Naoki Soga

Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function.

High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition

We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters

We present an attolitre-sized arrayed lipid bilayer chamber system (aL-ALBiC) for rapid and massively parallel single-molecule assay of membrane transporter activity. Because of the small reaction volume (200 aL), the aL-ALBiC performed fast detection of single transporter activity, thereby enhancing the sensitivity, throughput, and accuracy of the analysis. Thus, aL-ALBiC broadens the opportunities for single-molecule analysis of various membrane transporters and can be used in pharmaceutical applications such as drug screening.

Novel Nano-Device to Measure Voltage-Driven Membrane Transporter Activity

The use of an arrayed lipid bilayer chamber system (ALBiC) enables highly sensitive quantitative analysis of membrane transporter activity, a major target of pharmaceutical research. Although membrane voltage is one of the main driving forces of transporters, the versatility of ALBiC is limited to transporter assays in the absence of membrane voltage, owing to technical limitations with voltage modulation. Here, we report a novel nano-device based on ALBiC (el-ALBiC) containing sub-million lipid bilayer chambers, each equipped with nano-sized electrodes. Since the nano-sized electrodes enable quantitative modulation of membrane voltage, the el-ALBiC is capable of performing highly sensitive detection of the voltage-driven membrane transporter activity. Thus, the novel nano-device el-ALBiC extends the versatility of ALBiC and has potential for further analytical and pharmacological applications, such as drug screening.

Single-Molecule Analysis of Membrane Transporter Activity by Means of a Microsystem

Emerging microtechnologies are aimed at developing a microsystem with densely packed array structure, i.e., an array with a femtoliter reaction chamber, for highly sensitive and quantitative biological assays. Here, we describe a novel femtoliter chamber array system (arrayed lipid bilayer chambers, ALBiC) that contains approximately a million femtoliter chambers, each sealed with a phospholipid bilayer membrane with extremely high efficiency (>90%). This novel platform enables detection of membrane transporter activity at the single-molecule level and thus expands the applicability of femtoliter chamber arrays to highly sensitive assays of transporters.