Oxone

De-paraffinized sections were incubated for 30 min in 10% Oxone (catalog number 208036, Sigma, St Louis, MO), rinsed with water and 70% ethanol, stained for 1 h in aldehyde-fuchsin (pH 1) (#26328-01, Electron Microscopy Sciences, Hatfield, PA), rinsed in 95% ethanol and counterstained with eosin (Inoue et al., 2012 (link)).

L-ascorbic acid (99%), trehalose dihydrate, poly-ε-caprolactone, and chitosan (50,000–190,000 Da) were obtained from Sigma-Aldrich and were used without further purification. Acetonitrile and methanol (HPLC grade) were obtained from J.T. Baker. Low molecular weight chitosan and the chemicals used in the oxidation assays (Oxone® and EDTA) were obtained from Sigma-Aldrich. The AchE assays employed acetylthiocholine iodide and DTNB (5,5′-dithiobis (2-nitrobenzoic acid) acquired from Sigma-Aldrich.

AFB₁ was obtained from Toronto Research Chemicals (Toronto, ON, Canada). The LC-MS grade solvents H₂O, methanol, acetone and acetonitrile were purchased from Fisher Scientific (Ottawa, ON, Canada). DCM (anhydrous) was purchased from Fisher Scientific (Ottawa, ON, Canada). Blank serum (H4522; from human plasma, USA origin, sterile-filtered; Millipore Sigma Burlington, MA, USA), sodium bicarbonate and Oxone^® was purchased from Sigma-Aldrich (Oakville, ON, Canada). Labelled AFB₁-Lys adduct was prepared according to Renaud et al., (2022) [19 (link)]. In that work, which minimized the production of unwanted reaction by-products, quantitative NMR was used to demonstrate that the previously reported molar attenuation coefficient (ε₄₀₀ 30,866/M cm) [30 (link)] used for the generation of AFB₁-Lys standards is valid.

Staining was performed on 6-μm paraffin-embedded sections, which was previously described. The slides were oxidized with Oxone® (diluted with distilled water to 10% m/v, 228036, Sigma-Aldrich, Switzerland) or distilled water for 10 minutes at room temperature. After they were rinsed with three distilled water washes (2 minutes each time), the slides were stained with resorcin-fuchsin solution (26370, Electron Microscopy Sciences, USA) for 10 minutes. They were differentiated in pure ethanol (3 changes with 10 immersions each time). After rinsing with running water, they were dehydrated and mounted. The sections were observed using the stereomicroscope mentioned previously.

Eugenol (EU), allyl bromide and oxone (potassium peroxomonosulfate) were purchased from Sigma-Aldrich (Saint Louis, MI, USA) and used without further purification. Diglycidylether of bisphenol A resin (DGEBA, Araldite GY 240) had a weight per epoxy equivalent (EEW) of 182 g/mol and was purchased from Huntsman (Chocolate Bayou, TX, USA). Two different crosslinking agents were used for the preparation of thermosets: polyetheramine Jeffamine^® D-400 (JEF) (430 g/mol) with a weight per amine hydrogen equivalent of 115 g/mol, purchased from Huntsmann (Chocolate Bayou, TX, USA) and isophoronediamine (IPDA) (170.3 g/mol), purchased from Acros Organics (Pittsburgh, PA, USA).
The preparation of 3EPOEU was done according to the method described in a previous work [27 (link)], which consists in the allylation of the phenol group, followed by a Claisen rearrangement, a second allylation step of the phenol formed and final epoxidation of the three allyl groups in the formed compound. In the epoxidation step, oxone was used as an oxidizing agent instead of m-chloroperbenzoic acid (MCPBA), following the concept of green chemistry. Other alternatives to MCPBA can be found in the literature such as hydrogen peroxide (H₂O₂) [30 (link),31 (link)].

Dichloromethane (DCM, analytical grade, Carlo Erba, Barcelona, Spain), acetone (analytical grade, Carlo Erba, Italy), sodium hydrogen carbonate (NaHCO₃, ≥95% purity, Sigma-Aldrich, St. Louis, MO, USA), tetrabutylammonium hydrogen sulfate (Bu₄NHSO₄, ≥97% purity, Fluka, Chemie, GmbH, Buchs, Switzerland), epichlorohydrine (99% purity, Sigma-Aldrich), and Oxone^® (Sigma-Aldrich) were purchased and used as received. Natural isoeugenol (Sigma-Aldrich, 99%), diglycidyl ether of bisphenol A (DGEBA, Sigma-Aldrich, Steinheim, Germany), camphoric anhydride (CA, 98% purity, Alfa Aesar, Ward Hill, MA, USA), hexahydrophtalic anhydride (HHPA, 95% purity, Sigma-Aldrich) and 1,2-dimethylimidazole (DMID, 95% purity, Sigma-Aldrich) were purchased and used without any further purification. These reagents are depicted in Scheme 1.