2.3 Determination of TNBS
The methacrylation degree of GelMA was measured by 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich, USA) assay. GelMA was dissolved in sodium bicarbonate solution (pH = 8.5,0.1 m) to obtain a concentration of 200 μg/ml. 250 μL of 0.01% w/v TNBS reaction solution was added to the sample solution and reacted at 37 ℃ for 2 hours. The reaction was terminated with 250 μL 10% sodium dodecyl sulfate (SDS) and 125 μL 1 N hydrochloric acid (HCl). 100 ml mixture was added into 96 well plate and optical density (OD) was obtained by microplate reader (SpectraMax M5, Molecular, USA) at 335mm. Gelatin was used as a control.
Degree of the methacrylation = (1-OD of GelMA / OD of Gelatin) ×100 (%)