My MSc project aims at developing pH-responsive molecular tweezers for drug delivery applications. The project began with the synthesis of our 2nd generation tweezer, whose main objective was to improve our previous model, 1st generation tweezer, which contained a pH-responsive triad spacer and two naphthalene walls known to interact with hydrophobic drugs such as Mitoxantrone®. The naphthalene interaction sites were successfully modified to contain oligoethylene glycol chains to improve their water-solubility, in anticipation for more accurate measurements of pKa and binding constants in aqueous media. However, all attempts to convert such naphthalene derivatives into their corresponding boronic acid or ester through standard protocols (halogen-lithium exchange, palladium catalyzed borylation) failed. Without the required boronic acid/ester, the final Suzuki-Miyaura coupling with the di-bromo triad spacer was not achieved. Synthesis of the 3rd generation tweezer, which was modified to contain theophylline as the new interaction sites, was then attempted. The half-tweezer was successfully synthesized via copper (II) catalyzed coupling of theophylline with the 5-bromo-4-methoxyphenyl boronic acid. However, all attempts to convert it into the required boronic acid/ ester for the final Suzuki- Miyaura coupling reaction with 2,6-dibromopyridine failed. We then focused our attention on the conversion of the triad spacer into its corresponding diboronic acid. The synthesis of the triad diboronic acid was a success, however, the final copper (II) catalyzed reaction with theophylline to form the tweezer only yielded the mono-coupled product. Lastly, our 4th generation tweezer was engineered to avoid the synthetic difficulties encountered in the boronic acid/ ester synthesis stage. Using the commercially available 5-formyl-2-methoxyphenylboronic acid and o-phenylenediamine, we successfully synthesized a benzimidazole-derived “half tweezer” through ring condensation reaction. Alkylation of this half-tweezer was also successfully achieved, although purification of the alkylated product was not optimized. Using this crude product, we carried out the final tweezer reaction via Suzuki- Miyaura coupling with 2,6-dibromopyridine under microwave irradiation. 1H NMR results show formation of new species that is believed to be the 4th generation tweezer (although the presence of impurities made integration of the signals unreliable). Much work is needed in the purification of the alkylated half tweezer boronic acid in order to avoid complicated mixtures in the final tweezer reaction. / Thesis (Master, Chemistry) -- Queen's University, 2011-10-06 00:29:04.248

This research presents the development of an advanced, state-of-the-art optical trap for use in biological materials and nanosystems investigation. An optical trap is an instrument capable of manipulating microscopic particles using the inherent momentum of light. First introduced by Askin et al., the single beam gradient optical trap is capable of generating small forces (~1-100 pN) in a noninvasive manner. As a result, the optical trap is often used to manipulate biological specimen. This research presents the process for the construction of a custom optical trap, the methods to build a controllable optical trap through a traditional fixed gain controller as well as an adaptive controller, and also enables the application of torque to trapped particles. A method of using adaptive techniques for system identification and calibration is also presented. This research has the potential to use forces and torques to affect our understanding of the mechanics of single molecules and motor proteins. This instrument provides a more precise means of manipulating biological specimen as well as a tool for nanofabrication and has the potential to expand the knowledge base of DNA, chromosomes, biomotors, motor proteins, reversible polymers, and can be used to control chemical reactions. The research presented here documents the creation of an optical trap that is sensitive for applications requiring precise displacements and forces, adaptable to a variety of current and future research applications, and useable by anyone interested in researching micro- and nanosytems. / Dissertation

Integrated microfluidic devices for biomedical analysis attract lots of interest in the MEMS (Micro-Electro-Mechanical-Systems) research field. However, the characteristic Reynolds number for liquids flowing in these microchannels is very small (typically less than 10). At such low Reynolds numbers, turbulent mixing does not occur and homogenization of the solutions occurs through diffusion processes alone. Hence, a satisfactory mixing performance generally requires the use of extended flow channels and takes longer to accomplish such that the practical benefits of such devices are somewhat limited. Consequently, accomplishing the goal of u¡VTAS requires the development of enhanced mixing techniques for microfluidic structures. This study first presents a microfluidic mixer utilizing alternatively switching electroosmotic flow and proposes two microchannel designs of T-form and double-T-form micromixer. Switching DC field is used to generate the electroosmotic force to drive the fluid and also used for mixing of the fluids simultaneously, such that moving parts in the microfluidic device and delicate external control system are not required for the mixing purpose. Furthermore, this study also proposed a novel pinched-switching mode in the T-form microfluidic mixer, which could be effectively increase the perturbation within the fluid to promote the mixing efficiency. In this study, computer simulation for the operation conditions is used to predict the mixing outcomes and the mixing performance is also confirmed experimentally. Result shows the mixing performance can be as larger as 95% within the mixing distance of 1 mm downstream the common boundary between the different sample fluids. The novel method proposed in this study can be used for solving the mixing problem in a simple way in the field of micro-total-analysis-systems. Furthermore, in order to demonstrate the proposed micromixer is feasible for on-line bio-reaction, this study designs a fully integrated device for demonstration of DNA/enzyme reaction within the microfluidic chip. The microchip device contains a pre-column concentrating region, a micro mixer for DNA-enzyme mixing, an adjustable temperature control system and a post-column concentration channel. The integrated microfluidic chip has been used to implement the DNA digestion and extraction. Successfully digestion of £f-DNA using EcoRI restriction enzyme in the proposed device is demonstrated utilizing large-scale gel electrophoresis scheme. Results show that the reaction speed doubled while using the microfluidic system. In addition, on-line DNA digestion and capillary electrophoresis detection is also successfully demonstrated using a standard DNA-enzyme system of $X-174 and Hae III. Finally, this reasearch also proposes a novel cell/microparticle manipulation platform by integrating an optical tweezer system and a micro flow cytometer. During operation, electrokinetically driven sheath flows are utilized to focus microparticles to flow in the center of the sample stream then pass through an optical manipulation area. An IR diode laser is focused to generate force gradient in the optical manipulation area to manipulate the microparticles in the microfluidic device. Moving the particles at a static condition is demonstrated to confirm the feasibility of the home-built optical tweezer. The trapping force of the optical tweezer is measured using a novel method of Stocks-drag equilibrium. The proposed system can continuously catch moving microparticles in the flowing stream or switch them to flow into another sample flow within the microchannel. Target particles can be separated from the sample particles with this high efficient approach. More importantly, the system demonstrates a continuously manipulation of microparticles using non-contact force gradient such that moving parts and delicate fabrication processes can be excluded. The proposed system is feasible of high-throughput catching, moving, manipulation and sorting specific microparticles/cells within a mixed sample and results in a simple solution for cell/microparticle manipulation in the field of micro-total-analysis-systems. In this thesis, low-cost soda-lime glass substrates are adopted for the microchip fabrication using a simple and reliable fabrication process. Three kinds of novel microfluidic devices including an electrokinetically-driven microfluidic mixer, a high throughput DNA/enzyme reactor and an optically cell manipulation platform are successfully demonstrated. It is the author¡¦s believes that the results of this study will give important contributions in the development of micro-total-analysis-systems in the future. With the success of this study, we have a further step approaching to the dream of lab-on-a-chip system for bio-analytical applications.

付記する学位プログラム名: 京都大学卓越大学院プログラム「先端光・電子デバイス創成学」 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24123号 / 理博第4851号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 高橋 義朗, 教授 石田 憲二, 教授 田中 耕一郎 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The development of ultrasound contrast agents, or microbubbles, over the past 40 years has increased the possibilities for diagnostic imaging, although, more recently they have been proposed as a new vehicle for delivery of drugs and genes. However, there yet remains a considerable lack of fundamental understanding of microbubble behaviour under ultrasound excitation which has restricted their translation to therapeutic use. This project focussed on three key areas relating to the generation, observation, and bioeffects of microbubbles and the ultrasound used in their excitation. The experimental endeavour involved first, a full characterisation of the performance of a rotating mirror high-speed camera (Cordin 550-62) that was previously used by our group [and others] to investigate microbubble dynamics. Specifically, the investigation begins with an assessment of the frame-rate reporting accuracy of the system, a key aspect to the robustness of quantitative measurements extracted from recorded image sequences. This is then followed by the demonstration of a novel method of analysis for examining the image formation process in this type of camera, which facilitates a sensor-by-sensor assessment of performance that was not previously realised. Consolidating with previous work from within the group, this new analysis method was used to clarify previous data, and in the process suggested the presence of a temporal anomaly embedded within recorded images. In addition, the analysis also revealed empirical evidence for the mechanisms leading to this anomaly. Following on, a holographic optical tweezer system was developed for the purpose of exercising precise spatial control over microbubbles within their experimental environment. By positioning microbubbles in specific arrangements, interesting behaviours that were not previously achieved experimentally in the context of shelled microbubbles, were observed. Furthermore, by careful positioning of microbubbles within the imaging plane, it was possible to exploit the temporal anomaly present in the camera to greatly improve the integrity of data recorded, and to also operate in an enhanced imaging mode. Group aspirations to accelerate the development of therapeutic microbubbles had previously generated some early work on the in-house generation of bespoke bubble populations using microfluidic lab-on-a-chip techniques. In order to facilitate further development in this area, a finite-element computational model was herein developed to aid next generation chip design. Finally, in a slightly different context, considering not only the mechanical effect a microbubble may effect in a therapeutic treatment, a single biological cell assay was developed in order to probe any mechanical effects that were induced by the excitation ultrasound itself. Capitalising on the precise force control possible with atomic force spectroscopy, the elastic moduli of cells pre- and post-ultrasound insonation (sans microbubbles) were recorded. These new developments have extended the group capability and expertise in the areas of high-speed imaging, experimental observations of microbubble dynamics and with microfluidic generation of microbubbles. Additionally, the insights garnered have both served to consolidate the group's previous and as yet unpublished data, opening the way for circulation with absolute confidence in the integrity of that data.

abstract: In the frenzy of next generation genetic sequencing and proteomics, single-cell level analysis has begun to find its place in the crux of personalized medicine and cancer research. Single live cell 3D imaging technology is one of the most useful ways of providing spatial and morphological details inside living single cells. It provides a window to uncover the mysteries of protein structure and folding, as well as genetic expression over time, which will tremendously improve the state of the fields of biophysics and biomedical research. This thesis project specifically demonstrates a method for live single cell rotation required to image them in the single live cell CT imaging platform. The method of rotation proposed in this thesis uses dynamic optical traps generated by a phase-only spatial light modulator (SLM) to exert torque on a single mammalian cell. Laser patterns carrying the holographic information of the traps are delivered from the SLM through a transformation telescope into the objective lens and onto its focal plane to produce the desired optical trap "image". The phase information in the laser patterns being delivered are continuously altered by the SLM such that the structure of the wavefront produces two foci at opposite edges of the cell of interest that each moves along the circumference of the cell in opposite axial directions. Momentum generated by the motion of the foci exerts a torque on the cell, causing it to rotate. The viability of this method was demonstrated experimentally. Software was written using LabVIEW to control the display panel of the SLM. / Dissertation/Thesis / M.S. Bioengineering 2013

This thesis presents the development and application of nanoscale techniques to trap and / Chemistry and Chemical Biology

In this thesis, an approach for developing single-photon sources at the 1550nm wavelength will be demonstrated, based on optical trapping of luminescent upconverting nanoparticles. A single-photon source is a source that emits a single photon at a time, and hence it is a source of quantum bits that constitutes the basic building units in quantum computers and quantum communications. The approach exploits the plasmonic properties of gold films and the waveguiding characteristics of single mode optical fibers (SMFs). We start by planar nanofabrication of subwavelength nanoapertures in a thin gold film based on finite difference time domain simulations for a peak transmission at the wavelength in question. Subsequently, using ultraviolet curable epoxy adhesion material, a nanoaperture patterned on a gold film can be transferred to an SMF tip forming a nanoantenna enhanced optical fiber tweezer (NAFT). As a final step in building the optical tweezer, a test of the capability of the integrated optical fiber tweezer to trap 20 nm, and 30nm polystyrene nanospheres, as well as luminescent upconverting nanoparticles (UCNPs), has been experimentally realized with encouraging results. In addition to the optical trapping of the luminescent nanoparticles, the nano aperture antenna can improve light coupling into the low loss optical fiber guiding channel. Also, it could have a positive influence on enhancing the photon emission rate through the Purcell effect. Furthermore, we have combined NAFT with a low insertion loss wave splitter, a wavelength-division multiplexer (WDM), to allow measuring the 1550nm photon-emission statistics on a cooled superconducting nanowire single-photon detector (SNSPD) at ~ 2.4o K. Eventually, nanoantenna enhanced optical fiber tweezers can play an essential role in optical trapping towards developing single-photon sources and the emerging technology of quantum information processing, computation, and cryptography. / Graduate

<p>Microfluidics has established itself as a key technology in a wide range of fields including pharmaceuticals, point-of-care diagnostics, thermal management, and space technology. Most of these applications involve manipulation of small quantities (micro – nanoliters) of fluids and various particles or biological cells suspended in them. These platforms employ mechanical, thermal, acoustic, magnetic, optical, electric and many other means for creating particle and fluid motion. Many biological applications require handling cells that are vulnerable to getting damaged if proper physiological conditions are not maintained or if excessive force is applied on them. The non-invasive nature of optical and electrical micro-manipulation techniques such as rapid electrokinetic patterning (REP) has proven to be of great importance in such applications. These techniques enable handling, transportation, sorting and arrangement of fragile synthetic micro/nanoparticles and biological cells without compromising their structure and surface properties.</p> <p>REP is a recently developed micro-manipulation tool that employs optically induced electrothermal vortices to create custom flow patterns. Particle suspensions are entrained in these vortices and are trapped on an electrode surface through AC electrokinetic mechanisms. This work focuses on characterizing a REP trap and discusses its potential applications in handling biological cells. Polystyrene microparticles are confined in a REP trap and a MATLAB program is used to track their motion inside the trap. The tracked particle trajectory reveals that the potential energy of the trapped particle is parabolic and hence the trap is Hookean in nature. The trap is modelled as a spring-mass system and the stiffness coefficient of that system is found to be of the order of 10^-15 N/μm. The origin of the restoring force in the spring-mass model is found to be the drag force created by the electrothermal vortex. The ability to exert ultra-small forces in a stable trap enables REP to be used in various non-invasive particle manipulation applications.</p> <p>The transient nature of REP is studied using numerical modeling and particle image velocimetry (PIV) analysis of a vortex created by a moving laser spot. A numerical model suggests that custom-shaped steady state REP vortices can be created via superposition of multiple axisymmetric circular shaped vortices. However, the method of superposition cannot be extended to transient traps and a more involved 3D model is required to simulate them. The laser spot is scanned back-and-forth in a line with different speeds to create transient REP vortices. The PIV analysis, in agreement with the numerical model, shows that the location of the moving vortex is undiscernible at high speeds. Moreover, the circular shaped vortex is stretched out into a line when the laser scanning frequencies are more than 15 Hz.</p> <p>The particle-electrode attraction force, which entraps the particles at the electrode surface, is characterized using particle diffusometry (PD) and defocusing particle tracking. PD is used to measure the diffusion coefficient of polystyrene particles under different electric field parameters near an electrode surface. It is found that the particle diffusivity decreases with a decrease in the electric field frequency from 150 – 30 kHz and with an increase in the applied voltage from 4 – 8 V_pp. A MATLAB program is used to track the number of in-focus particles and their distance from the electrode surface. A histogram of the particles’ distance from the electrode surface shows an increase in the particle concentration near the electrode at low frequencies (30 – 60 kHz). These observations suggest that the average height of an entrapped particle decreases with a decrease in applied field frequency and an increase in applied voltage. This suggests that the attractive trapping force is significant at 30 kHz but diminishes at around 150 kHz.</p> <p>Salt and sugar-based isotonic media used for cell suspensions pose several challenges for electrokinetic mechanisms such as REP. Various solutions to overcome these challenges for bio-manipulation applications are discussed in this work. The presence of DC offset in the AC electric field is found to enhance particle entrapment in sugar-based media. The effect of DC offset on trapping performance in bio-relevant media is assessed by measuring the stability of the REP trap. This work also shows entrapment and manipulation of Mice pancreatic cancer cells (KPC2) suspended in the sugar-based isotonic media using REP. The biological applications of the REP technology are highly promising, but they have not yet been well-explored. This work lays the foundation of understanding how REP can be operated in high osmolarity media for bio-manipulation applications.</p>

The magnetic tweezer technique has become a versatile tool for unfolding or folding of individual molecules, mainly DNA. In addition to single molecule analysis, the magnetic tweezer can be used to analyze the mechanical properties of cells and extracellular matrices. We have established a magnetic tweezer that is capable of measuring the linear and non-linear viscoelastic behavior of a wide range of soft matter in precisely controlled environmental conditions, such as temperature, CO2 and humidity. The magnetic tweezer presented in this study is suitable to detect specific differences in the mechanical properties of different cell lines, such as human breast cancer cells and mouse embryonic fibroblasts, as well as collagen matrices of distinct concentrations in the presence and absence of fibronectin crosslinks. The precise calibration and control mechanism employed in the presented magnetic tweezer setup provides the ability to apply physiological force up to 5 nN on 4.5 µm superparamagnetic beads coated with fibronectin and coupled to the cells or collagen matrices. These measurements reveal specific local linear and non-linear viscoelastic behavior of the investigated samples. The viscoelastic response of cells and collagen matrices to the force application is best described by a weak power law behavior. Our results demonstrate that the stress stiffening response and the fluidization of cells is cell type specific and varies largely between differently invasive and aggressive cancer cells. Finally, we showed that the viscoelastic behavior of collagen matrices with and without fibronectin crosslinks measured by the magnetic tweezer can be related to the microstructure of these matrices.