Zinc chloride

IgG1 protein (Trastuzumab) producing CHO-GS(-/-) cell line was used and was provided by an industrial collaborator (Imgenex®, Bhubaneswar, India). The cell line is suitable for growth in glutamine-free media, once transfected with the vector containing glutamine synthetase gene (GS). A commercially available proprietary cell culture medium CD CHO® (Gibco™—12,490–001) was used as a basal medium, and Efficient Feed B medium (Gibco™—A1245605) as a medium feed supplement for fed-batch culture. Metal salts, copper (II) chloride dihydrate, magnesium sulfate heptahydrate, manganese chloride, iron sulfate heptahydrate, cobalt sulfate heptahydrate, zinc chloride, and nickel (II) chloride hexahydrate were purchased from Merck (Kenilworth, NJ, USA).

Whatman filter papers (N. 42, slow) with a reported mean pore size of 2.5 μm and a thickness of 0.39 mm were chosen as commercial cellulose filter papers. Zinc chloride, silver nitrate solution (0.1 M), sodium hydroxide, calcium hydroxide, ethylene glycol and dopamine hydrochloride were purchased from Merck Chemical Co. Polyethyleneimine (PEI, branched, MW 25,000 Da) was supplied by Sigma Aldrich Chemical Co. Graphene oxide (GO) sheets were bought from Platonic Nanotech Co.

Chemical compounds with their catalog number which used in this studied are DEAE- Cellulose (30477), Olive oil (75343), Sephadex G-50 column (S5897), Tween 20 (P9416), Tween-80 (P8074) and p-NPP (N2752), Sigma-Aldrich, Germany; Dialysis tubing cellulose membrane flat with 10 mm (D9277) and Rhodamine B (83689), Sigma, Germany; Millipore membrane filter 0.22 μm (GSWP02500) and 0.45 μm (HAWP04700), Merck Millipore, Germany; Acetone (100014), Ammonium sulphate (101217), Barium chloride (101716), Benzene (101783), Calcium chloride (102083), Chloroform (107024), Cobalt (II) chloride anhydrous (802540), Cyclohexane (102817), Ethanol (818760), Ethyl Acetate (100789), Hexane (104373), Isopropanol (113350), Magnesium Chloride (814733), Mercury(II) chloride (104417), Methanol (822283), Sodium deoxycholate (106504), Sodium dodecyl sulfate (817034), Toluene (108327), Triton X-100 (112298), Urea (108486) and Zinc chloride (108816), Merck, Germany. No human or animal was used in this research.

The investigation was conducted utilizing damp BCNF sheets obtained from Nano Novin Polymer Co. situated in Sari, Iran. From the local market, the fruits of jambolao were purchased (Bandar Abbas, Iran). The fruits were cleaned by submerging them in a sodium hypochlorite solution (100 g/mL) for 10 min. They were then washed in distilled water and allowed to air dry on perforated trays for two hours at 30 °C. Using stainless steel knives, the pulp skin-the edible portion of jambolao fruits-was separated from the seeds, frozen at -18 °C, and then shielded from light. Metallic salts such as lead nitrate (Pb(NO₃)₂), cobalt nitrate (Co(NO₃)₂), nickel sulfate (NiSO₄), aluminum chloride (AlCl₃), barium chloride (BaCl₂), manganese sulfate (MnSO₄), cadmium chloride (CdCl₂), copper nitrate trihydrate (Cu(NO₃)₂.3H₂O), zinc chloride (ZnCl₂), mercury chloride (HgCl₂), and sodium nitrate (NaNO₃) were provided by Merck. Adequate quantities of these salts were dissolved in deionized water to create stock solutions. Initially, the copper(II) nitrate stock solution was produced at a concentration of 1000 parts per million (ppm), after which gradual dilutions were made (10–1000 ppm). Solutions of acetate buffer were made in the lab by mixing sodium acetate and glacial acetic acid. The other substances (of analytical grade) were supplied by Sigma-Aldrich.

For the optimization of BoNT cleavage, the following reagents were used: Zinc chloride (Merck, Darmstadt, Germany), bovine serum albumin (BSA; Roth, Karlsruhe, Germany), Tween 20 (Merck, Darmstadt, Germany), trimethylamine N-oxide dihydrate (TMAO; Sigma-Aldrich, Taufkirchen, Germany), dithiothreitol (DTT; Roth, Karlsuhe, Germany), NaCl (neoLab Migge, Heidelberg, Germany). Purified botulinum neurotoxin serotypes A, B, C, D, E, and F were all obtained from Metabiologics (Madison, WI, USA). It was previously shown that BoNT/D from Metabiologics is actually the mosaic variant BoNT/DC [57 (link)]. However, this work only depicted the light chain activity, and the light chains of BoNT/D and BoNT/DC are almost identical [73 (link)]. Therefore, following the manufacturer, the designation used for the toxin in this manuscript was BoNT/D. Toxins were handled in a biosafety cabinet in a dedicated toxin laboratory and decontaminated with 5% NaOH for 24 h.

All the chemicals were of analytical reagent grade. Ferric Chloride (FeCl₃.6H₂O), Nickel chloride (NiCl₂.6H₂O), Zinc chloride (ZnCl₂) and Ammonium persulfate (APS) of Merck Germany were used. Sodium Hydroxide Pellets (NaOH), acetone and hydrochloric acid were procured from Fisher Scientific. Aniline, Sodium Hypochlorite and Orange II sodium salt dye were used taken from Sigma Aldrich. The double distillate water was used for experimentation.