Ethanol

Chitosan of medium molecular weight, M-Chit, (Mw: 190,000–375,000; 75–85% deacetylated chitin), chitosan of low molecular weight, L-Chit, (Mw: 50,000–190,000; ≥75% deacetylated chitin), poly (vinyl alcohol) (Mw: 89,000–98,000; 99+% hydrolyzed), and gelatin from cold-water fish skin (40–50% in H₂O) were purchased from Sigma-Aldrich (Darmstadt, Germany) and used as received. Acetic acid (min 99.8%, PA) and ethanol (min 99.8%, PA) from Riedel-de Haën (Munich, German), were used as chitosan solvent and neutralizer agent respectively.

Melatonin (Sigma, Steinheim, Germany, M5250) stock solution, 1 mg mL⁻¹, was prepared in ethanol (Riedel-de Haen, Seelze, Germany), protected from light and stored at 4 °C. All other used reagents, MeCN (Riedel-de Haen), chloroform (Sigma, 366919), methanol (Riedel-de Haen,), NaCl (Sigma, S7653), MgSO₄ (Sigma, M7506), sodium acetate (Sigma, S8750), and acetic acid (Sigma, A6283), were of analytical or chromatographic purity. The ultra-pure water was obtained with an Elix 3 (Millipore, Darmstad, Germany) system.

The PCR product was purified in 96-well plates using ethanol (Riedel de Haen; Munich, Germany). Approximately 60 µL of (100%) absolute ethanol was added to each well that contained the product of a sequencing reaction and was incubated at room temperature. After the mixture centrifugation at 1008 RCF for 30 min, 70 μL of 70% ethanol was added and centrifuged again. Then, 10 µL of formamide was added to each well and the plate was spun. The plate was incubated in PCR at 95 °C for 5 min. The reaction was done by using a Big Dye buffer (Thermo Fisher Scientific, Vienna, Austria). Mixture one was prepared, which contained 1.5 µL of Exosap and 3 µL of purified PCR product. The mixture was incubated at 37 °C for 15 min. The reaction was stopped by heating the mixture at 85 °C for 15 °C.

All the chemicals used in this study were of analytical grade. Fischer Scientific (Loughborough, UK) supplied acetylcholinesterase from electrical eel (AChE, EC 3.1.1.7) butyrylcholinesterase from equine serum (BChE, EC 3.1.1.8), butyrylthiocholine iodide, galanthamine, acetylthiocholine iodide, 5,5-dithiobis(2-nitrobenzoic) acid (DTNB), lipase from porcine pancreas, (EC 3.1.1.3), tyrosinase from mushroom (EC 1.14.18.1), and α-glucosidase from Saccharomyces cerevisiae (EC 232-604-7). Ethanol was purchased from Riedel de Haën (Buchs, Switzerland). Merck (Darmstadt, Germany) delivered Ethanol, mEthanol, and formic acid. Other solvents and chemicals were provided by VWR International (Leuven, Belgium).

The following chemicals and reagents were used: graphite (graphite powder, Aldrich, Milwaukee, WI, USA), sulfuric acid (95–98% H₂SO₄, Merck, Darmstadt, Germany), phosphoric acid (85% H₃PO₄, Sigma, St. Louis, MO, USA), potassium permanganate (KMnO₄ powder, Merck, Darmstadt, Germany), hydrogen peroxide (30% H₂O₂, Riedel-de Haën, Sleeze, Germany), hydrochloric acid (36.5–38% HCl, J.T. Baker, Phillipsburg, NJ, USA), sodium borohydride (98% NaBH₄, Aldrich, Milwaukee, WI, USA), titanium dioxide (TiO₂ ST01 powder, Ishihara Sangyo, Tokyo, Japan), sodium hydroxide (99% NaOH pellets, J.T. Baker, Phillipsburg, NJ, USA), ethanol (99.5–98% C₂H₆O, Riedel-de Haën, Sleeze, Germany), deionized (DI) water, carbon black (carbon powder, Uni-onward Corp., New Taipei, Taiwan), N-methyl-2-pyrrolidone (>99% NMP, Sigma-Aldrich, Milwaukee, WI, USA), polyvinylidenefluoride (PVDF, Aldrich, Milwaukee, WI, USA), and sodium sulfate (99% Na₂SO₄ powder, Merck, Darmstadt, Germany). All reagents were used as received without further purification.