Shadow electrochemiluminescence imaging of giant liposomes opening at polarized electrodes.

In this work, the release of giant liposome (∼100 µm in diameter) content was imaged by shadow electrochemiluminescence (ECL) microscopy. Giant unilamellar liposomes were pre-loaded with a sucrose solution and allowed to sediment at an ITO electrode surface immersed in a solution containing a luminophore ([Ru(bpy)3]2+) and a sacrificial co-reactant (tri-n-propylamine). Upon polarization, the electrode exhibited illumination over its entire surface thanks to the oxidation of ECL reagents. However, as soon as liposomes reached the electrode surface, dark spots appeared and then spread over time on the surface. This observation reflected a blockage of the electrode surface at the contact point between the liposome and the electrode surface, followed by the dilution of ECL reagents after the rupture of the liposome membrane and release of its internal ECL-inactive solution. Interestingly, ECL reappeared in areas where it initially faded, indicating back-diffusion of ECL reagents towards the previously diluted area and thus confirming liposome permeabilization. The whole process was analyzed qualitatively and quantitatively within the defined region of interest. Two mass transport regimes were identified: a gravity-driven spreading process when the liposome releases its content leading to ECL vanishing and a diffusive regime when ECL recovers. The reported shadow ECL microscopy should find promising applications for the imaging of transient events such as molecular species released by artificial or biological vesicles.

Electrochemiluminescent imaging of a NADH-based enzymatic reaction confined within giant liposomes.

Herein, transient releases either from NADH-loaded liposomes or enzymatic reactions confined in giant liposomes were imaged by electrochemiluminescence (ECL). NADH was first encapsulated with the [Ru(bpy)3]2+ luminophore inside giant liposomes (around 100 µm in diameter) made of DOPC/DOPG phospholipids (i.e., 1,2-dioleolyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycerol-3-phospho-(1'-rac-glycerol) sodium salt) on their inner- and outer-leaflet, respectively. Then, membrane permeabilization triggered upon contact between the liposome and a polarized ITO electrode surface and ECL was locally generated. Combination of amperometry, photoluminescence, and ECL provided a comprehensive monitoring of a single liposome opening and content release. In a second part, the work is focused on the ECL characterization of NADH produced by glucose dehydrogenase (GDH)-catalyzed oxidation of glucose in the confined environment delimited by the liposome membrane. This was achieved by encapsulating both the ECL and catalytic reagents (i.e., the GDH, glucose, NAD+, and [Ru(bpy)3]2+) in the liposome. In accordance with the results obtained, NADH can be used as a biologically compatible ECL co-reactant to image membrane permeabilization events of giant liposomes. Under these conditions, the ECL signal duration was rather long (around 10 s). Since many enzymatic reactions involve the NADH/NAD+ redox couple, this work opens up interesting prospects for the characterization of enzymatic reactions taking place notably in artificial cells and in confined environments.

Dynamic Electrochemiluminescence Imaging of Single Giant Liposome Opening at Polarized Electrodes.

In this work, the characterization of release events from liposomes has been addressed quantitatively by an electrochemiluminescence (ECL) imaging strategy. First, ECL reagents ([Ru(bpy)3]2+ and tripropylamine) were encapsulated in sealed giant asymmetrical liposomes (100 µm in diameter) made of DOPG/DOPC phospholipids. After sedimentation on an indium tin oxide electrode material, the opening of liposomes was triggered by polarization of the surface. Under these conditions, amperometry, epifluorescence imaging, and ECL imaging were combined and synchronized to monitor and image the rupture of giant liposomes during the release and subsequent ECL emission of their redox content. Amperometry allowed the quantification of the content released from single liposomes. The location and status of liposomes (closed or opened) were assessed by epifluorescence imaging. ECL provided the image of the efflux of matter after liposome opening. This original ECL imaging approach favorably compares with strictly photoluminescent or electrochemical techniques and appears to be adapted for the investigation of membrane rupture/permeation events.