Hiroki Yasuga

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.

Synthetic microfluidic paper

We introduce a polymer synthetic microfluidic paper for lateral flow devices. The aim is to combine the high surface area of paper, or nitrocellulose, with the repeatability, controlled structure, and transparency of polymer micropillars. Our synthetic paper consists of a dense, high aspect ratio array of transparent pillars that are slanted and mechanically interlocked. We describe the manufacturing using multidirectional UV lithography and demonstrate successful capillary pumping of whole blood.

Droplet microfluidics inside paper

Here, we demonstrate, for the first time: the self-digitization, i.e. spontaneous formation, of microdroplets during the imbibition of paper; the on-demand merging of individual microdroplets in paper; and the on-demand ejection of individual microdroplets from the paper. Two technical novelties underlie these novel functions: the formation of free-standing synthetic microfluidic paper, i.e. a porous matrix of slanted and interconnected micropillars without bottom layer; and the hydrophobic surface modification of the paper. The ease of manipulation and the direct access to the microdroplets from the environment makes this an extremely versatile tool, with potential applications in liquid sample digitisation and microparticle generation.

Self-generation of two-dimensional droplet array using oil–water immiscibility and replacement

Two-dimensional (2D) microdroplet arrays with indexed sample concentration gradients have been receiving considerable attention for high-throughput biological and medical analyses. However, the preparation of such an array by conventional methods mandates precise pipetting and/or pumping. In this paper, we introduce a method to spontaneously generate 2D-arrayed aqueous droplets using a well array, for which coarse pipetting is sufficient. The wells are connected in rows and columns via narrow channels. Aqueous solutions impregnated in the well array are split into droplets in every single well as a subsequently introduced immiscible solvent self-propagates and divides the solution at the channels. A concentration gradient of the samples can be formed across the connected solution in the well array; once droplets are generated, each droplet possesses a different sample concentration depending on its position in the array. We experimentally determined the optimal well dimensions and solvent species to obtain a high yield of droplet generation. We next demonstrated a 2D droplet array with a two-sample concentration gradient. Finally, the applicability of the system was demonstrated through a cell viability assay using a sample that induced apoptosis. We believe the proposed method contributes to simplification and miniaturization of the system to generate droplet arrays and thus is applicable to biological and medical analysis.

Adjustment of surface condition for self-generation of droplet array using oil-water replacement

This paper demonstrates a method to self-generate a two-dimensional (2D) array of nanoliter-scale droplets. 2D droplet arrays are powerful tools for high throughput and low-cost processes in the field of biology or medicine. We recently developed a self-generation method of droplet array without precise liquid control such as precise pipetting or pumping. The next challenge of the self-generation is the further miniaturization of generated droplets. Here, we addressed it by applying a photoresist off-stoichiometry thiol-ene (OSTE), of which surface energy can be tuned by polymer chain grafting. We experimentally found the surface conditions to self-generate an array of nanoliter-scale droplets.

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

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.

Vibration-triggered self-assembly of caged droplets to construct a droplet interface bilayer network

This paper proposes a self-assembly method to construct a droplet interface bilayer (DIB) network. The DIB network has been developed for biological logical circuits or chemical microreactors, using incorporated membrane proteins. In this work, the DIB network was constructed by using multi-geometric Droplet-Boxes (DBs), which captured and stabilized an aqueous droplet in organic solvent. The multi-geometry enabled DB tessellation, and DIB network was constructed along the tessellation. Vibration triggered self-assembly of the tessellation with a help of capillary force. DIBs were verified by electrical measurement of α-Hemolysin (αHL). The proposed method demonstrated the potential of DIB network formation with good configurability and yield; hence, facilitates design of biological logical circuits based on DIB network.

Connectable DNA-logic operation using droplets and rupture/reformation of bilayer lipid membranes

This paper experimentally demonstrated the connection of logic gates using DNA and membrane proteins channel reconstituted into bilayer lipid membranes (BLMs). In this system, droplets worked as the carrier of DNA to convey it as biological signal to next stage and reformation and rupture of BLM accelerated DNA transfer between droplets. Furthermore, DNA is detected based on the electrical and physical characters of DNA translocation via membrane proteins channel. In this study, we successfully demonstrated NOR operation by connecting OR and NOT gate. This system is readily applicable to develop high sensitive biosensor and computing system imitating biological information processing.