Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells. 2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo. 5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded. 7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations. 6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes. 11 Additionally, they have also tracked realtime changes in sodium concentrations in vivo when injected subcutaneously into mice. 3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.
Video LinkThe video component of this article can be found at https://www.jove.com/video/2896/ Protocol
Preparation of optodeBefore making the optode, aliquots of the components are need so that they can be easily measured and stored.1. A 50 mg Sodium Ionophore X (NaIX) vial and a 50 mg sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB) will each be brought up in tetrahyrdofuran (THF) and aliquoted into 1.5 ml polystyrene centrifuge tubes. In a chemical fume hood, dissolve the 50 mg of NaIX in 1 ml of THF in the shipping vial and mix to ensure that the solid has completely dissolved. Transfer 100 μl of this solution into 10 separate centrifuge tubes. Repeat for NaTFPB. Allow the THF to evaporate in a chemical fume hood overnight, label the centrifuge tubes and place them in a 4 degree refrigerator for storage. This creates 5 mg aliquots of dry NaIX and NaTFPB. 2. Dissolve the Chromoionophore III (CHIII) in 1 ml of THF and transfer to a 3 ml glass vial. Add another 1 ml of THF to the CHIII shipping vial to dissolve any residual CHIII and add this to the 3 ml glas...