In the second part of this study, we examined the role of the organic moiety in the assisted transport of carboxylates. To do this, we used a chloride ion-selective electrode to measure the efflux of Cl -due to Cl -/RCOO -antiport exchange (Cl-ISE assay, Figure , Table ). The Cl-ISE assay was chosen because it allowed us to focus on the transport of carboxylates assisted by a synthetic transporter, excluding contributions from the diffusion of the carboxylic acid. In addition, the Cl-ISE method enables the direct comparison of various carboxylates. In contrast, the different carboxylates induce different levels of quenching of lucigenin (Figures and). For these experiments, transporter T1 (1,1'-(1,2-phenylene)bis(3-(3,5-bis(trifluoromethyl) phenyl)urea)) was selected as it is readily synthesised and similar structures were previously reported to transport carboxylates. Accordingly, liposomes were loaded with 488 mM NaCl, 5 mM HEPES, pH 7 and suspended in a solution of 225 mM Na2SO4, 5 mM HEPES, pH 7. Then, T1 in methanol was post-incorporated to 3 mL of liposome solution with 1 mM lipids in a 1:1000 ratio (transporter:lipid), and the chloride efflux was monitored for 2 minutes. After that, the corresponding carboxylate pulse was added (25 mM), and the chloride efflux was measured over 4 minutes. Finally, liposomes were lysed with Triton X-100 solution to determine the 100% chloride efflux level. The Cl-ISE experiments showed that T1 is highly efficient in transporting aliphatic monocarboxylates (Figure ), including those with carboxylic acid or hydroxyl functional groups (Figure ). These results indicated that a polar functional group did not significantly impact the transport of aliphatic carboxylates. However, dicarboxylates such as malonate, succinate, and terephthalate, resulted in low chloride efflux measurements, highlighting the challenge of transporting multiply charged anions (Figure ). On the other hand, aromatic carboxylates gave large differences in transport. Interestingly, increasing the size of the aromatic rings from benzoate to 2-naphthoate and anthracene-9-carboxylate did not result in significant variations in the chloride efflux, despite the increase in lipophilicity (Figure and Table ). However, aromatic carboxylates with a hydroxyl group showed significantly higher Cl -/RCOO - exchange rates than their unfunctionalised analogues (Figures and).