Zncl2

Human Carbonic Anhydrase II (hCAII) was expressed and purified according to a protocol by Gaspari et al. [25 (link)]. with additional 60 µM ZnCl₂ (Sigma Aldrich, St. Louis, MO, USA) in the overnight culture and during cell growth, while protein expression was carried out with 1 mm ZnCl₂, based on the work of Cimmperman et al. [26 (link)]. Protein material was dialyzed against 10 mm HEPES (Carl Roth, Karlsruhe, Germany) buffer at pH 7.8 at 26 °C after the final purification step. The dialysis buffer was filtrated through a Thermo Scientific Nalgene Rapid-Flow PES Bottle Top Filter with a pore size of 0.2 µm (Thermo Fisher Scientific, Waltham, MA, USA) and used for the measurements. Dialyzed protein was aliquoted and stored at −80 °C.

A sterile 1% zinc chloride (ZnCl₂) (Sigma-Aldrich, St. Louis, MO, USA) solution was added to each M. aeruginosa UTEX LB 2385 culture to yield relative ZnCl₂ concentrations: 0 mg/L, 0.1 mg/L, 0.25 mg/L, and 0.5 mg/L (0 µM, 0.734 µM, 1.835 µM, 3.669 µM, respectively), as described previously [50 (link),70 (link)]. These ZnCl₂ concentrations were targeted through multiple growth curve analysis from past experiments, with concentrations as high as 10 mg/L ZnCl₂ (data not shown). The rationale was predicated on a previously studied cyanobacterium, Synechococcus sp. IU 625 with ZnCl₂. All ZnCl₂-treated cultures were maintained at the same culture growth parameters described above and were monitored by turbidity observation at optical density (O.D._{750 nm}) with an UltraSpec III (Pharmacia LKB—Pfizer, New York, NY, USA) for a predetermined time course of Days: 0, 1, 5, 8, 12, and 15. All M. aeruginosa UTEX LB 2385 culture cell counts were performed via hemocytometer at 400× magnification using an Olympus BH2 BHS-312 Trinocular Microscope (Olympus Corp, Waltham, MA, USA), and all experiments were repeated in triplicates. The standard deviations of duplicate set means were used to generate the growth curves.

Zn was given to the rats in drinking water as zinc chloride (ZnCl₂; Merck, Darmstadt, Germany) at the concentration of 30 or 60 mg Zn/L. The aqueous solutions containing 30 or 60 mg Zn/L were prepared by appropriate dilution, with redistilled water, of the solution of 1000 mg Zn/L (made up by dissolving a certain amount of ZnCl₂ in redistilled water). In the case of Zn administration under the exposure to Cd, the solutions containing together Cd (5 or 50 mg/L) and Zn (30 or 60 mg/L) were prepared using the 1000 mg Cd/L and 1000 mg Zn/L solutions and redistilled water for their appropriate dilution.

All chemicals for the preparation of NPs were of regent grade and were used without further purification. ZnCl₂ (12.8 mmol, Aldrich) and SnCl₄·5H₂O (6.4 mmol, Aldrich) were dissolved in deionized water (160 ml) under vigorous magnetic stirring. N₂H₄·H₂O (N₂H₄ molar ratio/Zn=4/1, 8/1, 24/1) was then added to the reaction solution. White precipitates formed immediately, and this solution, including the precipitate, was heated on a hot plate at 90 °C for 12 h. The obtained products were thoroughly washed with deionized water and ethanol and were then dispersed in 2-methoxy ethanol, resulting in a colloidal solution.

In a typical synthesis of zinc oxide nanorods, 0.3 M aqueous solutions of metal salts (ZnCl₂, Aldrich 99%) was prepared, then 0.8 M sodium hydroxide was added dropwise under stirring up to complete precipitation of zinc hydroxide. The resulting precipitate was irradiated with household microwave for 15 minutes followed by filtration, washing and drying at 80 °C overnight.

ZnO NRs were synthesized using a potentiodynamic electrodeposition process. The electrodeposition was carried out with a three-electrode system (Solartron 1280z). The working electrode was Cu foil (99.8 at%, Nippon Foil Mfg, Co.) with an exposed area of 1.5 × 1.5 cm². Pt wire (Aldrich) and KCl–saturated Ag/AgCl were used as the counter and reference electrodes, respectively. The electrolyte was composed of 100 μM ZnCl₂ (Aldrich) in deionized water and set at 40°C. The pH and conductivity of the electrolyte were 5.5 and 24 μS/cm, respectively. In the potentiodynamic mode, the reduction potential (V_R) and oxidation potential (V_O) were set to be −8 V and +0.5 V. The duty and frequency of the potentiodynamic mode were 50% and 1 Hz, respectively. For the deposition of Ni nanoparticles on the surface of ZnO NRs, we conducted electroless Ni plating in an aqueous solution of 95 mM NiSO₄·6H₂O and 284 mM NaPH₂O₂. The pH value of the solution was set to be 6.0 by adding 21.9 mM NaOH. Commercial sPVDF (Kynar HSV 900) nanoparticles were used. The sPVDF nanoparticles were dispersed in 15 wt% acetone and several spin-coatings were conducted until they were completely infiltrated between the NRs.