バーチャル電極ディスプレイ
Virtual Cathode Display for Biomolecules
分子スケールMR/AR (複合現実: mixed reality) 技術
現実の分子にあわせて電場をプロジェクションマッピングすることで，現実の分子機能を自在に拡張するような「分子スケールMR/AR（複合現実: mixed reality）技術」を目指しています．
Nano deposition and ablation was performed on living cell using an electron-beam induced chemical reaction in non-vacuum aqueous medium. Nanopattern was fabricated on living cell from 10-40 mM EDOT precursor in cultivation medium. And living cell was also successfully trimmed using ablation process with minimally invasion.
- 星野隆行, 宮廻裕樹, 応用物理, 92(5), 283, 2023, DOI:10.11470/oubutsu.92.5_283.
- T. Hoshino, Applied Physics Letters, 99, pp.174102, (2011).
- T. Hoshino, Biochem.B iophys. Res. Comm., 432 (2), 3459, (2013). DOI 10.1016/j.bbrc.2013.01.10
- T. Hoshino, Sens. Actuators B, 236, pp. 659, (2016).
Rapid pattern formation in model cell membranes when using an electron beam
H. Miyazako, T. Hoshino, “Rapid pattern formation in model cell membranes when using an electron beam,” Colloids and Surfaces B: Biointerfaces, vol. 220, p. 112967, Dec. 2022, DOI:10.1016/j.colsurfb.2022.112967.
人工細胞膜の流動を電子線により自在に制御するディスプレイを開発し、人工細胞膜の流れを制御し、二次元パターン（図形）を繰り返し描いたり消したりすることに成功しました。
また、このディスプレイ技術を用い、分子が自発的に集合して脂質膜内に島状のドメイン構造（脂質ラフト）を形成することに成功しました。これは、細胞膜で起きている相分離現象や自己組織構造の形成を模擬するものです。
このディスプレイは、人工細胞膜の流れや相分離現象をラピッドプロトタイピングすることができ、デジタル技術と分子現象をつなぐ技術として期待されます。
Patterning of supported lipid bilayers (SLBs) is a fundamental tool for mimicking and modeling of biological membranes, but it still remains a challenge to achieve resetting and retriggering for the membrane-mediated processes of the patterned SLBs. This paper reports that indirect scanning of an electron beam (EB) enables repeatable patterning and remodeling of SLBs. By indirect scanning of the EB onto SLBs through a 100-nm-thick silicon nitride membrane, chemical and electrical effects of scattered electrons can be applied to lipid molecules in SLBs; application of these effects enables formation and annihilation of diffusion barriers in SLBs. Two-dimensional patterning of SLBs was demonstrated to be controllable by changing the electron dose of the EB and two phenomena were applied to realize repeatable wiping off and wetting of SLB patterns. Moreover, respreading of SLBs was determined as able to induce remodeling of a raft domain in phase-separated SLBs. The proposed method will lead to a prototyping tool for mimicking cell membranes to design interactions of cells and membrane proteins as well as SLB-based microchips.
分子レベルの反応制御によるキネシン - 微小管運動制御
Surface-limited reactions for spatial control of kinesin–microtubule motility assays using indirect irradiation of an electron beam
Hiroki Miyazako, Ryuzo Kawamura, and Takayuki Hoshino, “Surface-limited reactions for spatial control of kinesin–microtubule motility assays using indirect irradiation of an electron beam,” Biomicrofluidics, vol. 16, no. 6, p. 064105, Dec. 2022, DOI:10.1063/5.0124921.
タンパク質分子モーターのひとつであるキネシン-微小管をアクチュエータとするナノデバイスの迅速な製造と評価のために、キネシンの活性と密度を制御することが重要です．従来の光学的な制御方法では，拡散過程に律速するため，長期的な空間制御性に欠けている．また，キネシン-微小管の高さは数十nmと微小であるため、キネシン分子を制御しようとした場合，キネシン分子だけでなく微小管も同時に損傷されてしまう問題がある．本研究では，電解液中の表面電界がデバイ遮蔽によりナノメートルスケールで遮蔽されることを利用し、電子ビームの間接照射によって誘導される表面限定の電気化学反応を使用して、デバイ長のなかにあるキネシン分子のみを制御し，外柄にある微小管には影響を与えない制御が可能であることを示した．これの成果は，キネシン密度と活性の細かい空間制御が可能であることを示しています。 これらのローカライズされた電気化学的効果は、EB 加速電圧を変更することによって、キネシンの除去とキネシン活性の運動制御の両方を引き起こすことを示しています。
Gliding of microtubules (MTs) on kinesins has been applied to lab-on-a-chip devices, which enable autonomous transportation and detection of biomolecules in the field of bioengineering. For rapid fabrication and evaluation of the kinesin–MT based devices, optical control techniques have been developed for control of kinesin activity and density; however, use of caged molecules lacks spatial controllability for long-term experiments, and direct irradiations of UV light onto kinesin-coated surfaces are inherently damaging to MTs due to their depth limit since the heights of the kinesin–MT systems are at the tens of a nanometer scale. Considering surface electric fields in electrolytic solutions are shielded at the nanometer scale due to Debye shielding, in this study, we show that fine spatial control of kinesin density and activity is enabled using surface-limited electrochemical reactions induced by indirect irradiations of an electron beam (EB). An EB is indirectly irradiated onto the kinesins through a 100-nm-thick silicon nitride membrane, and the electrons scattered in the membrane can cause localized electrochemical effects to the kinesins. We show that these localized electrochemical effects cause both ablation of kinesins and motility control of kinesin activity by changing the EB acceleration voltage. In particular, the latter is achieved without complete ablation of MTs, though the MTs are indirectly irradiated by the EB. As a demonstration of on-demand control of gliding MTs, we show the accumulation of the MTs on a target area by scanning the EB. The proposed accumulation technique will lead to rapid prototyping of microdevices based on MT–kinesin motility assay systems.
自在に動かせるナノ電極の周波数特性
Dielectric characteristics of deformable and maneuverable virtual cathode tool displayed by indirect electron beam drawing
Ken Sasaki and Takayuki HOSHINO, “Dielectric characteristics of deformable and maneuverable virtual cathode tool displayed by indirect electron beam drawing,” Japanese Journal of Applied Physics, 2022, DOI:10.35848/1347-4065/ac61ac.
誘電泳動法は，ソフトマターを操作する際に有用な手法の一つですが，生体分子をより自在に操作するためには，呈示電場を自在にかつ高速に操る電場呈示手法が必要となります．本論文では，電子ビームを用いたバーチャル電極（VC）ツールを設計し，これを自在に変形可能で操作性の高い電極として用い，分子操作が可能であることを明らかにしました．VCツールを適用して，YOYO-1で蛍光標識したDNA分子の電気化学的応答を調べ．そのパターン生成経路と周波数特性を評価しました．印加するVCツールが高い周波数成分をもつほど、ドーズ量が減っても効果範囲が拡大することが分かりました．これは，VCツールが誘電体であるSiN薄膜や電気二重層に起因するハイパス特性を有しており，誘電現象に律速していることを示唆しています．これらの結果から，VCツールは，ツールパスや描画周波数の設計を工夫することで，誘電現象を制御することができ，より柔軟な分子操作や加工が可能になると考えられます．
Dielectrophoretic manipulations are deft techniques for soft-matter processes. To actuate the target biomolecules more spatiotemporally, the manipulator which can maneuver the adjustable electric field at high speed is required. We have designed a virtual cathode (VC) tool drawn with an electron beam (EB), which is a deformable and maneuverable electrode. In this report, we investigated the electrochemical response of YOYO-1-labeled DNAs by applying the VC tool and evaluated dependency of its dielectric characteristics on pattern frequency. The specific fluorescent bleaching responses we obtained suggested that work lengths and strength of the VC-induced electric field were enhanced as the applied VC pattern has a high pattern frequency. Moreover, we validated the form of the EB-drawing pattern can also affect dielectric characteristics of the VC tool. These results therefore indicate that the VC tool can control the dielectric phenomenon by a well-tuned tool design, which will lead to more flexible manipulations.
電子のはたらきで脂質二重膜を押し引きし自在に形を制御 ～生体分子デバイスへ向けてナノディスプレイを開発～
Multi-Scale Lipid Membrane Flow by Electron Beam-Induced Electrowetting
Hiroki Miyazako, Kunihiko Mabuchi, and Takayuki Hoshino, “Multi‐Scale Lipid Membrane Flow by Electron Beam‐Induced Electrowetting,” Advanced Materials Interfaces, p. 2100257, Aug. 2021, DOI:10.1002/admi.202100257.
脂質二重膜は，細胞の内側と外側を隔てる細胞膜を構成し，我々の体にある細胞の情報伝達に深く関わっており，医学研究だけでなく人工脂質膜を基盤とする生体分子デバイスへの工学応用が期待されています。しかしながら，人工脂質膜はシャボン玉のように柔らかく形が定まっていないため，形を自在に制御して生体分子デバイスをつくることは困難でした。今回，表面のぬれ性を電場で変えるエレクトロウェッティング効果を自在に制御するバーチャル電極ディスプレイを用いて，人工脂質膜の形状を仮想的なナノ電極の呈示に従って自在に変形させることに成功しました。本手法を利用することで，これまでその場で形状を制御することが困難であった人工脂質膜を自在に変形させて，望みの形に脂質ネットワークをつくることができるようになり，人工脂質膜を基盤とした新しい生体分子デバイスの研究が加速できると期待されます。
A new control method is proposed for lipid membrane flow of supported lipid bilayers (SLBs) using an electron beam (EB). A focused electric field of an EB (virtual cathode, VC) induces local electrowetting effects and generates small-scale membrane flow only around the VC. Moreover, the VC can generate large-scale membrane flow by inducing surface instability.
ナノ接触界面の電気化学的な３次元観察に成功
Electrochemical imaging of contact boundary by using electron-beam addressing of a virtual cathode display
T. Hoshino, W. Tooyama, H. Miyazako, “Electrochemical imaging of contact boundary by using electron-beam addressing of a virtual cathode display,” Sensors and Actuators B: Chemical, p. 129558, Jan. 2021, DOI:10.1016/j.snb.2021.129558.
脂質膜は、細胞の内側と外側を隔てる細胞膜を構成し、われわれの体の中の細胞の機能や形をつくるうえで不可欠ですが、脂質膜が物と接触している界面がどのような状態で存在しているのか観察することは困難でした。今回、バーチャル電極ディスプレイの上に置いた試料との界面に仮想的なナノ電極を発生させ、3次元的な形態を電気化学的に観察する手法を開発し、水溶液中の脂質膜のナノ界面を観察することに成功しました。本手法を利用することで、これまで観察が困難であった細胞が物と接着する接触界面の状態を観察することができるようになり、新しい細胞組織工学の研究が加速できると期待されます。
A scanning electrochemical microscope (SECM) enables surface measurements of electrical properties of various wet samples including living cells, although its probe cannot reach the interface where the samples contact a substrate. This paper reports our innovative method for electrochemical imaging of the contact boundary in wet samples in water solution. We used electron-beam (EB) addressing of a virtual cathode (VC) for an inverted electron beam lithography system, and we carried out imaging of the contact boundaries which cannot be approached by conventional scanning probe microscopes. This VC was formed on the surface of a 100-nm thick silicon nitride (SiN) membrane by low-energy EB irradiation from the lower side of the membrane. The VC appeared at the contact boundary of the wet samples and the membrane; therefore the VC virtually worked at the boundary as the scanning probe of the SECM to observe the wet contact interface. We acquired topographic and electrochemical images of micro polystyrene spheres and a DPPC/TR-DPPE supported lipid bilayer by measuring the probe current in the sample interface. The imaging results suggested spatial resolutions of ~180 nm width and ~90 nm depth in a topographical image could be captured using this VC-SECM system.
自在にたんぱく質分子モーター・キネシンと微小管の運動機能を一時停止
Pause of the Target Gliding Microtubule
K. Hatazawa, H. Miyazako, R. Kawamura, T. Hoshino, "Pause of the Target Gliding Microtuble on the Virtual Cathode," Biochemical and Biophysical Research Communications, 514(3), 821. 2019.
たんぱく質分子モーターであるキネシンと微小管の滑走運動をピンポイントに一時的に停止・再滑走させることに成功した．SiN 上に自在に生じさせられる virtual cathode (VC) の垂直電場により，VC 直上にある微小管の滑走運動を一時的に停止できることを確認した．微小管滑走を一時的に停止させるには至適電流密度があり，至適条件では VC を解除すると微小管は再滑走する．埼玉大学 川村隆三先生との共同研究． We report the transient response of gliding microtubules on a virtual cathode. In vivo activities, microtubule-kinesin systems are known to act as motor proteins with respect to cell motility cytokinesis and cellular transport by hydrolyzing ATP molecules. With development of in vitro assays, motor proteins have been attracting much attention as a key component for highly efficient nano-transportation systems. The molecular functions based on structural states are affected by changing the ionic condition of the molecular functions and by changing the electrical field in solution because of electrical charges of the molecules. The virtual cathode, which was generated on the SiN display surface by a low energy electron beam, locally induced electrochemical reactions and electric field around the targeted molecules on the display surface, and then the gliding motions of the targeted microtubules were regulated. In this study, we demonstrated that the virtual cathode display temporally stops a selected gliding microtubule by only applying the virtual cathode to the microtubule. The pause mode of the microtubule was easily canceled by simply turning the virtual cathode off, and then the gliding motion was restarted.
ピンポイント分子導入
Pinpoint Delivery of Molecules
T. Hoshino, M. Yoshioka, A. Wagatsuma, H. Miyazako, and K. Mabuchi, “Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display,” IEEE Transactions on NanoBioscience, vol. 17, no. 1, pp. 62–69, Mar. 2018.
１細胞操作技術として任意に選択した標的細胞へ分子を導入する技術．バーチャル電極を標的細胞の所望の位置へ形成し，発生した局所的な高電界により細胞膜がピンポイントに破壊・穿孔される．このとき細胞外の物質が細胞内へ導入され，その後，穿孔した細胞膜は自律的に修復され閉じるので標的細胞は生存することを確認している．
Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). The transmembrane inflows depended on beam duration time and acceleration voltage. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.
細胞内の主ひずみ分布計測
Mechanical Strain Microscopy of Living Cell
T. Hoshino, H. Miyazako, A. Nakayama, A. Wagatsuma, and K. Mabuchi, “Electron beam induced fine virtual electrode for mechanical strain microscopy of living cell,” Sensors and Actuators B: Chemical, vol. 236, pp. 659–667, Nov. 2016.
細胞や生体分子がある電解質溶液の任意の時空間に局所電場を形成しうるバーチャル電極ディスプレイを構築した．電子線走査を原理として120nmの解像度で局所的な電気化学現象を操作できる．電場操作により細胞接着分子の操作を行い，細胞を局所に脱接着させ細胞に蓄積されているひすみ分布の可視化を可能にした．この結果は，本装置が生体分子の機能操作が可能であることを示している．
Low-invasive and fine virtual electrode is generated using focused low energy electron beam (EB). The virtual electrode induces local detachment of nanopartilces, and induced retraction of myoblast cell due to prestress. Distribution of prestressed mechanical strain of the cell were visualized and this indicated the elastic strain energy was stored around the nucleus and the lamellipodium. The spatial resolution of the virtual electrode was ~120-nm wide, and the repultion force was affected in <1µm in radius.
有機分子のその場パターニング
In-situ Patterning of Organic Molecules in Aqueous Solutions
電子線を照射することにより，水溶液中のMPCポリマーの脱離，ポリエチレンイミンなどの有機分子の堆積を“その場で”パターニングすることに成功した．細胞足場をその場で改変することで細胞運動を制御するなど，細胞の自己組織化現象の誘導に応用できると考えられる．
Low-invasive and fine virtual electrode is generated using focued low energy electron beam (EB). The virtual electode induecs local detachment of nanopartilces, and induced retraction of myoblast cell due to prestress. Distribution of prestressed mechanical strain of the cell were visualied and this indicated the elastic strain energy was stored arownd the nucleus and the lamellipodium. The spatial resolution of the viratual electrode was ∼120-nm wide, and the repultion force was affected in <1 µm in redius.
ナノポア上のナノ粒子操作
Reversible Concentration of Nanoparticles on a Nanopore
負のゼータ電位をもつナノ粒子の集合・解離を，ナノポアからの水の流出と局所電場の組み合わせで操作した．倒立電子線描画装置（I-EBL）は真空-水との隔膜にその場で任意の場所にナノポアと電場を形成でき，真空側への水流による求心力と電場による反発力によりナノ粒子の操作が可能である，ナノポアから半径3 μm 以下の領域のナノ粒子を集合でき，負電荷のタンパク質等の濃縮・析出に応用できる．
A reversible concentration of nanoparticles in water solution is produced on a nanopore fabricated in situ by inverted-electron beam lithography (I-EBL) in a SiN membrane. The SiN membrane separated the water solution and a vacuum chamber of the EB system. The nanopore led to a small leakage of water that flowed to the vacuum chamber. This flow led to the collection of the nanoparticles within an area of less than 3 µm surrounding the nanopore. Then, the EB-induced Coulomb force rapidly released the nanoparticles from only the targeted nanopore. This electron beam direct manipulation of the nanoparticles can provide simple in situ microfluidics in analytical methods for biochemistry.
ナノ流体の界面導電現象による時空間制御
Spatiotemporal Control of Electrokinetic Transport in Nanofluidics
厚さ100 nmのSiN薄膜で真空と液体試料を隔て，電子線を照射することにより，純水中において局所的に電気浸透流が生じることを示した．また，電気浸透流を用いることでナノ粒子の濃度の2次元パターニングをすることに成功した．化学走性の局所的な制御など，生化学反応系の時空間ダイナミクスの解明に応用できると考えられる．
By irradiating a 2.5 keV EB to a liquid sample through a 100-nm-thick SiN membrane, negative charges can be generated within the SiN membrane, and these negative charges can induce a highly focused electric field in the liquid sample. We showed that the EB-induced negative charges could induce fluid flow, which was strong enough to manipulate 240 nm nanoparticles in water, and we verified that the main dynamics of this EB-induced fluid flow was electroosmosis caused by changing the zeta potential of the SiN membrane surface. Moreover, we demonstrated manipulation of a single nanoparticle and concentration patterning of nanoparticles by scanning EB.
クローズドループ型倒立電子線描画システム
Closed-Looped in situ Nano Processing on a Culturing Cell Using an Inverted Electron Beam Lithography System
An electron beam lithography (EBL) was used as an in situ nano processing for a living cell. ► A synchronized optics was containing an inverted EBL and an optical microscope. ► This system visualized real-time images of the EB-induced nano processing. ► We demonstrated the nano processing for a culturing cell with 200–300 nm resolution. ► Our system would be able to provide high resolution display of virtual environments.