We introduce a nanofabricated silicon chip for massively multiplexed analysis of membrane channels and transporters in suspended lipid membranes that does not require any surface modification or organic solvent. Transport processes through single membrane complexes are monitored by fluorescence. The chip consists of an array of well-defined nanopores, addressing an individual pyramidal back-reflecting 30-fL compartment. The setup allows simultaneous analyses of ∼1,000 single transmembrane events in one field of view, observing translocation kinetics of transmembrane complexes.

Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250 000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.

We present the fabrication and characterization of nanopore microcavities for the investigation of transport processes in suspended lipid membranes. The cavities are situated below the surface of silicon-on-insulator (SOI) substrates. Single cavities and large area arrays were prepared using high resolution electron-beam lithography in combination with reactive ion etching (RIE) and wet chemical sacrificial underetching. The locally separated compartments have a circular shape and allow the enclosure of picoliter volume aqueous solutions. They are sealed at their top by a 250 nm thin Si membrane featuring pores with diameters from 2 µm down to 220 nm. The Si surface exhibits excellent smoothness and homogeneity as verified by AFM analysis. As biophysical test system we deposited lipid membranes by vesicle fusion, and demonstrated their fluid-like properties by fluorescence recovery after photobleaching. As clearly indicated by AFM measurements in aqueous buffer solution, intact lipid membranes successfully spanned the pores. The nanopore cavity arrays have potential applications in diagnostics and pharmaceutical research on transmembrane proteins.

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