Citric acid monohydrate

Acetaminophen (ACE), potassium hydroxide, dibasic sodium phosphate, ammonium heptamolybdate tetrahydrate (AHMo), thiourea (TU), molybdenum (VI) oxide (MoO₃) and sulfur powder, bicinchoninic acid (BCA), tetra-n-butylammonium bromide (TBAB), Nafion solution, and ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) reagents were purchased from Sigma-Aldrich. (Sigma Aldrich, Saint Louis, MO, USA) Citric acid monohydrate was purchased from Fisher Scientific (Thermo Fischer Scientific, Waltham, MA, USA). Toray Paper (TGP-H-060) was purchased from Fuel Cell Earth (Fuel Cell Earth, Woburn, MA, USA). All reagents were of analytical grade and were used without further purification, and all solutions were prepared using deionized water (18 mΩ·cm).

Acetonitrile (HPLC grade: ≥99.9%), water (HPLC grade: ≥99.9%), acetic acid (HPLC grade: ≥99.7%), citric acid monohydrate (HPLC grade) and dimethyl sulfoxide (reagent grade) were purchased from Fisher Scientific, Horsham, UK. Sodium phosphate dibasic (HPLC grade: ≥99.0%), sodium acetate (HPLC grade: ≥99.0%), phenol (reagent grade: >99%) and p-cresol (reagent grade: >98%) were purchased from Sigma-Aldrich, Gillingham, UK. PLE was purchased from Sigma-Aldrich, St. Louis, MO USA. 4-Nitrophenol (reagent grade: >97%), 4-Chlorophenol (reagent grade: >98%), 4-Hydroxybenzonitrile (reagent grade: >97%) and 4-Methoxyphenol (reagent grade: >99%) were sourced from Alfa Aesar, Haverhill, MA, USA. All reagents sourced from external suppliers were used without further purification. The TMZ esters were synthesised, purified and characterised following standard procedures [18 (link)]. The purity of each synthesised TMZ ester was also estimated to be at least 95%, using ¹H NMR.

Whey protein (Davisco, USA) was dissolved in Milli-Q water at 15% w/v, and stirred at 4 °C overnight. Afterwards, sodium citrate tribasic dihydrate (Sigma-Aldrich) and citric acid monohydrate (Fisher Scientific, USA) were mixed in the protein solution under vigorous stirring, to attain a citric concentration of 60 g/L and a pH value ~3.6. Then, the mixture was heated at 50 °C for 2h using a water bath, and incubated at room temperature for 24 h. After a slow (4 000 rpm, 20 min) and following fast centrifugation (14 000 rpm, 20 min) at room temperature, a nearly transparent solution and white sediment were obtained. The supernatant was collected and the heat-precipitation-centrifugation procedure was repeated for two more consecutive times before filtration by means of a 0.45 µm cellulose acetate membrane filter (Sigma-Aldrich). The filtered protein solution was dialyzed using a Spectra/Por dialysis membrane with 6–8 kDa MWCO (Spectrum Laboratories) against Milli-Q water at 4 °C, which was previously boiled for 10 min in 1 mM EDTA solution and extensively rinsed. Finally, the dialyzed solution was freeze-dried and kept at −20 °C for long-term storage.

Iron (III) chloride hexahydrate (FeCl₃·6 H₂O); iron chloride tetrahydrate (FeCl₂·4 H₂O; ammonium persulfate (APS); N,N,N′-Trimethylethylenediamine 97% (TEMED); triethyl amine (TEA); acryloyl chloride; potassium carbonate (K₂CO₃); dibasic sodium phosphate (Na₂HPO4); and glycine (C₂H₅NO₂) were obtained from Sigma Aldrich (St. Louis, MO, USA). Ammonium hydroxide (NH₄OH) was purchased from EMD Chemicals (Gibbstown, NJ, USA). Poly(ethylene glycol) 400 dimethacrylate (PEG400DMA) was obtained from Polysciences INC. (Warrington, PA, USA). Curcumin was purchased from Chem-Impex International, Inc. (Bensenville, IL, USA), and quercetin was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Citric acid monohydrate, sodium chloride (NaCl), calcium carbonate (CaCO₃), and sodium hydroxide (NaOH) were obtained from Fisher Scientific (Hannover Park, IL, USA). 3,3′,4,4′,5-Pentachlorobiphenyl (PCB-126) in Isooctane was purchased from Accustandard (New Haven, CT, USA). 5′-fluoro-3,3′,4,4′,5-pentachlorobiphenyl (F-PCB 126) was purchased from Resolution Systems Inc. (Holland, MI, USA). All solvents (Isooctane, ethanol HPLC grade, tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile (ACN), and acetone) were obtained from Fisher Scientific (Hannover Park, IL, USA). All materials were used as received.

The following chemicals, including their sources, purity, and CAS numbers, were used: citric acid monohydrate, Fisher, ACS grade, 5949-29-1; ᴅʟ-malic acid, Alfa Aesar, 98%, 6915-15-7; ᴅʟ-isocitric acid, trisodium salt hydrate, Acros Organics, 95%, 1637-73-6; ᴅʟ-lactic acid, TCI, >85%, 50-21-5; ᴅʟ-glyceric acid, TCI, 20% in water (ca. 2 mol/L), 473-81-4; ᴅʟ-tartaric acid, TCI, >99%, 133-37-9; glutaric acid, Acros Organics, 99% 110-94-1; tricarballylic acid, Alfa Aesar, 98%, 99-14-9; adipic acid, Sigma, 99%, 124-04-9; acetic acid, Sigma, ACS grade, 64-19-7; pimelic acid, Alfa Aesar; >98%, 111-16-0; succinic acid, TCI, >99%, 110-15-6; tartronic acid, Sigma, >97%, 80-69-3; sodium mesoxalate monohydrate, Chemodex, >98%, 31635-99-1; ᴅʟ-2-hydroxybutyric acid sodium salt, Alfa Aesar, >97%, 5094-24-6; ᴅʟ-3-hydroxybutyric acid sodium salt, Chem Impex Int’l Inc., 100.3%, 150-83-4/306-31-0; ascorbic acid, TCI, >99%, 50-81-7; ammonium nitrate, Fisher, ACS grade, 6484-52-2; sodium nitrate, VWR, ACS grade, 7631-99-4; sodium hydroxide, VWR, ACS grade, 1310-73-2; nitric acid, Sigma, ACS grade, 7697-37-2; and sodium azide, Sigma, 99.8%, 26628-22-8. Lacey carbon, 300 mesh, copper grids (product #01895) from Ted Pella, Inc. were used for electron microscopy.