D glucose glu

Cobalt (II) sulfate heptahydrate (CoSO₄·7H₂O), sodium phosphate dibasic (Na₂HPO₄), sodium phosphate monobasic (NaH₂PO₄), nickel (II) sulfate hexahydrate (NiSO₄·6H₂O), and sodium hypophosphite monohydrate (NaH₂PO₂·H₂O) were purchased from Alfa Aesar (Ward Hill, MA, USA). Glycerol and anhydrous ethanol (C₂H₅OH, 99.9%) were purchased from J.T. Baker (Phillipsburg, NJ, USA). Hydrogen peroxide (H₂O₂), urea, Nafion solution (5 wt% in mixture of lower aliphatic alcohols and water), uric acid (UA), L-Ascorbic acid (AA), dopamine hydrochloride (DA), D-(+)-Glucose (Glu), and sodium chloride (NaCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The deionized water (DI water) was produced from a Milli-Q water purification system (Millipore, Milford, MA, USA). All chemicals were analytical grade and were used as received without further purification.

The human neuroblastoma cell line SH-SY5Y was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) containing 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate; 10% fetal bovine serum, and penicillin and streptomycin at 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and kept at 37 °C in a humidified 5% CO₂ incubator. SH-SY5Y cells were obtained from ATCC, and all experiments were carried out up to 12 passages. In total, 80,000 cells/well were seeded on 12-well tissue culture plates. After 24 h, cells were treated with different concentrations of D-Glucose (GLU, Sigma-Aldrich, St. Louis, MO, USA) (75, 100, 150, 200, 250, and 300 mM) for 24, 48, and 72 h to find out the maximum concentration that induce cytotoxicity. At the same time, different concentrations of ammonium glycyrrhizate (AG, Sigma-Aldrich, St. Louis, MO, USA) (200 μg/mL) were used to select the concentration able to counteract the effects of high glucose. As control, we used cells treated with mannitol (MAN, Sigma-Aldrich, St. Louis, MO, USA), an osmotic sugar alcohol that is metabolically inert in humans, at the same concentration as GLU.