Figure 3.

Determination of optimal conditions for the enzymatic digestion of Red BSA. The proteinase K concentration was varied from 0–10 units/mL with a 30 min reaction (A). The dose-response curves obtained showed that although little fluorescent signal was observed in the absence of protease (no protease), the signal at a constant cTnI dose increased with increasing protease concentration from 0.1 to 10 units/mL. Using the optimum protease concentration of 10 units/mL the detection limit was ~0.19 ng/mL cTnI. The digestion time was then optimized in the range 0–120 min (B). The fluorescent signal measured at a constant analyte dose increased over time, and was significantly enhanced at 25 ng/mL cTnI when the reaction time was ≥30 min. Therefore, digestion was carried out using 10 units/mL proteinase K for 30 min.