Determination of protamine using microtiter plate-format optodes Original Research Article

The microtiter plate-format optodes prepared with an optimized composition of poly(vinyl chloride) (PVC), bis(2-ethylhexyl)sebacate (DOS), dinonylnaphthalene sulfonic acid (DNNS) and 9-(diethylamino)-5-octadecanoylimino-5H-benzo[α]phenoxazine (ETH 5294) exhibited reproducible and sensitive absorbance changes in response to the varying protamine concentrations. Since the variations in absorbance changes in limited sample volume measurement mode are proportional to the initial concentration of protamine, the error tends to propagate with increasing protamine concentrations. However, the imprecision of microtiter plate-format optodes for protamine determination could be minimized by taking the data at a designated time interval (e.g. 10 min). Furthermore, taking the averages of several measurements obtained from one plate, the accuracy of the determination could be greatly improved. This method has been applied for the determination of protamine in plasma samples, and the results were compared with those obtained using a competitive binding displacement mechanism between protamine and heparin-azure A dye complex.