), ibuprofen sodium salt (98%), and salicylic acid (99%) were purchased from Alfa Aesar, Tokyo Chemical Industry Co, Sigma-Aldrich, and BDH, respectively.

Four model molecules (theophylline, dextran blue, Salicylic acid, insulin) were selected for their differences in molecular weight (from 138 to 2.106 g/mol), aqueous solubility and pKa value. Theophylline was supplied from Pierre Fabre Medicament Laboratory (Labège, France). Insulin solution was provided from Lilly France SAS. Blue dextran was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Salicylic acid was purchased from Fisher Scientific (Illkirch, France).

Prior to crystallization, purified CAII was concentrated to 10 mg/mL via Amicon Ultra-15 centrifugal filters. CAII was crystallized via the hanging drop vapor diffusion method; 2.5 μL of 10 mg/mL protein was added to siliconized glass cover slips along with 2.5 μL of mother liquor consisting of 1.6 M sodium citrate and 50 mM Tris at pH 7.8; 500 μL of mother liquor was added to the wells and grease was used to seal the glass clover slips to the wells [28 (link)]. Crystals formed within 24 h. The 500 mM Aspirin was purchased through Sigma Aldrich (St. Louis, MI, USA) and salicylic acid was purchased through Fisher Scientific (Lenexa, KA, USA). Each chemical was determined to be >99% purity through NMR and other assays. Aspirin was dissolved in 100% ethanol and a 1:10 dilution was made for a final concentration of 50 mM Aspirin in 10% ethanol; 1 μL of the Aspirin solution was added to the CAII drops and allowed to soak for 20 min.

Sodium deoxycholate (NaDC; 97%), sodium taurodeoxycholate (NaTDC; 95%), sodium taurocholate (NaTC; ≥97%), sodium cholate (NaC; 97%), sodium glycocholate (GC; ≥97%), sodium glycodeoxycholate (GDC; ≥97%) and l‐α‐phosphatidylcholine from dried egg yolk (≥50%) were used as purchased from Sigma Aldrich, Dorset, UK for the preparation of stock solutions of mobile phase. Analytical grade 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES buffer) was purchased from Sigma Aldrich, Dorset, UK. The compounds considered in this work were caffeine 97% (Sigma Aldrich, Dorset, UK), fenoprofen 97% (Fluka, Dorset, UK), acetaminophen 99% (Sigma Aldrich, Dorset, UK), ketoprofen 98% (Sigma Aldrich, Dorset, UK), phenylbutazone 99% (Sigma Aldrich, Dorset, UK), fluconazole 98% (Sigma Aldrich, Dorset, UK), carbamazepine 99% (Sigma Aldrich, Dorset, UK), cimetidine (Sigma Aldrich, Dorset, UK), naproxen 98% (Sigma Aldrich, Dorset, UK), terbutaline 96% (Sigma Aldrich, Dorset, UK), zolmitriptan >98% (Sigma Aldrich, Dorset, UK), salicylic acid 99% (Fisher Scientific, Loughborough, UK), ibuprofen 98% (BASF, Cheshire, UK), acetyl salicylic acid 99% (Acros Organics, Geel, Belgium), diclofenac 98% (TCI Europe, Zwijndrecht, Belgium), flurbiprofen 98% (TCI Europe), nicotinic acid >98% (Sigma Aldrich, Dorset, UK) and theophylline 98%, (TCI, Oxford, UK).