Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers [1][2][3][4][5] to form networks [4][5][6] . Droplet interface bilayers have been used for rapid screening of membrane proteins 7,8 and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores 9 . Networks, meanwhile, have been used to form microscale batteries and to detect light 4 . Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.To obtain directional ionic current flows in droplet networks ( Fig. 1), we constructed a diode-like pore from staphylococcal a-haemolysin (aHL). aHL forms a heptameric protein pore 10 that inserts vectorially into lipid bilayers 11 . The crystal structure of the pore reveals a 14-stranded transmembrane b barrel capped by an extramembraneous domain, which contains a roughly spherical cavity 10 ( Fig. 2a, left). The wild-type (WT) pore is a 'blank slate' for protein engineering with properties similar to those of an electrolyte-filled tube; it is weakly rectifying and weakly anion selective and gates only at extreme applied potentials of either polarity 12 .aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials. Therefore, in an attempt to obtain a fully rectifying pore, we tested an extreme version of aHL in which seven residues were replaced with arginines (7R-aHL) to yield a heptameric pore in which 49 additional positively charged side chains were located within the barrel (Fig. 2a, right). In 1 M KCl, 25 mM Tris HCl at pH 8.0, 100 mM, in planar lipid bilayers, the 7R-aHL pore has a unitary conductance of 0.95 + 0.01 nS (þ50 mV, n ¼ 8). The conductance of the WT pore under the same conditions is similar (0.99 + 0.02 nS, n ¼ 4), which suggests, surprisingly, that the drastically altered 7R-aHL pore is properly formed. The current-voltage (I-V) characteristics of 7R-aHL in 1 M KCl, however, showed virtually complete current rectification (Fig. 2b,c). At positive applied potentials, 7R-aHL remained in an open form with a stable steady-state current and infrequent short-lived closures of less than 10 ms. By contrast, at negative applied potentials, the pore was closed, with occasional brief current spikes ascriba...
