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 (<i>n</i> = 256 within 30 min) and channel inhibition experiments by contacting different types of the droplets in a short time frame

Frequency-dependent power density and diagrams of two different electrode systems: inserted and bottom-well, (a).

<p>Time required for αHL reconstitution using the drop-off (b) and repainting (c) methods, (b,c).</p

Photograph of the recording system, DW chip with an amplifier connected to a PC, at a high-altitude site (near the summit of Mount Fuji), (a).

<p>Current time frequency histogram of actual data taken at the field site, (b). Power density spectra of the open channel current of αHL in the field and the laboratory, (c).</p

Portable system for ion channel current recordings.

<p>Droplet contact method for reproducible and stable lipid bilayer formation, (a). Illustration of the double-well (DW) chip used for ion channel measurement with the droplet contact method, (b). Schematic diagram of the channel current recordings for alpha-hemolysin reconstituted in the lipid bilayer, (c). Photograph of our portable system containing the DW chip with a handheld amplifier connected to a laptop PC, (d).</p

Serial DNA relay in DNA logic gates by electrical fusion and mechanical splitting of droplets

<div><p>DNA logic circuits utilizing DNA hybridization and/or enzymatic reactions have drawn increasing attention for their potential applications in the diagnosis and treatment of cellular diseases. The compartmentalization of such a system into a microdroplet considerably helps to precisely regulate local interactions and reactions between molecules. In this study, we introduced a relay approach for enabling the transfer of DNA from one droplet to another to implement multi-step sequential logic operations. We proposed electrical fusion and mechanical splitting of droplets to facilitate the DNA flow at the inputs, logic operation, output, and serial connection between two logic gates. We developed Negative-OR operations integrated by a serial connection of the OR gate and NOT gate incorporated in a series of droplets. The four types of input defined by the presence/absence of DNA in the input droplet pair were correctly reflected in the readout at the Negative-OR gate. The proposed approach potentially allows for serial and parallel logic operations that could be used for complex diagnostic applications.</p></div

Droplet contact method (DCM) and the binary system based on DNA blocking and translocation.

<p>(a) Definition of the binary system based on the presence of DNA in an aqueous droplet coated with a lipid monolayer. (b) A schematic view of the bilayer lipid membrane (BLM) formed via DCM. (c) The system for DNA transfer among droplets by DNA translocation through αHL nanopores. (d–f) Electrical detection of DNA translocation and determination of the presence of DNA constructs in the droplet. (d) Output 1 (translocation): A single-stranded DNA (ssDNA) is translocated through an αHL nanopore with a short current blockade. (e) Output 0 (blocking): A double-stranded DNA (dsDNA) is not translocated through the αHL nanopore owing to its larger diameter, inducing a long current blockade. (f) Output 0 (No DNA strand): No DNA strand is present for translocation, and thus no current blockade is generated.</p

A schematic view of the four-well chip, the device used for the four-droplet network.

<p>(a) A four-droplet network for Negative-AND operation. Input (1, 0) was selected as an example. The input DNA strands were injected into the input droplets and the C_DNA strands were prepared in the operation droplet. (b) An overall view of the 4WC. The electrodes in the wells were connected to a patch-clamp amplifier. (c) The wiring to the four wells. The electrodes were embedded on the bottom of the wells.</p

Procedures and results of the NOR operation.

<p>(a–i) Procedures of the NOR gate. (j–m) Electrical signals and outputs of the NOR operation. (j) Input (0, 0). Peak-like current inhibitions were predominant, and the readout criterion number (R_TB) was 0.68 ± 0.20 (i.e., output 1). (k–m) Inputs (0, 1), (1, 0), and (1, 1), respectively. Long current inhibitions were predominant, and the R_TB values were −0.36 ± 0.16, −0.76 ± 0.16, and −0.28 ± 0.08, respectively. Thus, these results exhibited output 0.</p

Experiment to determine mixing duration.

<p>(a–c) Fluorescence images and corresponding schematics at 0, 30, and 60 min after electrical fusion of the droplets. (d) Time-course monitoring of the fluorescence intensity of the donor and acceptor droplets. Error bars: standard deviations (N = 3).</p

Negative-AND (NAND) operation system based on DNA translocation.

<p>(a) NAND logic gate concept based on αHL nanopores and three types of DNA strands, A_DNA, B_DNA, and complementary DNA (C_DNA) in a droplet network. (b) A schematic table of NAND operations with the DNA structures and nanopore result for each input.</p