MicroBioRobots for Single Cell Manipulation

One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable micro actuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a bio-integrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 μm coated with a monolayer of the swarming Serratia marcescens. The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments, which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples of sub-micron scale transport and assembly as well as computer-based closed-loop control of MBRs are presented. We demonstrate experimentally that vision-based feedback control allows a four-electrode experimental device to steer MBRs along arbitrary paths with micrometer precision. At each time instant, the system identifies the current location of the robot, a control algorithm determines the power supply voltages that will move the charged robot from its current location toward its next desired position, and the necessary electric field is then created. Second, we develop biosensors for the MBRs. Microscopic devices with sensing capabilities could significantly improve single cell analysis, especially in high-resolution detection of patterns of chemicals released from cells in vitro. Two different types of sensing mechanisms are employed. The first method is based on harnessing bacterial power, and in the second method we use genetically engineered bacteria. The small size of the devices gives them access to individual cells, and their large numbers permit simultaneous monitoring of many cells. In the second part, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic micro transporters driven by external magnetic fields capable of exerting forces at the pico Newton scale. We develop micro transporters using a simple, single step micro fabrication technique that allows us to produce large numbers in the same step. We also fabricate microgels to deliver drugs. We demonstrate that the micro transporters can be navigated to separate single cells with micron-size precision and localize microgels without disturbing the local environment

Liquid Metal-Based Multifunctional Micropipette for 4D Single Cell Manipulation.

A novel manufacturing approach to fabricate liquid metal-based, multifunctional microcapillary pipettes able to provide electrodes with high electrical conductivity for high-frequency electrical stimulation and measurement is proposed. 4D single cell manipulation is realized by applying multifrequency, multiamplitude, and multiphase electrical signals to the microelectrodes near the pipette tip to create 3D dielectrophoretic trap and 1D electrorotation, simultaneously. Functions such as single cell trapping, patterning, transfer, and rotation are accomplished. Cell viability and multiday proliferation characterization has confirmed the biocompatibility of this approach. This is a simple, low-cost, and fast fabrication process that requires no cleanroom and photolithography step to manufacture 3D microelectrodes and microchannels for easy access to a wide user base for broad applications

A microgripper for single cell manipulation

This thesis presents the development of an electrothermally actuated microgripper for the manipulation of cells and other biological particles. The microgripper has been fabricated using a combination of surface and bulk micromachining techniques in a three mask process. All of the fabrication details have been chosen to enable a tri-layer, polymer (SU8) - metal (Au) - polymer (SU8), membrane to be released from the substrate stress free and without the need for sacrificial layers. An actuator design, which completely eliminates the parasitic resistance of the cold arm, is presented. When compared to standard U-shaped actuators, it improves the thermal efficiency threefold. This enables larger displacements at lower voltages and temperatures. The microgripper is demonstrated in three different configurations: normally open mode, normally closed mode, and normally open/closed mode. It has-been modelled using two coupled analytical models - electrothermal and thermomechanical - which have been custom developed for this application. Unlike previously reported models, the electrothermal model presented here includes the heat exchange between hot and cold arms of the actuators that are separated by a small air gap. A detailed electrothermomechanical characterisation of selected devices has permitted the validation of the models (also performed using finite element analysis) and the assessment of device performance. The device testing includes electrical, deflection, and temperature measurements using infrared (IR) thermography, its use in polymeric actuators reported here for the first time. Successful manipulation experiments have been conducted in both air and liquid environments. Manipulation of live cells (mice oocytes) in a standard biomanipulation station has validated the microgripper as a complementary and unique tool for the single cell experiments that are to be conducted by future generations of biologists in the areas of human reproduction and stem cell research

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

High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution

マルチ スケール キノウ ヲ ユウスル コウソク ジドウ マイクロ マニピュレーション システム

Ebubekir Avci, Chanh-Nghiem Nguyen, Kenichi Ohara, Yasushi Mae, Tatsuo Arai, Analysis and suppression of residual vibration in microhand for high-speed single-cell manipulation, International Journal of Mechatronics and Automation, 2013-Vol.3, No.2, pp.110-11