Sulphuric acid

Dichloromethane (DCM), n-hexane, and isooctane used in this study were obtained from Merck, while sodium sulphate (anhydrous), sodium hydroxide, sulphuric acid, and silica gel (60–200 mm) were obtained from Sigma Aldrich, South Africa, respectively. Pentachloronitrobenzene and 50 μg/mL individual PBDE standards were purchased from Cambridge Isotope Laboratories, USA through Industrial Analytical Pty, South Africa. Helium (99.99%) and argon were obtained from Afrox Pty., South Africa.

Natural graphite flakes (99.8% purity) were purchased from VWR (UK). Sulphuric acid (98%), phosphoric acid (85%), hydrogen peroxide (35%), potassium permanganate (99%), Ni(II) chloride and SDS were purchased from Sigma Aldrich (UK). All reagents were used without further purification. Deionized (DI) water produced by a Nanopure (Thermo Scientific, USA) purification system was used in all the experiments.

The oil produced from C. finmarchicus (Calanus oil) was provided by the company Zooca^® formerly named Calanus^® AS (Tromsø, Norway). Heptane (99,8%), isopropanol (100%), diethyl ether (≥99,8%) and ethanol (96%) were purchased from VWR, Darmstadt, Germany. Acetic acid (99,8%) and hydrochloric acid (37%) were purchased from Honeywell, Seelze, Germany. Chloroform (99,0–99,4%), phosphoric acid 99,99%, potassium bicarbonate ≥99,95%, sodium hydroxide, copper (II) sulphate pentahydrate ≥98,0%, sulphuric acid and toluene were obtained from Sigma-Aldrich, Darmstadt, Germany. N-Hexane was obtained from Merck KgaA, Darmstadt, Germany.

Sulphuric acid (ACS reagent, 95–98%), human serum from human male AB plasma (USA origin, sterile filtered, ref. 4522, Lot# SLBJ3904V), hydrogen peroxide (H₂O₂ 30%), (3-glycidyloxypropyl)trimethoxysilane (98%), Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (97%, TLC), dry toluene (99.8%), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), N-(3-dimethylamino propyl)-N’-ethylcarbodiimide hydrochloride (EDC), (Aminoethyl)polyethylene glycol (5,000 Da), 2-(N-Morpholino)ethanesulfonic acid (MES), bicarbonate buffer solution, phosphate buffered saline (PBS), sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic and Tween® 20 were purchased from Sigma-Aldrich (St. Louis, USA). Spherical 100-nm-diameter gold nanoparticles coated with 5 nm thick carboxyl polymer (C11-100-TC-50) were acquired from Nanopartz (USA). Silicon cantilever arrays were purchased from Micromotive GmbH (Mainz, Germany). The nominal length, width and thickness of the cantilever are 500, 90 and 1 μm, respectively. The immunoassays were carried out with purified monoclonal anti-HIV-1/2 (HIV-018-48304, Capricorn Immunoreagents Perfected, USA), monoclonal Anti-HIV type1 p24 clone 39/5.4A (ZeptoMetrix, USA) and recombinant HIV-1 gag p24 antigen (Virogen, USA). 96-well microtiter plate format from Eppendorf Deepwell Plates.

Sulphuric acid, hydrochloric acid, ethanol, methanol, sodium tetraborate, carbazole, cysteine hydrochloride, ferric chloride, sodium phosphate dibasic, triton™ X-100, and potassium hydroxide were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Acetic acid and sodium hydroxide were purchased from VWR (VWR International, Radnor, PA, USA). Standards such as Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), DPPH (1,1-diphenyl-2-picryl-hydrazyl), TPTZ (2,4,6-tripyridyl-s-triazine), ascorbic acid, alginic acid, mannitol, and commercial fucoidan (from Fucus vesiculosus), and laminarin (from Laminaria digitata) standards were also purchased from Sigma. Enzyme-based kits for mannitol and glucan assay were purchased from Megazyme International Ltd., (Bray, Ireland). All reagents used were of analytical grade.

Individual inositol phosphates (IP3–IP6) were extracted and determined by HPLC and refractive index detection according to Burbano, et al. [19 (link)]. A total of 10 μL of Aliquots were injected on a PRP-1 column (150 × 4.1 mm i.d., 5 μm, Hamilton, Reno, Nevada, USA), and was maintained at 45 °C. The mobile phase was a mixture of methanol/water (52/48, v/v) with 0.8% of tetrabutyl ammonium hydroxide (40% in water, Sigma, St. Louis, MO, USA), 0.05% of 91% formic acid (Sigma, St. Louis, MO, USA), 100 μL of a phytic acid hydrolysate (6 mg/mL), and the pH was adjusted to 4.3 with 5 M of sulphuric acid. The flow rate was 1 mL/min. The quantification was developed by using a calibration curve obtained from sodium phytate (0–5 mg/g, y = 0.144x + 0.016, R² = 0.99) (Sigma, St. Louis, MO, USA). Original HPLC chromatograms of IP3-IP6 of all the analysed samples are presented in supplementary Figure S4.