Ethanol

Acrylamide (≥ 95% for HPLC) and Acrylamide-d3 standard solution (1000 mg L⁻¹ in acetonitrile) were purchased from Sigma-Aldrich (Steinheim, Germany). Methanol (for UHPLC), ethanol (99.5%), and dichloromethane (for HPLC) were from Panreac (Barcelona, Spain). High-purity water from a Millipore Simplicity 185 water purification system (Millipore Iberian S. A., Madrid, Spain) was used for all chemical analyses and glassware washing. The solvents employed for HPLC were filtered through a Nylon filter of 0.45 μm pore size (Whatman, Clifton, NJ, USA) and degasified for 10 min in an ultrasound bath.

Neodymium nitrate hexahydrate (Nd(NO₃)·6H₂O Sigma-Aldrich, St Louis, MO, USA), chitosan (acetylation degree of chitin 0.13, Sigma-Aldrich, St Louis, MO, USA), manganese(II) sulphate (MnSO₄·H₂O Sigma-Aldrich, St Louis, MO, USA), acetic acid (CH₃COOH, 99.7%, 6H₂O Sigma-Aldrich, St Louis, MO, USA), hydrochloric acid (HCl, 37.4%, J.T. Baker, Phillipsburg, NJ, USA), sodium hydroxide (NaOH, 97%, Panreac, Barcelona, Spain), nitric acid (HNO₃, 69.0%, J.T. Baker, Radnor, PA, USA), sodium chloride (NaCl, 99.5%, Prolabo, Fontenay-sous-Bois CEDEX, France), iron(III) chloride (FeCl₃·6H₂O, 99–102%, Sigma-Aldrich, St Louis, MO, USA), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA, 99% Panreac, Barcelona, Spain), methanol (98% Panreac), ethanol (etOH, 96% Panreac), methanol (meOH, UV-IR-HPLC isocratic, Panreac), and deionized water type II laboratory water were used.

Acetone, ethanol, methanol, sodium carbonate, calcium chloride and gallic acid were purchased from Panreac (Barcelona, Spain). Butylated hydroxytoluene (BHT) were purchased from Acros (Hampton, NH, USA). Folin reagent, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ascorbic acid, chlorophyll a, chlorophyll b, lutein, β-carotene and fucoxanthin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solvents including ethanol, methanol and acetonitrile of high-performance liquid chromatography (HPLC) purity were purchased from Lab-Scan (Lisbon, Portugal).

Sulphuric acid (95–98%), nitric acid (69%), ethanol (99%) and chloroform were purchased from PANREAC, S.A.U. (Barcelona, Spain). N,N′-Diisopropylcarbodiimide (DIC, 98%), N-hydroxysuccinimide (NHS, 97%), triethylamine (TEA, 99%), cysteamine hydrochloride (98%), N-Boc-ethylenediamine (Boc-EDA, 98%), cellulose microcrystalline (50 μm particle size), acetone (99.5%), 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF₄, 97%), dimethyl sulfoxide (DMSO, 99.9%), sodium carbonate (99.95%). 5-Aminosalicylic acid and the diisocyanates purchased from Sigma–Aldrich, while the multi walled carbon nanotubes were purchased from Bayer (Germany). NMR spectra were measured on a Bruker Avance-400.

Rosmarinic acid, apigenin-7-O-glucoside, luteolin-7-O-glucoside, eriodictyol-7-O-glucoside, luteolin-8-C-glucoside, quinic acid, caffeic acid, and t-5-O-caffeoylquinic acid were obtained from Extrasynthese (Genay Cedex, France). Gallic acid and nisin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteu reagent, Na₂CO₃, formic acid, and ethanol were purchased from Panreac (Barcelona, Spain). n-hexane, methanol, and acetonitrile with HPLC purity were purchased from Lab-Scan (Lisbon, Portugal). Mueller-Hinton agar was obtained from VWR, Prolabo Chemicals, West Chester, PA, USA. Water was treated in a Direct-Q^® water purification system (Merck Life Science, Germany).

Quantification of biofilm production was carried out by adapting the microtiter biofilm formation protocol described by Stepanovic et al. [49 (link)]. Briefly, in a flat bottom 96 microplate, wells were filled with 200 μL of test solutions at sub-MIC concentrations (or 20 mg/mL, according section 2.2) with inoculum being added at 2% (v/v). Following this the microplate was incubated at 37 °C for 48 h. To visualize biofilms, the contents of each well were discarded and the well washed 3 times with sterile deionized water in order to remove non-adherent cells. The remaining attached bacteria were fixed with 200 μL of ethanol (Panreac, Barcelona, Spain) for 15 min. ethanol was then discarded and the wells air dried. After that, 200 μL of crystal violet solution (Merck, Darmstadt, Germany) were added to the wells for 5 min. Excess stain was removed by rinsing the plate under tap water and the air dried.
Adherence was quantified by measuring the Optical Density (OD) at 660 nm using a microplate reader (FlUOstar, OPTIMA, BGM Labtech, Ortenberg, Germany).
Results for this test were given as percentage of biofilm formation inhibition applying the following formula:

All assays were done in triplicate.

Sodium carbonate, sodium acetate, potassium persulphate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (Madrid, Spain). Hydrochloric acid, ethanol, glycine, Folin reagent, and iron trichloride (FeCl₃·6H₂O) were obtained from Panreac (Barcelona, Spain). ABTS (2,2-azino-bis-(3 ethylbenzothiazolne-6-sulfonic acid) diammonium salt) and Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) were purchased from Fluka (Madrid, Spain).