Ultrasensitive Diamond Microelectrode Application in the Detection of Ca²⁺ Transport by AnnexinA5-Containing...

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca²⁺ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca²⁺ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca²⁺ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca²⁺ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca²⁺. The ion transport appears Ca²⁺-selective, since dispensing proteoliposomes in the presence of Mg²⁺ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca²⁺, thus confirming that the potential reduction was not only due to the binding of Ca²⁺ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca²⁺ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca²⁺ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.