Metal salts

Thermal denaturation assays of purified and reconstituted Cgr2 were prepared on ice in 0.2 mL skirted 96-well PCR plates (VWR, Radnor, PA) sealed with optical adhesive covers (Life Technologies, Woburn, MA). Each reaction contained 10 µg of purified or reconstituted Cgr2, Sypro Orange protein gel stain (Thermo Fisher Scientific, Waltham, MA) diluted 5000-fold, and buffer containing 100 mM buffering agent and 100 mM NaCl in a total volume of 30 µL. The following buffering agents were used: acetate/acetic acid for pH 4–6, HEPES for pH 7, Tris-HCl for pH 8–9, and glycine-NaOH for pH 10. For metal binding assays, metal salts (Sigma-Aldrich, St. Louis, MO) were dissolved in pH 8 buffer to generate 100 mM stock solutions and added to a final concentration of 48 µM (8 equivalents relative to Cgr2). Data was collected on a CFX96 Touch Real-Time PCR machine (Bio-Rad, Hercules, CA) using the ‘FRET’ filter setting with FAM excitation and HEX emission channels (485 nm and 556 nm respectively). The following temperature-scan protocol was used: 25°C for 30 s, then ramp from 25°C to 100°C at a rate of 0.1 °C/ s.

Media, sodium chloride, metal salts, H₂SO₄, HCl and NaCl were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Antibiotic discs were from (Oxoid, Limited, Basingstoke, Hampshire, England).

2-Hydroxy-5-methylbenzene-1, 3-dialdehyde, and 2-aminothiophenol were used as starting materials in the synthesis of the ligand. The solvents used in this project, which include ethanol, acetonitrile, and N,N dimethylformamide, are of high purity and manufactured by Merck. Metal salts were also obtained from Sigma-Aldrich. The available IR spectrum was taken by a Jasco FT/IR-4200 instrument. NMR spectra were taken by a Bruker NMR spectrometer (400 MH). All frequencies are in units of 1 cm wave number. UV–Vis spectra were recorded using a Varian Eclipse 300 spectrophotometer. A quartz cell with a path length of 10 mm was used in all experiments.
The Mass spectrometry experiments were performed on a Thermo LXQ ESI-MS with an ion trap mass separator. A solution of synthesized ligand (NC) was made at 1 mg mL⁻¹ in LC/MS grade CH₃CN. After a blank was recorded, the solution containing a ligand was injected into the instrument, and the mass spectra were recorded. The Mass spectrum of the chemosensor was reported in positive ion mode.

The starting chemicals were metal salts, ammonium acetate, hydrazine hydrate, acetyl thiophene, 4-methoxy benzaldehyde, ethyl cyanoacetate, ethyl chloroacetate, and potassium carbonate that were purchased from Sigma Aldrich and utilized without additional purification. Fisher Scientific (Loughborough, UK) supplied the solvents, which were ethanol (99%) and N,N-Dimethylformamide (99%).

Metal salts were obtained from Sigma Chemical Company (St. Louis, U.S.A.) for the various metal(loids): selenite (Na₂SeO₃), tellurite (Na₂TeO₃), vanadate (NaVO₃), chromate (Na₂CrO₄), arsenite (NaAsO₂), cadmium (CdCl₂), and mercury (HgCl₂) were diluted in sterile water at twice the highest concentration to be tested (Turner et al., 2001 (link)). Metal solutions were passed through a 0.22 μm syringe into sterile vials and stored at room temperature.