Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, cloning mammalian species through somatic cell nuclear transfer has been simply inefficient in all species in which live clones have been produced, such as ‘Dolly’ the sheep, and ‘Samrupa’ the buffalo. Most of the experiments resulted failure, and the success rate ranges from 0.1% to 3%. Developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Researchers have tried strategies to improve the efficiency of nuclear transfer. However, significant breakthroughs are yet to happen. The enucleation procedure consisting of extracting reprogrammable genetic material during nuclear transfer has been linked to inefficiencies due to manual error, lack of repeatability and decreased high throughput. Conventional manual enucleation process requires a series of complicated cell rotation in three-dimensional (3D) spaces using a blunt or sharp tipped pipette, and can puncture the cell during genetic material extraction. Current methods frequently damage the cell via physical or chemical contact, and thus have low throughput. Therefore, there is a need for simple, readily automated, non-contact methods for controlled cell rotation. Precise rotation of the suspended cells is one of the many fundamental manipulations in a wide range of biotechnological applications, such as cell injection and enucleation. Noticeably scarce from the existing rotation techniques is 3D rotation of cells on one single chip. To bridge this gap, this research presents a means of controlled cell rotation for bovine oocytes around both the in-plane (yaw) and out-of-plane (pitch) axes using a simple, low cost biochip fabricated using a mixture of conventional lithography and low-cost micro-milling. It uses a phase varying dielectrophoresis (DEP)-based electrorotation (EROT) biochip platform, which has an open-top sub-millimetre square chamber enclosed by four sidewall electrodes and two bottom electrodes to induce torque to rotate the cells about two axes, thus 3D cell rotation for the first time. Before fabrication, phase varying DEP-based rotational electric field simulations were carried out in the designed rotation chamber. For this analysis, initial rotational fields are characterised for both in-plane and out-of-plane axes using multi-physics finite element software. Electrode shape and chamber design were optimised using realistic parameters for the medium and electrode material properties. Results showed remarkable promise to rotate dielectric particles in rotational field strengths of the order of 104 V/m. From simulations, a basic biochip design was optimised. Within the fabricated biochip, controlled rotations around the in-plane and out-of-plane axes were demonstrated, and the electric field activation frequency range and electrokinetic properties of the bovine oocytes were characterised. Rotation was measured via video image processing with data included on electronic annex. Results show controllable rotation in steps of 5 degrees around both axes with the same chip. In experiments, the maximum rotation rate reached 150°/s in yaw axis and 45-50°/s during pitch axis, while a smooth, stable and controllable rotation rate was found below 30-40°/s. Optimum rotation rates are found for inputs of 10 Vp-p at 500-800 kHz AC frequency for yaw-axis rotation, and 10-20 Vp-p and 10-100 kHz for pitch-axis rotation. In addition, zona intact and zona free oocytes are shown to have electrical equivalence and found no noticeable difference, generalising the bio-chips capability and results. Further, experimental results were used to validate the numerical solid shell model used in design and it was found that the bovine oocytes are highly polarizable than the surrounding medium. Finally, the dielectric properties of the oocytes were fully characterised enabling further design optimization in future, if desired. The biochip was successfully designed, optimised and experimentally validated, and successful rotation of bovine oocytes in 3D spaces was demonstrated. These results create a platform tool for biologists to utilise enucleation with high throughput efficiency and ease. In summary, this simple, transparent, low-cost, open-top, and biocompatible biochip platform, allows further function modules to be integrated and is the foundation for more powerful cell manipulation systems. In brief key novel aspects of the research were: • Rotation of suspended spherical oocytes in multiple axes (3D rotation) was obtained by AC induced electric fields. • An open top biochip was successfully fabricated to enable further processing of the rotated cell in 3D spaces. • Bovine oocyte dielectric spectra were analysed in both in-plane and out-of-plane axes for the first time. • Bovine oocytes were determined to behave as solid spherical spheres, rather than single spherical shells.

No description available.

Nanoparticle based devices are becoming of great interest because of their single-electron transport behavior, and high surface charge density. Nanoparticle based devices operate at low power, and are potentially highly stable and extremely robust. Making interconnections to nanoparticle devices, however, has been an impending issue. Also percolating/conductive array of nanoparticles is not easy to build since repulsion between the charged nanoparticles causes them to deposit at distance significantly larger for electron tunneling. In this study, we resolve these challenges to make nanoparticle based electronic devices. Using biological (bacteria) or physical (polyelectrolyte fiber) scaffolds, we selectively deposited percolating array of 30 nm Au nanoparticles, to produce a highly versatile nanoparticle-organic hybrid device. The device is based on electron tunneling phenomena, which is highly sensitive to change in inter-particle distance and dielectric constant between nanoparticles. The key to building this structure is the molecular brushes on the surface of the scaffold, which shield the charge on nanoparticle to allow for percolating deposition. The electrostatic attraction for such a deposition on bacteria was measured to be so strong (0.038 N/m) that it could bend a 400 nm long and 25 nm wide gold nanorod. Once the device is built, the hygroscopic scaffolds were actuated by humidity, to modulate the electron tunneling barrier width (or height) between the metallic nanoparticles. A decrease in inter-particle separation by < 0.2 nm or a change in the dielectric constant from ~ 40 to 3 (for humidity excursion from 20% to ~0%), causes a 40-150 fold increase in electron tunneling current. The coupling between the underlying scaffold and the Au particle structure is essential to achieving such a high and robust change in current. In contrast to most humidity sensors, the sensitivity is extremely high at low humidity. This device is >10-fold better than standard microelectronic and MEMS technology based humidity sensors. After the deposition, the "live" bacterial scaffold retains its biological construct, providing an avenue for active integration of biological functions with electronic transport in nanoparticle device. Such hybrids will be the key to conceptually new electronic devices that can be integrated with power and function of microorganisms, on flexible plastic-like substrates using simple beaker chemistry. The technology has broad potential based on variety of nanoparticles (for example, magnetic, metallic and semi-conducting) to make electro-optical and inorganic devices, bringing a prominent advancement in the present technology. Our work is published in, Angewandte Chemie, JACS and Nano Letters, and featured in places such as, Discover Magazine, Science News and Nature. / Ph. D.

Micromanipulation is considered a challenging task which requires high precision motion and measurement at the micro scale. When micromanipulation is concerned with living organisms important considerations need to be addressed. These include the physical or chemical properties of micro-organisms, living conditions, responses to the environment and achieving suitably delicate manipulation. Bio-micromanipulation can include micro surgery or cell injection operations, or to determine interaction forces as the basis to investigate behavior and properties of living micro-organisms. In order to achieve suitable bio-micromanipulation appropriate processes and/or sensory systems need to be investigated. This thesis aims to look into the force interaction and sensing addressing two distinctive challenges in the field of bio-micromanipulation. To this end, this thesis presents two major contributions to advancing bio-micromanipulation. Firstly, a novel Haptic Microrobotic Cell Injection System is introduced which is able to assist a bio-operator through haptic interaction. The system introduces a mapping framework which provides an intuitive method for the bio-operator to maneuver the micropipette in a manner similar to handheld needle insertion. To accurately control the microrobot, a neuro-fuzzy modeling and control scheme has been developed. Volumetric, axial and planar haptic virtual fixtures are introduced to guide the bio-operator during cell injection. Aside from improving real-time operator performance using the physical system, the system is novel in facilitating virtual offline operator training. Secondly, a first-of-its-kind micro-pillar based on-chip system for dynamic force measurement of C. elegans motion is introduced. The system comprises a microfabricated PDMS device to direct C. elegans into a matrix of micropillars within a channel mimicking its dwelling environment. An image processing algorithm is able to track the interaction of the C. elegans with the pillars and estimate contact forces based on micropillar deflections. The developed micropillar system is capable of measuring the force with sub-micron resolution while providing a continuous force output spectrum.

In this work, MEMS based fabrication is used to engineer multifaceted enhancements to microfluidic systems such as lab-on-a-chip devices. Two specific elements of microfluidic systems are the focus of this study: microfluidic chambers and microneedles. Microfluidic chambers, which are back-end passive elements, via proposed material and structural modifications, are shown to exhibit reduced non-specific DNA binding and enable increased cell lysis efficiency. Microneedles, which are front-end interfacing elements, have been fabricated in silicon and in silicon dioxide varieties. The geometry of silicon microneedles has been varied via DRIE processing to yield sharpened tips. Sharpening of microneedle tips provides reduced skin insertion force without compromising structural strength. Variation of skin insertion force of microneedles with change in tip sharpness has been studied, and toughness of human skin derived to be approximately 26 kJ/m². The axial and shear fracture limits of the microneedles have also been studied. Axial fracture of 36 gauge silicon needles takes place at an average force of 740gf. Shear fracture force of silicon needles varies from 275gf (33 gauge needles) to 35.6gf (36 gauge needles). Fracture limits of circular and square shaped silicon dioxide needles show reduced strength of square needles; which is pronounced in the case of shear fracture.

La mesure de cellules biologiques constitue une étape de routine dans de nombreuses investigations en biologie. Les techniques actuelles utilisées par les biologistes sont principalement basées sur l'utilisation marqueurs optiques de coloration ou fluorescents, qui fournissent des observations moléculaires et cellulaires très précises et efficaces. Dans ce contexte, la spectroscopie diélectrique micro-ondes pour analyse cellulaire constitue une méthode nouvelle et attrayante, en raison du manque de préparation et manipulation des cellules, sans besoin d'ajout de produits chimiques, qui pourraient interférer avec d'autres constituants cellulaires. Sa compatibilité avec l'analyse de cellules uniques, potentiellement en temps réel, constitue également deux atouts importants de la technique d'analyse. Les travaux de cette thèse ont donc porté sur l'optimisation d'un biocapteur hyperfréquence microfluidique, dédié à la spectroscopie diélectrique de cellules biologiques uniques, et au développement de sa métrologie pour accéder au comportement diélectrique de cellule soumise à des stimuli chimique. Après un état de l'art sur les techniques courantes d'analyse de cellule individuelle, nous nous sommes attachés à optimiser le biocapteur hyperfréquence pour en améliorer les performances en sensibilité et en répétabilité. Ces optimisations ont porté sur le procédé de micro-fabrication, l'architecture du composant, que ce soit au niveau mécanique vis à vis de l'efficacité de blocage d'une cellule unique, mais aussi d'un point de vue électromagnétique avec une étude paramétrique. Ces études ont été validées dans un premier temps expérimentalement par la mesure de billes de polystyrène, modèle diélectrique simplifié par rapport à la complexité d'une cellule biologique, puis sur cellules individuelles vivantes dans leur milieu de culture. Le banc de caractérisation a également été optimisé afin de permettre la mesure diélectrique de cellules au cours du temps, et notamment en réaction à un stimulus d'ordre chimique. La cinétique de réaction d'une cellule unique soumise à de la saponine a été enregistrée automatiquement pour différentes cellules. Ces travaux ouvrent ainsi la voie à l'analyse à l'échelle cellulaire par spectroscopie diélectrique micro-onde de processus biologiques complexes en temps réel. / The measurement of biological cells is a routine step in many biological investigations. Current techniques used by biologists are mainly based on staining or fluorescent labelings, which provide very precise and effective molecular and cellular observations. Within this context, the microwave dielectric spectroscopy for cell analysis represents a new and attractive method, due to the lack of cells preparation and manipulation, without adding chemicals that could interfere with other cellular constituents. Its compatibility with the analysis of single-cells, potentially in real-time monitoring, constitute also two major assets of the analysis technique. This PhD thesis therefore focused on the optimization of a microfluidic and microwave based biosensor, which is dedicated to the dielectric spectroscopy of individual biological cells, and the development of its metrology to assess the dielectric behavior of cells subjected to chemical stimuli. After a state of the art on the current techniques available to analyze single cells, we focused on the optimization of the microwave biosensor to improve its performances in terms of sensitivity and repeatability. These optimizations dealt with the microfabrication process, the component architecture through the investigation of single cell loading efficacy as well as an electromagnetic parametric study. These developments were validated first experimentally with the measurement of polystyrene beads, which present a simplified dielectric model compared to the complexity of a biological cell, followed then by living individual cells in their culture medium. The test bench was also optimized to allow the dielectric measurement of cells over time, and especially in response to a chemical stimulus. The reaction kinetics of a single-cell subjected to saponin was recorded automatically for different cells. This work opens the door to single-cell analysis with microwave dielectric spectroscopy of complex biological processes in real-time.

abstract: Demand for biosensor research applications is growing steadily. According to a new report by Frost & Sullivan, the biosensor market is expected to reach $14.42 billion by 2016. Clinical diagnostic applications continue to be the largest market for biosensors, and this demand is likely to continue through 2016 and beyond. Biosensor technology for use in clinical diagnostics, however, requires translational research that moves bench science and theoretical knowledge toward marketable products. Despite the high volume of academic research to date, only a handful of biomedical devices have become viable commercial applications. Academic research must increase its focus on practical uses for biosensors. This dissertation is an example of this increased focus, and discusses work to advance microfluidic-based protein biosensor technologies for practical use in clinical diagnostics. Four areas of work are discussed: The first involved work to develop reusable/reconfigurable biosensors that are useful in applications like biochemical science and analytical chemistry that require detailed sensor calibration. This work resulted in a prototype sensor and an in-situ electrochemical surface regeneration technique that can be used to produce microfluidic-based reusable biosensors. The second area of work looked at non-specific adsorption (NSA) of biomolecules, which is a persistent challenge in conventional microfluidic biosensors. The results of this work produced design methods that reduce the NSA. The third area of work involved a novel microfluidic sensing platform that was designed to detect target biomarkers using competitive protein adsorption. This technique uses physical adsorption of proteins to a surface rather than complex and time-consuming immobilization procedures. This method enabled us to selectively detect a thyroid cancer biomarker, thyroglobulin, in a controlled-proteins cocktail and a cardiovascular biomarker, fibrinogen, in undiluted human serum. The fourth area of work involved expanding the technique to produce a unique protein identification method; Pattern-recognition. A sample mixture of proteins generates a distinctive composite pattern upon interaction with a sensing platform consisting of multiple surfaces whereby each surface consists of a distinct type of protein pre-adsorbed on the surface. The utility of the "pattern-recognition" sensing mechanism was then verified via recognition of a particular biomarker, C-reactive protein, in the cocktail sample mixture. / Dissertation/Thesis / Ph.D. Electrical Engineering 2011

Diseases of the heart have been the most common cause of death in the United States since the middle of the 20th century. The development of engineered cardiac tissue over the last three decades has yielded human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes (CMs), microscale “heart-on-a-chip” platforms, optical interrogation techniques, and more. Having spawned its own scientific field, ongoing research promises lofty goals to address the heart disease burden around the world, such as patient-specific disease models, and clinical trials on chip-based platforms. The greatest academic pursuit for engineered cardiac tissues is to increase their maturity, thereby increasing relevance to native adult tissue. Investigation of cardiomyocyte maturity necessitates the development of 3D-tissue compatible techniques for measuring and perturbing cardiac biology with enhanced precision. This dissertation focuses on the development of biological microelectromechanical systems (BioMEMS) for precision measurement and perturbation of cardiac tissue. We discuss three unique approaches to interfacing MEMS-based tools with cardiac biology. The first is a high resolution magnetic sensor, which directly measures the spatial gradient of a magnetic field. This has an ideal application in magnetocardiography (MCG), as the flux of ions during cardiac contractions produces measurable magnetic signals around the tissue and can be leveraged for noncontact diagnosis. The second is a highly functionalized heart-on-a-chip platform, wherein the mechanical contractions of cardiac microtissues can be simultaneously recorded and actuated. Contractile dynamics are leading indicators of maturity in engineered cardiac tissue and mechanical conditioning has shown recent promise as a critical component of cardiac maturation. The third is the imaging of contractile nanostructures in engineered cardiomyocytes at depth in a 3D microtissue. We use small angle X-ray scattering (SAXS) to discern the periodic arrangement of myofilaments in their native 3D environment. We enable a significant structural analysis to provide insight for functional maturation. Enabling these three thrusts required developing two supporting technologies. The first is the engineered control of dynamic second order systems, a foundational element of all our MEMS and magnetic techniques. We demonstrate numerous algorithms to improve settling time or decrease dead-time such that samples with fast temporal effects can be measured. The second is a microscale gluing technique for integrating myriad of materials with MEMS devices, yielding unique sensors and actuators. / 2022-05-15T00:00:00Z

Intravenously delivered cancer drugs face transport limitations at the tumor site and cannot reach all parts of tumors at therapeutically effective concentrations. Transport limitations also prevent oxygen from distributing evenly in tumors resulting in hypoxia, which plays a critical role in cancer progression. In this dissertation, I present the development of micro-devices that mimic transport limitations of drugs and nutrients on three dimensional tumor tissues, enable visualization and quantification of the ensuing gradients, and enable simple analysis and mathematical modeling of obtained data. To measure the independent effects of oxygen gradients on tumor tissues, an oxygen delivery device that used microelectrodes to generate oxygen directly underneath three-dimensional tumor cylindroids was developed. Supplying oxygen for 60 hours eliminated the necrotic region typically found in the center of cylindroids. Dead cells were observed moving away from the location of oxygen delivery. These measurements show that oxygen gradients are an important determinant of cell viability and rearrangement. Another micro-device was developed to mimic the delivery and systemic clearance of therapeutic agents. This microfluidic device consisted of cuboidal tumor tissue subjected to continuous medium perfusion along one face. The device was used to measure the spatiotemporal dynamics of the accumulation of therapeutic bacteria in tumors. Suspensions of Salmonella typhimurium and Escherichia coli strains were delivered to tumor tissues for 1 hour. Bacterial motility strongly correlated (R2 = 99.3%) with the extent of tissue accumulation. Based on spatio-temporal profiles and a mathematical model of motility and growth, bacterial dispersion was found to be necessary for deep penetration into tissue. These results show that motility is critical for effective distribution of bacteria in tumors. The microfluidic device was further used to mimic the delivery and clearance of equal concentrations of doxorubicin and liposome-encapsulated doxorubicin (Doxil). A pharmacokinetic/pharmacodynamic model incorporating mechanisms of tissue-level diffusion and binding was developed and experimental data was fit to this model. Doxorubicin was found to have the optimal diffusivity and binding for maximizing therapeutic effect. Doxil was severely limited by low intratumor drug release. These results show that in-vitro models mimicking tissue-level transport limitations more accurately predict the therapeutic response of drugs.

Aux petites échelles, les équilibres physiques sont bouleversés. En particulier, les forces de contact, de friction et d'adhésion deviennent prépondérantes au regard des autres effets, perturbant ainsi la manipulation des entités appartenant au micro-monde.<br />Ce travail apporte une contribution aux techniques de micromanipulation sans contact dans les microsystèmes intégrés. En nous appuyant sur le principe de la lévitation diamagnétique, qui bénéficie très favorablement de la réduction d'échelle, nous supprimons tout contact physique entre les dispositifs et les microparticules manipulées.<br />Ce point clé nous a permis de démontrer, à travers des structures intégrées et/ou prototypes, la faisabilité d'une microfluidique digitale dans l'air et sans contact, et entre autres le confinement, le micropositionnement et l'actionnement sans contact de divers bioparticules. Ces réalisations ouvrent des perspectives intéressantes au développement de microréacteurs biochimiques sans contamination.