Copper 2 sulfate

Materials used in this research were of high quality such as BTC (1, 3, 5-benzene tri-carboxylic acid, 98%), Copper (II) nitrate (Cu(NO₃)₂·3H₂O, 99%), Copper (II) sulfate (Cu(SO₄)₂·3H₂O, 99%), phosphate buffer saline (PBS), NaHCO₃, Cu plate, NaOH, H₂SO₄, melamine and PVDF (Polyvinyldifluoridone) as purchased from Merck. The solvent used in the preparation of materials was NMP (N-methyl pyrrolidone), DMF(N, N-dimethylformamide), and DI (deionized) water.

These study bacteria isolates were subjected to heavy metals (mercury Hg²⁺, chromium Cr⁶⁺, zinc Zn²⁺, and copper Cu²⁺) tolerance assay through a series of two-fold agar dilutions method. The bacterial suspensions were prepared as described at a concentration of 10⁹ CFU. The bacterial suspensions were then spread onto the trypticase soy agar medium that incorporated with mercury II chloride, potassium dichromate, zinc sulfate, and copper II sulfate (Merck, Germany) in five different concentrations each. The concentrations for Cr⁶⁺ and Zn²⁺ were ranging from 25 to 400 μg/mL while the two-fold concentrations of Hg²⁺ and Cu²⁺ were ranging from 2.5 to 40 μg/mL and 150 to 2400 μg/mL, respectively. The inoculated media was incubated for 24 h at room temperature. The bacteria were considered resistant to the tested heavy metals if they grow at concentrations of 10 μg/mL Hg²⁺, 100 μg/mL for Zn²⁺ and Cr⁶⁺, and 600 μg/mLCu²⁺. The operational definition of tolerance as used in this study was based on positive bacterial growth when the concentration of heavy metals was above the stated concentration for resistance [10 (link)-12 ].

Palm kernel shell (PKS) as the precursor for CDs synthesis was obtained from Tennamaram palm oil mill located at Bestari Jaya, Selangor, Malaysia. Diethylene glycol (DEG) was purchased from Labchem Sdn. Bhd. (Malaysia). Chitosan and quinine sulfate was purchased from Sigma-Aldrich (M) Sdn. Bhd. (Malaysia). Ultrapure water (UPW) (~ 18.2 MΩ m, 25 °C) obtained from Sartorius Arium PRO (Fisher Scientific, USA) water purification system was used throughout the experiment. Copper (II) sulfate and phosphate buffer solutions (PBS) were purchased from Merck, Malaysia.

Urea (99%), citric acid (99.5%), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) (99%), N-hydroxysuccinimide (NHS) (98%), ethanol (99.9%), phosphate buffered saline (PBS) pH 7.4, anhydrous N,N-dimethylformamide (DMF), rhodamine (99.5%), copper (II) sulfate (99%), ascorbic acid (99.5%), azide-PEG300-biotin, irinotecan hydrochloride (IT), 4-Pentynoic acid, and Sephadex G10, G15, and G25, dialysis tubing MWCO 2 kDa were purchased from Sigma Aldrich and used as received. NH2-PEG2000-CC was obtained as previously described [29 (link)].
MCF-7 and MDA-MB-231 cell lines were purchased from Sigma Aldrich and cultured in supplemented Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, EuroClone, Milan, Italy), 1% of penicillin/streptomycin (10,000 U mL⁻¹ and 10 mg mL⁻¹ respectively, EuroClone), and 1% of l-glutamine (EuroClone), at 37 °C in 5% CO₂ humidified atmosphere. Cell Titer 96 Aqueous One Solution Cell Proliferation assay (MTS solution) was purchased from Promega (Madison, WI, USA).

A bovine serum albumin (BSA) solution at 1 mg/mL in PBS was used to prepare a protein standard curve ranging from 0 to 650 µg/mL. A total 10 µL of OMVs’ suspension was mixed with 2 µL of 1.5% Triton X100 (Sigma, St. Louis, MI, United States) and 13 µL of PBS. The mix was vortexed and incubated at room temperature for 5 min in order to solubilize the OMVs’ membrane. A mix constituted of 50 parts of bicinchoninic acid (BCA) (Sigma) and 1 part of copper II sulfate (Sigma) was prepared extemporaneously, and 200 µL of this mix were added to each well of a transparent, flat-bottom 96-well plate (Corning, Glendale, CA, United States). A total of 25 µL of a BSA standard solution or the OMV preparations were added in wells containing the BCA/Copper II mix. After incubation at 37 °C for 30 min to allow the development of the purple color, the absorbance of each well was measured at 536 nm using a Varioskan spectrophotometer (Thermofisher, Waltham, MA, United States). All the standard and sample solutions were prepared in triplicates.