Determination of protamine using microtiter plate-format optodes

The microtiter plate-format optodes prepared with an optimized composition of poly(vinyl chloride) (PVC), bis(2-ethylhexyl)sebacate (DOS), dinonylnaph thalene sulfonic acid (DNNS) and 9-(diethylamino)-5-octadecanoylimino-5H-be nzo[alpha]-phenoxazine (ETH 5294) exhibited reproducible and sensitive abso rbance changes in response to the varying protamine concentrations. Since t he variations in absorbance changes in limited sample volume measurement mo de are proportional to the initial concentration of protamine, the error te nds to propagate with increasing protamine concentrations. However, the imp recision of microtiter plate-format optodes for protamine determination cou ld be minimized by taking the data at a designated time interval (e.g. 10 m in). Furthermore, taking the averages of several measurements obtained from one plate, the accuracy of the determination could be greatly improved. Th is method has been applied for the determination of protamine in plasma sam ples, and the results were compared with those obtained using a competitive binding displacement mechanism between protamine and heparin-azure A dye c omplex. (C) 2001 Elsevier Science B.V. All rights reserved.