Single Cell Manipulation Research Articles

Functionalized nanopipettes: toward label-free, single cell biosensors

Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms.

Magnetic nanostructures for the manipulation of individual nanoscale particles in liquid environments (invited)

The manipulation of geometrically constrained magnetic domain walls (DWs) in nanoscale magnetic strips attracted much interest recently, with proposals for prospective memory and logic devices. Here we demonstrate that the high controllability of the motion of geometrically constrained DWs allows for the manipulation of individual nanoparticles in solution on a chip with the active control of position at the nanometer scale. Our approach exploits the fact that magnetic nanoparticles in suspension can be captured by a DW, whose position can be manipulated with nanometer scale accuracy in specifically designed magnetic nanowire structures. We hereby show that the precise control over DW nucleation, displacement, and annihilation processes in such nanostructures allows for the capture, transport, and release of magnetic nanoparticles. As magnetic nanoparticles with functionalized surfaces are widely used as molecule carriers or labels for single molecule studies, cell manipulation, and biomagnetic sensing, the accurate control over the handling of the single magnetic nanoparticle in suspension is a crucial building block for several applications in biotechnology, nanochemistry, and nanomedicine.

Manipulation of Suspended Single Cells by Microfluidics and Optical Tweezers.

Chondrocytes and osteoblasts experience multiple stresses in vivo. The optimum mechanical conditions for cell health are not fully understood. This paper describes the optical and microfluidic mechanical manipulation of single suspended cells enabled by the μPIVOT, an integrated micron resolution particle image velocimeter (μPIV) and dual optical tweezers instrument (OT). In this study, we examine the viability and trap stiffness of cartilage cells, identify the maximum fluid-induced stresses possible in uniform and extensional flows, and compare the deformation characteristics of bone and muscle cells. These results indicate cell photodamage of chondrocytes is negligible for at least 20 min for laser powers below 30 mW, a dead cell presents less resistance to internal organelle rearrangement and deforms globally more than a viable cell, the maximum fluid-induced shear stresses are limited to ~15 mPa for uniform flows but may exceed 1 Pa for extensional flows, and osteoblasts show no deformation for shear stresses up to 250 mPa while myoblasts are more easily deformed and exhibit a modulated response to increasing stress. This suggests that global and/or local stresses can be applied to single cells without physical contact. Coupled with microfluidic sensors, these manipulations may provide unique methods to explore single cell biomechanics.

Drop-on-Demand Patterning of Bacterial Cells Using Pulsed Jet Electrospraying

In this study, the drop-on-demand patterning of bacterial cells on a raw silicon wafer was newly conducted with an electrospray pulsed jet. We produced various sized line patterns and spot patterns of bacterial cells on the silicon wafer under varying experimental conditions of frequency, flow rate, and translational speed of the electrospray system in one and two-dimensional ways. Especially, the electrospray's pulsed jet of bacterial solution produced regular patterns of around 10 microm diameter spots, each of which contained a single bacterial cell. By enabling drop-on-demand patterning and manipulation of single bacterial cells on a raw wafer in the gas phase, this new technique broadens the scope of biological experiments beyond liquid phase treatments to solid and gas-phase treatments.

Single cell manipulation using ferromagnetic composite microtransporters

For biomedical applications, such as single cell manipulation, it is important to fabricate microstructures that can be powered and controlled wirelessly in fluidic environments. In this letter, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic microtransporters driven by external magnetic fields. Microtransporters were fabricated using a simple, single step fabrication method and can be produced in large numbers. We demonstrate that they can be navigated to manipulate single cells with micron-size precision without disturbing the local environment.

Hydrodynamic Trap for Single Cells and Particles

Particle trapping and micromanipulation techniques have revolutionized biological sciences during the last two decades. Proteins, enzymes and cells have been studied extensively through manipulation methods based on optical, magnetic and electric fields. In this work, we present an alternative trapping method called the hydrodynamic trap which is based solely on hydrodynamic forces generated in a microfluidic device. The hydrodynamic trap is based on a purely extensional flow field created at the junction of two perpendicular microchannels where opposing laminar flow streams converge. The flow field in the vicinity of the microchannel junction can be described as a potential flow with a semi-stable potential well and a stagnation point. We implement an automated feedback-control mechanism to adjust the location of the stagnation point, thereby actively trapping arbitrary particles in free solution. Using the hydrodynamic trap, we successfully demonstrate trapping and manipulation of single cells and single particles with micron and sub-micron dimensions for arbitrarily long observation times. Brownian dynamics simulations show that the trap stiffness is comparable to alternative trapping techniques including magnetic traps. Overall, this new technique offers a venue for observation of biological materials without surface immobilization, eliminates potentially perturbative optical, magnetic and electric fields, and enables the ability to vary the surrounding medium conditions of the trapped object in real-time.

2P1-B12 感熱ゲルアクチュエーションによる噴出・吸引プローブを用いた単一細胞操作

In this paper, we fabricate a thermal gel actuated probe which uses the volume change of the thermal gel as an actuator. The thermal gel actuated probe can be used for single cell analysis, especially single cell manipulation. The thermal gel actuated probe realizes spout and suction in several nano liters (nl) only to switch the heater turning on and off. It is difficult to realize solution spout and suction less than several dozen nl by the conventional probe type devices which use air or oil pressure. The volume change of the thermal gel was evaluated in this paper. The target single cell harvesting was demonstrated by this thermal gel actuated probe. It is considered that the thermal gel actuated probe can contribute to the single cell analysis from the aspect of single cell manipulation.

Human mammalian cell sorting using a highly integrated micro-fabricated fluorescence-activated cell sorter (microFACS).

We demonstrate a high performance microfabricated FACS system with highly integrated microfluidics, optics, acoustics, and electronics. Single cell manipulation at a high speed is made possible by the fast response time (approximately 0.1 ms) of the integrated PZT actuator and the nozzle structure at the sorting junction. A Teflon AF-coated optofluidic waveguide along the microfluidic channel guides the illumination light, enabling multi-spot detection, while a novel space-time coding technology enhances the detection sensitivity of the microFACS system. The real-time control loop system is implemented using a field-programmable-gate-array (FPGA) for automated and accurate sorting. The microFACS achieves a high purification enrichment factor: up to approximately 230 fold for both polystyrene microbeads and suspended human mammalian cells (K562) at a high throughput (>1000 cells s(-1)). The sorting mechanism is independent of cell properties such as size, density, and shape, thus the presented system can be applied to sort out any pure sub-populations. This new lab-on-a-chip FACS system, therefore, holds promise to revolutionize microfluidic cytometers to meet cost, size, and performance goals.

2P232 1C1520 フェムト秒レーザー誘起衝撃力による単一細胞の操作と刺激(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

2P232 1C1520 フェムト秒レーザー誘起衝撃力による単一細胞の操作と刺激(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

Optoelectronic Tweezers as a Tool for Parallel Single-Cell Manipulation and Stimulation

Optoelectronic tweezers (OET) is a promising approach for the parallel manipulation of single cells for a variety of biological applications. By combining the manipulation capabilities of OET with other relevant biological techniques (such as cell lysis and electroporation), one can realize a true parallel, single-cell diagnostic and stimulation tool. Here, we demonstrate the utility of the OET device by integrating it onto single-chip systems capable of performing in-situ, electrode-based electroporation/lysis, individual cell, light-induced lysis, and light-induced electroporation.

Electrodes on a budget: Micropatterned electrode fabrication by wet chemical deposition

Precise patterning of metals is required for diverse microfluidic and microelectromechanical system (MEMS) applications ranging from the separation of proteins to the manipulation of single cells and drops of water-in-oil emulsions. Here we present a very simple, inexpensive method for fabricating micropatterned electrodes. We deposit a thin metal layer of controlled thickness using wet chemistry, thus eliminating the need for expensive equipment typically required for metal deposition. We demonstrate that the resulting deposited metal can be used to fabricate functional electrodes: The wet-deposited metal film can sustain patterning by photolithography down to micron-sized features required for MEMS and microfluidic applications, and its properties are suitable for operative electrodes used in a wide range of microfluidic applications for biological studies.

A rapid and efficient single-cell manipulation method for screening antigen-specific antibody–secreting cells from human peripheral blood

Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody-secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.

Electrical characterization of a single cell electroporation biochip with the 2-D scanning vibrating electrode technology

Advancements in microfabrication technology have lead to the development of planar micro-pore electroporation technology. This technology has been shown to provide greater control in single cell manipulation, and electroporation which is independent from cell size. In this work we report direct and spatially resolved characterization of electric currents within a planar micropore electroporation biochip to better understand this phenomenon at the cellular level. This work was performed using a two-dimensional (2-D) vibrating probe (VP). Analysis of the spatial patterns of current density yielded a 4th order polynomial profile in the planes parallel to the biochip's surface and a three parameter hyperbolic decay profile in the planes perpendicular to the chip surface. A finite element model was developed which correlates with actual measurements on the micropore. Preliminary VP current density measurements of electroporated HepG2 cells revealed a significantly high current density minutes after electroporation even with non-electroporative pulses. These results indicate that cells take a considerable amount of time for complete electrophysiological recovery and indicate the use of the VP as a cell viability indicator for optimized electroporation.

Single-Cell Injectoassay for ES Cell Engineering

It is important to analyze the intensity dependent action manners of differentiation associated genes in ES cell. For this purpose, single-cell injectoassay was performed by femto-injection method using a single-cell manipulation supporting robot. An Oct3/4 expression vector was introduced into mouse ES cell quantitatively and its effect was evaluated by a cell shape change indicator defined by the perimeter to radius ratio (PR ratio). In the cases of no injection control and the injection control, PR ratio was 7.3-8.0 until 72h. In contrast, when the vector concentration in the injector capillary was 300 ng/micro-L, the rate of differentiation was 33% (2/6). This rate further increased up to 86% (6/7) at 1200 ng/micro-L. These indicate the feasibility of the single-cell injectoassay.

Chemistry and Biology in Femtoliter and Picoliter Volume Droplets

The basic unit of any biological system is the cell, and malfunctions at the single-cell level can result in devastating diseases; in cancer metastasis, for example, a single cell seeds the formation of a distant tumor. Although tiny, a cell is a highly heterogeneous and compartmentalized structure: proteins, lipids, RNA, and small-molecule metabolites constantly traffic among intracellular organelles. Gaining detailed information about the spatiotemporal distribution of these biomolecules is crucial to our understanding of cellular function and dysfunction. To access this information, we need sensitive tools that are capable of extracting comprehensive biochemical information from single cells and subcellular organelles. In this Account, we outline our approach and highlight our progress toward mapping the spatiotemporal organization of information flow in single cells. Our technique is centered on the use of femtoliter- and picoliter-sized droplets as nanolabs for manipulating single cells and subcellular compartments. We have developed a single-cell nanosurgical technique for isolating select subcellular structures from live cells, a capability that is needed for the high-resolution manipulation and chemical analysis of single cells. Our microfluidic approaches for generating single femtoliter-sized droplets on demand include both pressure and electric field methods; we have also explored a design for the on-demand generation of multiple aqueous droplets to increase throughput. Droplet formation is only the first step in a sequence that requires manipulation, fusion, transport, and analysis. Optical approaches provide the most convenient and precise control over the formed droplets with our technology platform; we describe aqueous droplet manipulation with optical vortex traps, which enable the remarkable ability to dynamically "tune" the concentration of the contents. Integration of thermoelectric manipulations with these techniques affords further control. The amount of chemical information that can be gleaned from single cells and organelles is critically dependent on the methods available for analyzing droplet contents. We describe three techniques we have developed: (i) droplet encapsulation, rapid cell lysis, and fluorescence-based single-cell assays, (ii) physical sizing of the subcellular organelles and nanoparticles in droplets, and (iii) capillary electrophoresis (CE) analysis of droplet contents. For biological studies, we are working to integrate the different components of our technology into a robust, automated device; we are also addressing an anticipated need for higher throughput. With progress in these areas, we hope to cement our technique as a new tool for studying single cells and organelles with unprecedented molecular detail.

Antifouling coatings for optoelectronic tweezers

Optoelectronic tweezers enables parallel manipulation of individual single cells using optical addressing and optically induced dielectrophoretic force. This provides a useful platform for performing a variety of biological functions, such as cell manipulation, cell sorting, and cell electroporation. However, in order to obtain more reliable cellular manipulation, especially of adherent mammalian cells, antifouling coatings need to be used to avoid non-specific cell adherence. Two antifouling coatings are discussed here, which can reduce the amount of non-specific adherence by as much as a factor of 30.

2A2-L12 力覚フィードバック系を持つ光駆動ナノロボットの開発

We realized remote manipulation of single cell in liquid with simultaneous reaction force sensing by using a "Optically Driven Nanomachine". We developed the system which we can measure the cell 2 direction reaction force for a long time and feel the reaction force while operating Nanomachine. Intuitive cell operation and force measurement was enabled by this system.

Parallel single-cell light-induced electroporation and dielectrophoretic manipulation

Electroporation is a common technique for the introduction of exogenous molecules across the, otherwise, impermeant cell membrane. Conventional techniques are limited by either low throughput or limited selectivity. Here we present a novel technique whereby we use patterned light to create virtual electrodes which can induce the parallel electroporation of single cells. This technique seamlessly integrates with optoelectronic tweezers to provide a single cell manipulation platform as well. We present evidence of parallel, single cell electroporation using this method through use of fluorescent dyes and dielectrophoretic responses. Additionally, through the use of integrated microfluidic channels, we show that cells remain viable following treatment in the device. Finally, we determine the optimal field dosage to inject propidium iodide into a HeLa cell and maintain cellular viability.

Development of a poly-dimethylsiloxane microfluidic device for single cell isolation and incubation

Although single cell manipulation has been developed for precise understanding of cell biology, it is still difficult to handle single cells because of their morphology including the size variation and the flexibility. We developed a single cell manipulation device featuring microchannels and micropockets for single cell capture and cultivation. Single cells were captured noninvasively and sequentially, and each cell was repeatedly sub-cultured to four generations. The single cell manipulation device was microfabricated with two main parallel channels allowing the cell suspension and the carrier flow to be injected separately. Those channels, that are main channel and buffer channel, were connected with a narrow (3 μm) drain channel, and single cell capture pockets were placed at the point where the main channels and the drain channel connected. A gentle flow was produced in the drain channel because of the difference in the flow rate between the main channel and buffer channel, realized the individual single cell isolation in the capture pocket. When a single cell was captured in a single cell pocket, the captured cell capped the drain channel and caused the other cells that were flowing through the channel to go to the next capture pockets. The ratio between the cells that were captured and the cells that passed through the main channel was about 70%. The captured singe cell was cultured successively in the same pocket, and the cells divided into four cells. The doubling times of two cells that grew in the first cell division were slightly different (10 min). These fundamental single cell manipulations were carried out mainly by controlling the flow rate, essentially the pressure on each channel. Occasionally the manipulations also were carried out by changing the shapes and the sizes of the micropockets in the microfluidic device. Since this device was used successfully for the noninvasive isolation and long-term cultivation of single cells, it can contribute to various biological analyses, such as biopsy, noninvasive bioanalysis, and the morphology of single cells.

Nanoscale dispensing in liquid environment of streptavidin on a biotin-functionalized surface using hollow atomic force microscopy probes

A microfabricated nanoscale dispenser (NADIS) based on an atomic force microscopy probe has been developed to enable the delivery of ultrasmall amounts of liquid material with a high lateral accuracy in liquid or gaseous surroundings. The dispenser consists of a hollow cantilever with a tip with an opening at its apex. The hollow cantilever, which acts as a microfluidic channel, is connected to a reservoir in the chip. The chip, in turn, is fixed to a modified probe holder with fluidics connections to the reservoir. By applying a pressure to the chip reservoir, liquid from the cantilever can be ejected through the tip aperture into the surroundings. The system was demonstrated by writing streptavidin spots on a biotin-functionalized surface in HEPES buffer. The applications foreseen include manipulation of single cells, such as force controlled intracellular injections.