Selective Leaflet-Anchored DNA Nanoprobes for Simultaneous Monitoring of Juxta-Plasma Membrane Environments

The cell membrane functions as a bidirectional interface that coordinates the selective transport of substances and information between the interior and exterior of the cell. Simultaneous monitoring of both the inner and outer local environments surrounding this lipid bilayer is crucial for elucidating various cellular activities but significantly challenged by the lack of technologies capable of precisely engineering biosensing probes on both membrane leaflets. In this work, by developing fusogenic nanoliposomes with high cell fusion efficiency, we successfully anchored amphiphilic DNA tetrahedral probes onto the inner leaflet of the plasma membrane. By integrating this with the direct anchoring of amphiphilic probes on the outer leaflet, we achieved precise functionalization of both leaflets of the cell membrane, thus enabling simultaneous monitoring of localized targets within their respective juxta-plasma membrane environments, avoiding signal contamination caused by the optical diffraction limit. Using selectively dual-leaflet-anchored tetrahedral DNAzyme probes, we revealed that the transmembrane ion channel SLC41A1 synergistically modulated the influx of Na⁺ and the efflux of Mg²⁺ in live cells. With a modular design, this membrane-anchored DNA nanoplatform can be readily extended for the study of bilateral interface-dominant cellular processes, shifting the paradigm toward a more localized and subtle perspective.