In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 +/- 0.06 in units of mol fraction, implying that the nonannular sites will only be approximately 70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from approximately 2.5% in the presence of 25 mol % phosphatidylglycerol to approximately 62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K(+) close to the membrane surface.

The fabrication of ordered nanoelectrode arrays using both electron-beam lithography and nanoimprint lithography is described. Arrays of nanoelectrodes with varying individual electrode diameters were produced and characterised electrochemically. Whilst both methods are highly reproducibile, nanoimprint lithography has the potential to produce devices rapidly and at low-cost. To our knowledge, this is the first report where nanoimprint lithography is employed for the production of nanoelectrode arrays for electroanalytical sensors.

The diversity of mitochondrial arrangements, which arise from the organelle being static or moving, or fusing and dividing in a dynamically reshaping network, is only beginning to be appreciated. While significant progress has been made in understanding the proteins that reorganise mitochondria, the physiological significance of the various arrangements is poorly understood. The lack of understanding may occur partly because mitochondrial morphology is studied most often in cultured cells. The simple anatomy of cultured cells presents an attractive model for visualizing mitochondrial behaviour but contrasts with the complexity of native cells in which elaborate mitochondrial movements and morphologies may not occur. Mitochondrial changes may take place in native cells (in response to stress and proliferation), but over a slow time-course and the cellular function contributed is unclear. To determine the role mitochondrial arrangements play in cell function, a crucial first step is characterisation of the interactions among mitochondrial components. Three aspects of mitochondrial behaviour are described in this review: (1) morphology, (2) motion and (3) rapid shape changes. The proposed physiological roles to which various mitochondrial arrangements contribute and difficulties in interpreting some of the physiological conclusions are also outlined.

We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.Artificial BLM techniques are of great importance in membrane protein research. Electrophysiological studies of proteins reconstituted into BLMs can generate detailed information at the single molecule level on protein activity, ligand-binding and kinetics. 1 Classical BLM techniques have been in use for over 40 years, but are not amenable to applications where high-throughput processes and reproducibility are required (e.g. drug screening and biosensing applications). 2 New labon-chip technologies are being developed which allow for ever greater integration of complex functions with precise control of fluids on the micro-scale. 3 This technology can be exploited to combine parallelization, achieved through microfluidics, with the sensitivity and selectivity of BLM approaches. Although various miniaturized devices have been reported, only a few BLM formation methods 4-7 have been developed on-chip that are reproducible, potentially automatable and suitable for singlechannel recording.An alternative technique was recently described by Holden et al.,[8][9][10][11][12] which uses an alkane-lipid solution as the bulk phase, as opposed to an aqueous electrolyte. Droplets of buffer are immersed in the organic solution and at the interface between the two phases lipid monolayers form. When two droplets are brought into contact, a BLM forms at the interface between the droplets. As well as forming single BLMs, this approach allows for the creation of BLM networks. However, the method required manual manipulation of the droplets using micromanipulators.Electrical methods for droplet manipulation exist, based on electrodynamic techniques, such as dielectrophoresis (DEP) and electro-wetting on dielectric (EWOD).

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This paper describes the systematic investigation of a range of microelectrode arrays with varying dimensions fabricated by standard photolithographic and reactive-ion etching techniques. As expected from theory, the electrochemical behavior of microelectrode arrays with a constant individual diameter varied strongly with center-to-center spacing, the larger the spacing the more sigmoidal the recorded voltammogram. Furthermore, the behavior of arrays with a constant relative center-to-center spacing is shown to vary with individual electrode diameter, the arrays with the smallest electrodes producing strongly peaked voltammograms. Peak current densities and signal-to-noise ratios were also obtained for a variety of array geometries, and the use of electrodeposited platinum black electrodes was investigated. To demonstrate one advantage of using a loosely packed microelectrode array in electroanalysis, a ferrocene-mediated enzyme-linked assay involving the biocatalytic reduction of H2O2 was investigated. Results showed an improved temporal response, with current-time transients reaching a steady-state response more quickly using arrays with increased center-center spacings.

To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.