An assay of adenosine 5′-triphosphate (ATP) is important to estimate the total amount of living cellar material (biomass) as well as the activity or metabolic state of the cells, since all types of living cells use ATP for storage of energy produced via metabolic processes. The firefly bioluminescence (BL) method has been widely used for the determination of ATP. In the firefly luciferin-luciferase reaction, luciferase catalyses the oxidative decarboxylation of luciferin in the presence of ATP and Mg 2+ , with concomitant BL:Firefly BL provides a rapid and very sensitive method for the determination of ATP. 1 Recently, the enhancement of light emission has been noted in view of improving the sensitivity of the firefly BL assay for ATP. The presence of apporopriate aqueous micellar media can increase the maximum BL intensity. Aqueous nonionic and zwitterionic micellar media increased the BL peak height intensity as well as prolonged the dulation of the BL.2 In addition, cationic detergents such as benzalkonium chloride increased the firefly luciferin-luciferase reaction rate several-fold, with a sharply defined optimum concentration of detergent. 3 However, the enhancement of light emission in the presence of surfactant micelles was observed only when higher ATP concentrations were used. luciferin+ATP+Mg 2+ +O2 → oxyluciferin+AMP+PPi+CO2+light luciferase Previously, we found that a cationic polysaccharide such as diethylaminoethyl dextran was more effective than other types of charged polysaccharides in enhancing the BL intensity. 4 The detection limit for ATP was improved by a factor of three times compared to that in water alone. In addition, the effect of different chargetypes of liposome on the BL intensity was investigated.5 Cationic liposome containing stearylamine (SA) was superior to anionic and zwitterionic liposomes as an enhancer in the firefly luciferinluciferase BL reaction. The sensitivity for ATP in the presence of cationc liposome containing SA was improved by a factor of five times compared to that in water alone.The aim of this work was to prepare newly cationic liposomes using stearyltrimethylammonium chloride and tetracaine as a cationic component for improving the sensitivity of firefly BL assay for ATP and to clarify the differences in the BL enhancement between cationic components by measuring membrane surface potential of the cationic liposomes. Experimental MaterialsLuciferase from firefly (Photinus pyralis: product number L9009), D-luciferin, The effect of cationic components in liposome on the bioluminescence (BL) intensity from the firefly luciferin-luciferase reaction with ATP was investigated by use of vesicles formed by the extrusion technique (VET). Stearyltrimethylammonium chloride (STAC), stearylamine (SA) and tetracaine (TC) were used as cationic components. The VET containing STAC and SA enhanced the maximum BL intensity. In contrast, a lowering of the maximum BL intensity was observed in the presence of the VET containing TC. The sensitivity for ATP in the presence o...