Arrayed Lipid Membranes on Femtoliter Chambers Allow Highly Sensitive Detection of Ion Translocation Catalyzed by Transporter Protein

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Femtoliter reaction chamber array enables highly sensitive and quantitative biological assays such as single-molecule enzymatic assay, and digital PCR. Although femtoliter chamber arrays are very powerful for protein science as well as for biomedical applications, most of them have been in general limited to use for water-soluble proteins, due to the technical difficulties in preparing uniform and stable lipid bilayers. Here, we report an arrayed lipid bilayer chamber system (ALBiC) that displays a sub-million of femtoliter chambers, each equipped with micron-size electrodes, and sealed with a stable lipid bilayer membrane with extremely high efficiency of over 90 %. When reconstituted an ion transporter, FoF1-ATP synthase (FoF1), into the bilayers of ALBiC, proton translocation driven by catalysis or membrane voltage were observed, showing that the highly sensitive detection of ion translocation is performed in ALBiC. Next, to explore the feasibility of single-molecule detection of transporter activity, we conducted the same assay in a condition where only a few molecules (0, 1, or 2 molecules) of FoF1 were reconstituted into each bilayer. The results showed that the response to proton translocation was no longer homogeneous between chambers, i.e. stochastic and quantized proton translocation was observed, demonstrating that the single molecule analysis of ion translocation catalyzed by transporter protein is first achieved in this study. Thus, the new platform, ALBiC, largely extended the versatility of femtoliter chamber arrays, and holds promise for understanding the working mechanism of transporter proteins as well as for further analytical and pharmacological applications.