Yutaro Tsuji

Automated Parallel Recordings of Topologically Identified Single Ion Channels

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Droplet Split-and-Contact Method for High-Throughput Transmembrane Electrical Recording

This paper describes the rapid and repetitive formation of planar lipid bilayers via a mechanical droplet contact method for high-throughput ion channel analysis. In this method, first, an aqueous droplet delivered in a lipid-in-oil solution is mechanically divided into two small droplets. Second, the two small droplets contact each other, resulting in the lipid bilayer formation. Third, an ion channel is immediately reconstituted into the bilayer and the transmembrane current signals are measured. By repeating this procedure, massive data sets of the channel signals can be obtained. This method allowed us to perform statistical analysis of α-hemolysin conductance (n = 256 within 30 min) and channel inhibition experiments by contacting different types of the droplets in a short time frame.

Droplet-based lipid bilayer system integrated with microfluidic channels for solution exchange

This paper proposes a solution exchange of a droplet-based lipid bilayer system, in which the inner solution of a droplet is replaced for the purpose of efficient ion channel analyses. In our previous report, we successfully recorded the channel conductance of alpha-hemolysin in a bilayer lipid membrane using a droplet contact method that can create a spontaneous lipid bilayer at the interface of contacting droplets; this method is widely used as highly efficient method for preparing planar lipid membranes. When only pipetting droplets of the solution, this method is highly efficient for preparing lipid membranes. However, the drawback of droplet-based systems is their inability to exchange the solution within the droplets. To study the effect of inhibitors and promoters of ion channels in drug discovery, it would be beneficial to conduct a solution exchange of droplets to introduce membrane proteins and to apply or wash-out the chemicals. In this study, we propose a droplet contact method that allows for the solution exchange of droplets via microfluidic channels. We experimentally and numerically investigated the bilayer stability with respect to exchanging flow rates, and then demonstrated a binding assay of an alpha-hemolysin using one of its blockers. The solution exchange in this system was conducted in less than 20 s without rupturing the membrane. We believe that the proposed system will enhance the efficiency of ion channel analyses.

A portable lipid bilayer system for environmental sensing with a transmembrane protein

This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin) was achieved in the field at a high-altitude (∼3623 m). This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.

Logic gate operation by DNA translocation through biological nanopores

Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs “1” and “0” as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND) operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.

Logic gate using artificial cell-membrane: NAND operation by transmembrane DNA via a biological nanopore

This paper describes microfluidic logic gates which use DNA and biological nanopores. Single-stranded DNA (ssDNA) can pass through αHL, a biological nanopore, incorporated in bilayer lipid membranes (BLMs), whereas double-stranded DNA (dsDNA) cannot. In this study, these passing and non-passing phenomena were applied as the binary system and logic gates. Two types of ssDNA were used as inputs, while the output was obtained by electrical signals across the nanopores, which recognizes whether ssDNA passed through the nanopores or not. NAND gate was successfully demonstrated by exploiting the mechanism. The proposed approach herein is significantly different from the conventional computation using DNA in the respect that electrical signals are directly obtained as the output, which drastically facilities the microfluidic system to connect to electrical systems for fast and accurate computing. In addition, it is not required to use fluorescence, enzyme or PCR in order to obtain outputs. We believe that this method leads to a rapid computing system using biomolecules.

Split-and-contact device to form planar lipid bilayers

This paper describes a method to form bilayer lipid membranes by splitting, sliding and contacting water droplets in oil containing lipid. A droplet surrounded by a lipid monolayer, which is easily formed by injecting an aqueous droplet into an organic solvent that contains lipids, is split and then, brought into contact each other. A bilayer lipid membrane is immediately formed at the interface of the split droplets. These processes are conducted on a micro device which we termed as split-and-contact device. The aqueous droplet with a lipid monolayer is split mechanically by either spinning or sliding motions of the device. We experimentally verified the effectiveness of the proposed split-and-contact device by using α-hemolysin and its blockers (single-stranded DNA and hepta-6-sulfato β-cyclodextrin). This device can be readily applicable to highly efficient biological sensor or drug discovery.

Easy and stable lipid bilayer formation: A droplets-contacting-method in parylene mircopores for multiple ion channel recordings

This paper describes simultaneou us 16 channels ion-channel recordings through membrane proteins reconstituted in bilayer lipid membrannes (BLMs) as shown in Figure 1. Although multiple ion-channel recording is necessary for high-throughput screening (HTS) of drug [1], there are no reports of such systems as far as the authors are aware. We have developed several strategies to construct BLMs arrray for multiple ion-channel recording. The “droplets contacting method” which forms BLMs at the interface of two contacting droplets of lipid monolayers is one of the simplest and most efficient methods. Since it does not require skilled techniques, it is highly reproducible and can be applied to an automated system. [2]. In this study we particularly used a double well chip (DWC) (Figure 1a [3]) with parylene micro-pores in thedroplets contacting method. The parylene micro-pores confined the BLMs forming areas and subsequently, augmented the mechanical stability [4]. Using the droplets contacting method and parylene micro-pores, we succeeded in the multiple recording in 14 channels, which is the most multiple recording as far as we know. This method can be applied to fully automated membrane protein reconstitution for HTS system using a spotting robot [5].

Solution exchange of droplet contacting lipid bilayer system

This paper describes a bilayer lipid membranes (BLMs) chip capable of solution exchange. In the previous report, we successfully reconstituted α-hemolysin (αHL) in BLMs using droplets contacting method with an array device. For drug screening, solution exchange is required to test the effect of inhibitors or promoters for proteins. In this study, we attempted to exchange the solution in a droplet using a microfluidic channel (Figure 1). We could successfully inject desired solution into the droplet forming BLMs and to withdraw the initial solution out of a droplet. The electrical signal of αHL with its some blockers which was injected later instead of the initial signal was successfully recorded. We consider that this method will enable more accurate and efficient drug screening system using artificially BLMs and reconstituted membrane protein.

Intra/extracellular investigation for ion channels with lipid bilayer array at the single molecule level

This paper describes an investigation of drug effects for ion channels with recognizing the intra/extracellular directions. We have previously reported the ion channel recordings with bilayer lipid membranes (BLMs) which formed on a parylene micropore using droplets contact method. However, the low reconstitution probability of ion channels into BLMs has been a major issue. In this study, BLMs area and position are regulated with changing the numbers and positions of the parylene micropores. As a result, the simultaneous single channel recordings of K+ channel expressed in nerve system with BLM array were able to be achieved at the high probability.