Sodium carbonate

The standard of rutin, galic acid, Folin–Ciocalteu’s phenol reagent, HPLC grade methanol, 2,2-diphenyl-1-picrylhydrazyl and ethanol were obtained from Sigma Aldrich (St. Louis, MO, USA), stevioside and rebaudioside A were from TransMIT (Geiben, Germany), HPLC-grade acetonitrile and sodium acetate were from Sharlau Chemie S. A. (Sentmenat, Spain), HCl, sodium carbonate and acetic acid were from Carl Roth (Karlsruhe, Germany), hexamethylenetetramine and aluminium chloride were from Thermo Fisher Scientific (Lancashire, UK). All solutions were prepared with ultrapure 18.2 MΩ water from a Watek ultrapure water purification system (Watek Ltd., Ledeč nad Sázavou, Czech Republic).

Ultrapure water was obtained by purifying demineralized water in a Direct-Q 3 UV-R system (Merck, Millipore, Darmstadt, Germany). LC-MS-grade acetonitrile, methanol, ammonium formate, and HPLC-grade chloroform were purchased from Carl Roth GmbH and Co. KG (Karlsruhe, Germany). LC-MS-grade isopropanol was from Merck KgaA (Darmstadt, Germany). LC-MS tuning mix and hexamethoxyphosphazine, used as tuning standards, as well as hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine, and purine, used as lock masses, were purchased from Agilent Technologies (City of Santa Clara, CA, USA). Acetic acid, ethanol, sodium carbonate, disodium hydrogen phosphate, sodium chloride, potassium chloride, and potassium dihydrogen phosphate (all analytical-grade) were from Carl Roth GmbH and Co. KG (Karlsruhe, Germany). 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), fluorescein, and trolox (all analytical-grade) were from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). 2,2’-Azobis(2-methylpropionamidine) dihydrochloride (AAPH), potassium peroxodisulphate, and gallic acid (both of analytical-grade) were purchased from Fisher Scientific GmbH (Schwerte, Germany). Folin-Ciocalteu phenol reagent (analytical-grade) was obtained from Merck KgaA (Darmstadt, Germany). Acetone (analytical-grade) was obtained from VWR International LLC (Fontenay-sous-Bois, France).

Chitosan with an average molecular weight of 15 kDa and with minimum 85% degree of deacetylation (Polysciences, Inc.), vanillin (Sigma-Aldrich), syringaldehyde (Acros Organics), 4-hydroxybenzaldehyde (Sigma-Aldrich), sodium cyanoborohydride (Acros Organics), sodium carbonate (Carl Roth), kraft lignin from southern pine trees (Domtar), carbon black AB-520 (MTI Corporation), and carbon paper Spectracarb 2050A-0550 (Fuel Cell Store) were used as received. Solvents acetic acid (VWR International), ethanol (Fisher), propylene glycol (Acros Organics), and deuterated solvents water (Sigma-Aldrich) and acetic acid (Sigma-Aldrich) were used without further purification. The surface morphology of carbon black, ChiVan–CB composite and the composite after grinding was investigated by SEM using a Zeiss Leo Gemini 1550 microscope. Nitrogen adsorption measurements were performed using a Quantachrome Quadrasorb SI porosimeter with N2 at 77 K. ¹H NMR samples were prepared by dissolving the material (10 mg) in D₂O (1 mL) and D₃CCOOD (10 μL). The spectra were measured using an Ascend 400 MHz NMR spectrometer (Bruker). FT-IR measurements were performed at a Nicolet iS 5 FT-IR Spectrometer (ThermoFisher Scientific). ICP-OES was conducted using a Horiba Ultra 2 instrument equipped with photomultiplier tube detection. Samples were dissolved in aqua regia and filtered prior to analysis.

For spectrophotometric analysis acetonitrile (99.9%) and Folin–Ciocalteu reagent were purchased from Merck, Darmstadt, Germany, methanol (99.9%) and trifluoroacetic acid (98%) were from Sigma-Aldrich, Saint Louis, MO, USA; acetic acid (99%) was from Lachema, Neratovice, Czech Republic, aluminum chloride was from Chempur, Piekary Sląskie, Poland; sodium carbonate and hexamethylenetetramine (C₆H₁₂N₂) (99.9%) were from Roth, Karlsruhe, Germany; 2.2-diphenyl-1-picrylhydrazyl (DPPH) was from Sigma, Germany. Standards for HPLC qualitative analysis: trans-synapic acid, rosmarinic acid, and hesperetin were purchased from Fluka, Steinheim, Germany, trans-p-coumaric acid was from Chromadex, Irvine, CA, USA and rutin was from Sigma, Steinheim, Germany. Bidistilled water was prepared using distillation system Firstreem^TM Cyclon^TM, Loughborough, UK.

The reagents used in the assays met all quality requirements and were of analytical grade. The following reagents were used in the study: ethanol 96% (v/v) (manufactured by SC Vilniaus degtinė, Vilnius, Lithuania), Folin–Ciocalteu reagent, gallic acid monohydrate, ferric chloride hexahydrate (FeCl₃ × 6H₂O), sodium acetate trihydrate (CH₃COONa × 3H₂O), DPPH (2,2-diphenyl-1-picrylhydrazyl), Trolox ((±) -6-hydroxy-2,5,7,8-tetramethylchromano-2-carboxylic acid), hexamethylenetetramine, chlorogenic acid, caffeic acid, phloridzin, quercetin, quercitrin, kaempferol-3-O-glycoside (+)-catechin, (−)-epicatechin, and (−)-epicatechin gallate (Sigma-Aldrich, Steinheim, Germany), sodium carbonate (Na₂CO₃), rutin (Carl Roth GmbH, Karlsruhe, Germany), glacial acetic acid 99.8% (Lachner, Neratovice, Czech Republic), 2,4,6-tripyridyl-s-triazine (TPTZ) (Alfa Aesar, Karlsruhe, Germany), concentrated hydrochloric acid, aluminum chloride hexahydrate (Fluka-Chemie, Buchs, Switzerland), sodium molybdate, sodium nitrite, and sodium hydroxide (Chempur, Tarnowskie Gory, Poland). Purified water was prepared, using the Milli-Q^® water-purification system (Millipore Co., Bedford, MA, USA).

Purified MAbs were labeled with HRP. MAbs were dialyzed against sodium carbonate (Carl Roth, P028.1) buffer (pH 9.5) overnight at 4 °C. Peroxidase (Merck KGaA, P8375) and 0.2 M sodium periodate (Merck KGaA, 71859) were mixed together and incubated for 30 min at RT in the dark. The activated Peroxidase was desalted using Sephadex G-25 column (GE Healthcare Life Sciences, 17085101), mixed with each dialyzed MAbs and incubated for 2 h at RT in the dark. The pH of 9.5 was attained by adding 0.2 M sodium carbonate buffer to the mixture. Sodium borohydride (Merck KGaA, 213462) was added to the final concentration 0.2–0.4 mg/mL and incubated for 2 h at 4 °C in the dark. The mixture was then dialyzed against PBS overnight at 4 °C. After dialysis, HRP-labeled MAbs were supplemented with BSA and glycerin (Carl Roth, 3783.1) to the final concentrations of 2% and 50%, respectively, and stored at −20 °C.