Fabrication of Functional Gel-Microbead for Local Environment Measurement in Microchip

Proceedings of the 2008 IEEE International Conference on Robotics and Automation / 2008 IEEE International Conference on Robotics and Automation, Pasadena, CA, USA, May 19-23, 200

Gel-tool Sensor Positioned by Optical Tweezers for Local pH Measurement in a Microchip

2007 IEEE International Conference on Robotics and Automation, Roma, Italy, 10-14 April 200

Microbead-Based Biosensing in Microfluidic Devices

Microbeads are frequently used as a solid support to capture target analytes of interest, such as proteins and nucleic acids, from a biological sample. The integration of microbeads into microfluidic systems for biological testing is an area of growing interest. Such lab-on-chip systems are designed to integrate several functions of a conventional laboratory onto a single chip. As a platform to capture targets, beads offer several advantages over planar surfaces such as large surface areas to support biological interactions (increasing sensitivity), the availability of libraries of beads of various types from many vendors, and array-based formats capable of detecting multiple targets simultaneously (multiplexing). This dissertation describes the development and characterization of microbead-based biosensing devices. A customized hot embossing technique was used to stamp an array of microwells in a thin plastic substrate where appropriately functionalized agarose microbeads were selectively placed within a conduit. Functionalized quantum dot nanoparticles were pumped through the conduit and used as a fluorescent label to monitor binding to the bead. Three-dimensional finite element simulations were carried out to model the mass transfer and binding kinetics on the beads’ surfaces and within the porous beads. The theoretical predictions were critically compared and favorably agreed with experimental observations. A novel method of bead pulsation was shown to improve binding kinetics in porous beads. In addition, the dissertation discusses other types of bead arrays and demonstrates alternative bead-based target capture and detection strategies. This work enhances our understanding of bead-based microfluidic systems and provides a design and optimization tool for developers of point-of-care, lab-on-chip devices for medical diagnosis, food and water quality inspection, and environmental monitoring

A disposable bio-nano-chip usuing agarose beads for protein analysis

This thesis reports on the fabrication of a disposable bio-nano-chip (BNC), a microfluidic device composed of polydimethylsiloxane (PDMS) and thiolene-based optical epoxy which is both cost-effective and suitable for high performance immunoassays. A novel room temperature (RT) bonding technique was utilized so as to achieve irreversible covalent bonding between PDMS and thiolene-based epoxy layers, while at the same time being compatible with the insertion of agarose bead sensors, selectively arranged in an array of pyramidal microcavities replicated in the thiolene thin film layer. In the sealed device, the bead-supporting epoxy film is sandwiched between two PDMS layers comprising of fluidic injection and drain channels. The agarose bead sensors used in the device are sensitized with anti-C-reactive protein (CRP) antibody, and a fluorescent sandwich-type immunoassay was run to characterize the performance of this device. Computational fluid dynamics (CFD) was used based on the device specifications to model the bead penetration. Experimental data revealed analyte penetration of the immunocomplex to 100μm into the 280μm diameter agarose beads, which correlated well with the simulation. A dose response curve was obtained and the linear dynamic range of the assay was established over 1ng/mL to 50ng/mL with a limit of detection less than 1ng/mL

Development of new adaptive materials based on spiropyran molecular photoswitches

Spiropyrans are a family of photochromic compounds that can be reversibly switched between two states: a colourless, non-polar, uncharged spiro form (SP) and a highly coloured, polar, conjugated, zwitterionic merocyanine form (MC), i.e. SP → MC (UV light), MC → SP (green, white light). Furthermore, the MC form possesses a phenolate group that can reversibly bind metal ions and this ion binding is also photo-reversible, as when the MC-ion complex reverts to the passive SP form (upon green/white light exposure), it releases the bound ions. Using molecular-switches based on spiropyran-like molecules, new materials were produced whose properties such as metal-ion uptake/release and polarity can be controlled under external photonic stimulation. Spiropyran derivatives were immobilised on polystyrene and silica microbeads and evaluated for their reversible photoswitchable metal ion binding behaviour. When in the MC form, in the presence of metal ions such as Cu2+ and Zn2+, further spectral and colour changes occurred that were found to vary according which metal ion was bound. Subsequent irradiation with white light caused reformation of the SP form and release of the metal ion. This process was shown to be repeatable at least several times. The spiropyran functionalised silica microbeads were packed into a capillary column and the new stationary phase demonstrated to form the basis of a photodynamic system for retention, detection and release of metal ions pumped into the capillary. In parallel other spiropyran derivatives were incorporated into monolithic stationary phases. The spiropyran-functionalised polymer was switched between a protonated MC form and a neutral SP form upon white light irradiation. The monoliths were encased on a microfluidic chip and the system filled with an acid electrolyte. When a voltage was applied at the two ends of the encased monolith, electro-osmotic flow was generated and the flow rate shown to be variable upon white light exposure as a consequence of the formation of the SP form and the formation/disruption of the surface charge necessary for efficient electro-osmotic pumping. In this thesis the synthesis and the characterisation of these new materials is described and the switchable optical properties evaluated

Towards Single Bacterium Detection: A Microelectronic/Microfluidic Hybrid System Based on a CMOS Technology

RÉSUMÉ Cette thèse porte sur le développement d'un biocapteur hybride CMOS microfluidique capable de détecter des bactéries pathogènes une à une en temps réel basé sur un principe de spectroscope impédimétrique. Le biocapteur proposé se compose d'une matrice de capteurs qui comportent une matrice de microélectrodes, desmultiplexeurs à commande numérique, et des circuits de détection intégrés sur une puce de silicium CMOS. Cette recherche propose une nouvelle structure de microélectrodes qui permet à une structure de microélectrodes face à face à haute densité intégrable par post-traitement d’une puce CMOS. Au lieu d’être créée par le dépôt et la gravure de couches métalliques supplémentaires, la structure de microélectrodes face à face est construite en exploitant un empilement de couches métalliques disponible avec la technologie CMOS adoptée. Les détecteurs sont obtenus en construisant des microcanaux qui traversent le substrat. Ces microcanaux passent entre les microélectrodes face à face. Lorsque les fluides où se trouvent les échantillons traversent le microcanal, le système détecte de façon continue les changements d'impédance entre les microélectrodes induits par le passage de chaque bactérie . Cette thèse étudie le processus de microfabrication qui permet de libérer la matrice de microélectrodes et de fabriquer les microcanaux traversant le substrat. Les techniques dites de FIB (pours Focused Ion Beam) et de DRIE (pour Deep Reactive Ion Etching) sont utilisées. Les forces et faiblesses de chaque technologie sont analysées et des recettes de processus optimisés sont étudiées. La matrice de microélectrodes a été réalisée avec succès par les deux technologies. Comme preuve de concept, plusieurs microcanaux traversant le substrat sont également formés en utilisant la technologie FIB. Cette thèse propose également un nouveau circuit de détection. Réalisé grâce à la micro-électronique, ce circuit est capable de détecter les changements d'impédance causés par le passage d’une seule bactérie dans un milieu conducteur. Sans conditionnement de signaux et de circuit de traitement complexes, tels que des amplificateurs de haute précision, des filtres ou des convertisseurs analogue à numérique ou numérique à analogique, les circuits de détection sont conçus pour offrir une bonne sensibilité et une configurabilité qui permet de l'adapter aux différentes conditions de détection. Une technique de mise en boîtier biocompatible est également mise en oeuvre pour encapsuler le capteur intégré tout en fournissant des interfaces fluidiques et électriques pour l'injection d'échantillons et de signaux électriques. Une nouvelle approche pour améliorer la sélectivité de détection basée sur l’utilisation de bactéries magnétotactiques est également proposée dans cette thèse. Sous le contrôle d’un champ magnétique extérieur, les bactéries magnétotactiques sont utilisées comme bio-transporteurs, qui peuvent chercher activement et capturer les bactéries pathogènes cibles afin de les amener à la zone de détection. Une puce microfluidique est fabriquée grâce à des techniques de prototypage rapide afin de valider les idées proposées et de fournir des guides de conception d'une puce plus avancés. Les résultats de microfabrication et les résultats des tests préliminaires montrent que l'intégration monolithique des technologies CMOS et microfluidique est possible et qu’elle permet la réalisation de microélectrodes face à face dans une plate-forme capable de détecter le passage d’une seule bactérie en isolation.----------ABSTRACT This thesis reports on the development of a CMOS Microfluidic hybrid biosensor technology that is proposed to detect single pathogenic bacterium in real time based on impedimetric spectroscopy. The proposed biosensor consists of a CMOS silicon die that incorporates a microelectrode array, digitally controlled multiplexers, and sensing circuits. This research proposes a novel microelectrode structure, which is obtained by first manufacturing high density face to face microelectrodes on a CMOS die, possible by a relatively simple CMOS post-processing. Instead of deposition and patterning of additional metal layers, the face to face microelectrode array is constructed by stacking metal and via layers of the adopted CMOS technology. By constructing through substrate microchannels in between pairs of face to face microelectrodes, when a fluid sample flows through the microchannel, the microelectrodes on the wall detect the impedance change induced by bacterium in the fluid in a continuous way. This thesis investigates the microfabrication process of releasing microelectrode arrays and constructing through substrate microchannels. FIB (Focused Ion Beam) and DRIE (Deep Reactive Ion Etching) technologies are utilized. The strength and weakness of each technology are analyzed and optimized process recipes are investigated. Microelectrode array were successfully released using both process technologies. As a proof of concept, several through substrate microchannels were also formed by using the FIB technology. This thesis also proposes a novel sensing microelectronic circuit, which is able to sense the impedance change caused by a single bacterium in a conductive medium. The system does not require complex signal conditioning and processing circuits, such as high precision amplifiers, filters or ADC/DAC. The proposed simple sensing structure offer high sensitivity, reliability and configurability. A dedicated biocompatible packaging is also implemented to encapsulate the CMOS die and provide a microchamber, fluidic and electrical interfaces for sample injection and signal interfaces. A new approach to achieve detection selectivity or specificity assisted by magnetotactic bacterium is also proposed in this thesis. Under the control of an external magnetic field, the viii magnetotactic bacteria are used as bio-carriers, which can actively search and capture some target pathogenic bacteria and bring them to the sensing area. A microfluidic chip is fabricated by rapid prototyping techniques to validate the proposed idea and to provide design guides for a more advanced and highly integrated CMOS chip. The achieved microfabrication results and preliminary testing results show that the monolithic integration of CMOS and microfluidic technology, especially the face to face microelectrode structure is a suitable platform for single bacterium detection and analysis

Cell encapsulation systems based on hybrid hydrogels

The entrapment of cells into biomaterials is one of the most appealing and usefulness tool in tissue engineering and cell based therapy applications. Cell encapsulation procedures allow the immunoisolation of cells from the surrounding environment, after their transplantation and the maintenance of the normal cellular physiology. In the current PhD work, various microencapsulation cell procedures are reported, based on a gas driven mono-jet device, a vibrating-nozzle procedure and microfluidics. All the analysed procedures were critically evaluated and applied to cells from different sources. The obtained microcapsules were characterized by excellent morphological characteristics and a very narrow size distribution. Interestingly, the results demonstrated that the microencapsulation procedures did not alter the morphology, viability and functions of the embedded cells. Moreover, the production of engineered microcapsules or microfibres has been also developed with the aim of enhancing mechanical characteristics, viability and functional life-span of the entrapped cells. In conclusion, the encapsulation technologies, here presented, represent a promising strategy for the treatment of many pathologies open to further development and scaling up towards regulatory agencies approval

Microfluidics for Biosensing

There are 12 papers published with 8 research articles, 3 review articles and 1 perspective. The topics cover: Biomedical microfluidics Lab-on-a-chip Miniaturized systems for chemistry and life science (MicroTAS) Biosensor development and characteristics Imaging and other detection technologies Imaging and signal processing Point-of-care testing microdevices Food and water quality testing and control We hope this collection could promote the development of microfluidics and point-of-care testing (POCT) devices for biosensing

Genetic Analysis and Cell Manipulation on Microfluidic Surfaces

Personalized cancer medicine is a cancer care paradigm in which diagnostic and therapeutic strategies are customized for individual patients. Microsystems that are created by Micro-Electro-Mechanical Systems (MEMS) technology and integrate various diagnostic and therapeutic methods on a single chip hold great potential to enable personalized cancer medicine. Toward ultimate realization of such microsystems, this thesis focuses on developing critical functional building blocks that perform genetic variation identification (single-nucleotide polymorphism (SNP) genotyping) and specific, efficient and flexible cell manipulation on microfluidic surfaces. For the identification of genetic variations, we first present a bead-based approach to detect single-base mutations by performing single-base extension (SBE) of SNP specific primers on solid surfaces. Successful genotyping of the SNP on exon 1 of HBB gene demonstrates the potential of the device for simple, rapid, and accurate detection of SNPs. In addition, a multi-step solution-based approach, which integrates SBE with mass-tagged dideoxynucleotides and solid-phase purification of extension products, is also presented. Rapid, accurate and simultaneous detection of 4 loci on a synthetic template demonstrates the capability of multiplex genotyping with reduced consumption of samples and reagents. For cell manipulation, we first present a microfluidic device for cell purification with surface-immobilized aptamers, exploiting the strong temperature dependence of the affinity binding between aptamers and cells. Further, we demonstrate the feasibility of using aptamers to specifically separate target cells from a heterogeneous solution and employing environmental changes to retrieve purified cells. Moreover, spatially specific capture and selective temperature-mediated release of cells on design-specified areas is presented, which demonstrates the ability to establish cell arrays on pre-defined regions and to collect only specifically selected cell groups for downstream analysis. We also investigate tunable microfluidic trapping of cells by exploiting the large compliance of elastomers to create an array of cell-trapping microstructures, whose dimensions can be mechanically modulated by inducing uniform strain via the application of external force. Cell trapping under different strain modulations has been studied, and capture of a predetermined number of cells, from single cells to multiple cells, has been achieved. In addition, to address the lack of aptamers for targets of interest, which is a major hindrance to aptamer-based cell manipulation, we present a microfluidic device for synthetically isolating cell-targeting aptamers from a randomized single-strand DNA (ssDNA) library, integrating cell culturing with affinity selection and amplification of cell-binding ssDNA. Multi-round aptamer isolation on a single chip has also been realized by using pressure-driven flow. Finally, some perspectives on future work are presented, and strategies and notable issues are discussed for further development of MEMS/microfluidics-based devices for personalized cancer medicine

Engineering Micro/Nanorobots Towards Biomedical Applications: Targeted Delivery, Surgery and Detoxification

Over the past decade, micro/nanomotors have emerged as novel and versatile nanotools demonstrating considerable promise for many environmental and biological applications. This dissertation aims to demonstrate unique advantages of micro/nanorobot platforms.The first theme focuses on developing the use of ultrasound propelled nanostructures for diverse applications, including “on-the-move” capture and the transport of multiple cargoes and internalization and movement inside live MCF-7 cancer cells.The second theme explores the use of acoustically triggered microcannons. This principle was tested towards mechanochemical blood clot degradation and enhancing drug permeability through the epidermis.The third theme describes the use of biohybrid robotics systems for detoxification and decontamination of environmental pollutants, including bacteria (E. coli), nerve agent (methyl paraoxon) and heavy metal ions (Cd and Pb) from aqueous solutions.In the not-so-distant future, Micro/nanorobots could serve as robust and versatile platform for potentially improving medical diagnosis and treatment