Evaluating EGCNP anti-glycation potential

The ability of EGCNPs to prevent protein glycation was determined by measuring the distinctive fluorescence of advanced glycation end products (AGEs) as previously reported [24 (link)]. Briefly, two solutions of bovine serum albumin (BSA, 10 mg/mL) and D-glucose (10 mg/mL) were separately made in PBS (pH = 7). Sodium azide was added to each solution (0.02%, Sigma Aldrich, Paisley, UK) as a preservative. Equal volumes of BSA and glucose solution were mixed in 2 mL Eppendorf tubes with or without treatments. The treatments were as follows: EGCNPs (50, 100 and 200 µg/mL) and aminoguanidine as an anti-glycating agent (10 and 20 mM). The samples were incubated for 3 days at 40 °C. After the incubation period, aliquots were taken from all samples (100 µL) and placed in the wells of F-bottom black 96-well plates (Greiner). The fluorescence intensity of AGEs was measured at ex/em: 300 ± 9 nm/400 + 20 nm using a plate reader (TECAN infinite 200 PRO, Männedorf, Switzerland). The same steps were repeated using bovine α-crystallin (1 mg/mL, Sigma Aldrich, Paisley, UK) and glucose (5 mg/mL) to check for protection against glycation in a lens protein. All experiments were run in triplicate (n = 3).