Directed Placement for mVLSI Devices

Continuous-flow microfluidic devices based on integrated channel networks are becoming increasingly prevalent in research in the biological sciences. At present, these devices are physically laid out by hand by domain experts who understand both the underlying technology and the biological functions that will execute on fabricated devices. The lack of a design science that is specific to microfluidic technology creates a substantial barrier to entry. To address this concern, this article introduces Directed Placement, a physical design algorithm that leverages the natural "directedness" in most modern microfluidic designs: fluid enters at designated inputs, flows through a linear or tree-based network of channels and fluidic components, and exits the device at dedicated outputs. Directed placement creates physical layouts that share many principle similarities to those created by domain experts. Directed placement allows components to be placed closer to their neighbors compared to existing layout algorithms based on planar graph embedding or simulated annealing, leading to an average reduction in laid-out fluid channel length of 91% while improving area utilization by 8% on average. Directed placement is compatible with both passive and active microfluidic devices and is compatible with a variety of mainstream manufacturing technologies

Single-Molecule Detection of Unique Genome Signatures: Applications in Molecular Diagnostics and Homeland Security

Single-molecule detection (SMD) offers an attractive approach for identifying the presence of certain markers that can be used for in vitro molecular diagnostics in a near real-time format. The ability to eliminate sample processing steps afforded by the ultra-high sensitivity associated with SMD yields an increased sampling pipeline. When SMD and microfluidics are used in conjunction with nucleic acid-based assays such as the ligase detection reaction coupled with single-pair fluorescent resonance energy transfer (LDR-spFRET), complete molecular profiling and screening of certain cancers, pathogenic bacteria, and other biomarkers becomes possible at remarkable speeds and sensitivities with high specificity. The merging of these technologies and techniques into two different novel instrument formats has been investigated. (1) The use of a charge-coupled device (CCD) in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement by simultaneously tracking and detecting single-molecules in multiple microfluidic channels was demonstrated. The CCD/TDI approach allowed increasing the sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s-1 per channel and 2208 molecules s-1 for an eight channel microfluidic system was achieved. A cyclic olefin copolymer (COC) waveguide was designed and fabricated in a pre-cast poly(dimethylsiloxane) stencil to increase the SNR by controlling the excitation geometry. The waveguide showed an attenuation of 0.67 dB/cm and the launch angle was optimized to increase the depth of penetration of the evanescent wave. (2) A compact SMD (cSMD) instrument was designed and built for the reporting of molecular signatures associated with bacteria. The optical waveguides were poised within the fluidic chip at orientation of 90° with respect to each other for the interrogation of single-molecule events. Molecular beacons (MB) were designed to probe bacteria for the classification of Gram +. MBs were mixed with bacterial cells and pumped though the cSMD which allowed S. aureus to be classified with 2,000 cells in 1 min. Finally, the integration of the LDR-spFRET assay on the cSMD was explored with the future direction of designing a molecular screening approach for stroke diagnostics

Development of DNA assembly and error correction protocols for a digital microfluidic device

Customized production of synthetic DNA from oligonucleotides is in high demand. However, current technologies are costly and labor-intensive. A microfluidic technology can significantly decrease cost and labor. The purpose of this study was to develop a gene assembly protocol that was utilized on the Mondrian™ SP digital microfluidic device. The fragment of the human influenza virus hemagglutinin (HA) gene (339 bp) was assembled from 12 oligonucleotides by the Gibson assembly method and error corrected with CorrectASE™ enzyme twice. The samples were analyzed by Sanger sequencing to verify the final accuracy of the assembly. A complete automation of droplet generation and movement on digital microfluidic droplet technology was achieved in the study. The reactions were scaled down to 0.6-1.2 µL. Gibson assembly, PCR, and enzymatic error correction reactions were optimized and combined in a single protocol. The microfluidic assembly demonstrated approximately 3 errors/kb error frequency. Polymerase chain reaction supplemented with additional MgCl2, Phusion, and PEG 8000 provided amplification of the assembly and error correction products. The lowest error frequency of 0.3 errors/kb was achieved after one CorrectASE™ treatment. However, microfluidic error correction was not reliable due to CorrectASE™ interactions with the microfluidic surface, which need to be the subject of future work

Microparticle Array on Gel Microstructure Chip for Multiplexed Biochemical Assays

Ph.DDOCTOR OF PHILOSOPH

Coplanar Electrowetting-Induced Droplet Ejection for 3D Digital Microfluidic Devices

Digital microfluidics is a promising fluid processing technology used in lab-on-achip applications to perform chemical synthesis, particle filtration, immunoassays, and other biological protocols. Traditional digital microfluidic (DMF) devices consist of a 2D grid of coated electrodes over which droplets are manipulated. Selective activation of the electrodes results in an electrowetting effect that deforms the droplets and can move them around the electrode grid. More recently, electrowetting on dielectric has also been used to eject droplets and transfer them between opposing surfaces. This has given rise to new 3D DMF devices capable of more sophisticated routing patterns that can minimize crosscontamination between different biological reagents used during operation. A better understanding of electrowetting-induced droplet ejection is critical for the future development of efficient 3D DMF devices. The focus of this work was to better predict electrowetting-induced droplet ejection and to determine how droplet selection and electrode design influence the process. An improved model of droplet gravitational potential and interfacial energy throughout ejection was developed that predicts a critical electrowetting number necessary for successful detachment. Predictions using the new model agreed more closely with experimentally observed thresholds than previous models, especially for larger droplet volumes. Droplet ejection experiments were also performed with a variety of coplanar electrode designs featuring different numbers of electrode pieces and different spacings between features. The critical voltage for ejection was observed to be approximately the same for all designs, despite the poor predicted performance for the case with the widest spacing (200 ) where nearly 25% of the area beneath the droplet was dead space. Findings indicated that a critical electrowetting for ejection must be achieved at the contact line of a droplet rather than over the entire droplet region. Droplets were also ejected for the first time from devices with inkjet-printed electrodes, demonstrating the feasibility of future low-cost 3D DMF systems

Optimization of Continuous Flow Polymerase Chain Reaction with Microfluidic Reactors

The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific ~gion ofDNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to cont,amination due its inherent efficiency and high sensitivity. By employing microfluidic' systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR . in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and ,' temperature control/determination are the main focus within this work. Microchannels and microreactors are char:acterized by extremely high surface-tovolume ratios. This dictates that surfaces play a dominant role in defining the efficiency ofPCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within??????? this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing Copper oxide and Zinc oxide' nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to singlestranded DNA and the catalytic extension of this primer to fonn the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton e'xcitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.Imperial Users onl

Manipulation of magnetic microparticles in liquid phases for on-chip biomedical analysis methods

Magnetic microparticles and their application in bioanalytical microfluidic systems have been steadily gaining interest in recent years. This progress is fueled by the comparatively large and long range magnetic forces that can be obtained independently of the fluidic flow pattern. This thesis work presents new approaches for using magnetic microparticles in Lab-on-a-Chip systems. The first approach deals with the design of a magnetic droplet manipulation system and the second combines magnetic particle actuation with integrated optical detection. The applicability of both systems for miniaturized bioanalysis will be shown, demonstrating the potential of magnetic particle based Lab-on-a-Chip systems. The magnetic droplet manipulation system tackles the handling of small liquid volumes, which is an important task in miniaturized analytical systems. The careful adjustment of hydrophilic/hydrophobic surface properties and interfacial tensions leads to the design of a system, where small droplets are manipulated in a controllable fashion. The system's setup permits the direct implementation of bioanalytical protocols and two different procedures are in consequence examined. Based on a commercial laboratory kit, a platform for the on-chip extraction and purification of DNA will be designed. The miniaturized setup allows the user to capture and clean the DNA obtained from a raw cell sample containing as little as 10 cells, which is several orders of magnitude lower than known for macroscopic systems. A similar performance is observed for the colorimetric antibody detection further-on evaluated in the droplet manipulation system, where the small sample volumes permit a significant reduction of the reaction times. With the possibility of concentrating the biomolecules of interest on the particle surface, a sensitive and fast immunosorbent assay can be devised. A further miniaturization is examined in a CMOS system, which combines magnetic actuation and optical detection. The small dimensions of the actuation system allow the manipulation of single magnetic microparticles and the integration of Single Photon Avalanche Diodes (SPADs) enables their optical detection. An innovative detection algorithm permits hereby to distinguish the particles in size and, in combination with a velocity measurement, to evaluate the magnetic properties of the detected particles. In consequence, bioanalysis on a single magnetic particle using fluorescent measurements can be performed, as is shown by preliminary experiments

Actionnements électriques de fluides dédiés aux microsystèmes

La récente progression des techniques de fabrication et la nécessité de miniaturiser des systèmes de détection et de diagnostic a permis l'émergence d'un nouveau champ de recherche appelé laboratoires sur puces. Le déplacement et la manipulation de fluides d'intérêt biologique à l'échelle micrométrique a ouvert la voie à de nombreux phénomènes basés sur des champs électriques. Parmi ceux-ci, l'électromouillage a été développé en tant qu'actionnement de volumes finis de liquides, et parallèlement, l'électroosmose comme actionnement de flux. Les travaux de cette thèse traitent de l'intégration de ces deux phénomènes sur des microsystèmes d'ores et déjà existant. Dans un premier temps, nous présentons l'utilisation de l'électromouillage dans un système de dépôts par contact (à base de microleviers), liquides de dimensions caractéristiques micrométriques. L'application d'une tension électrique entre le substrat conducteur et un fluide, modifie l'énergie de ce dernier provoquant son étalement sur le solide et préférentiellement dans les canaux de l'outil de dépôt. Nous obtenons, ainsi, une méthode originale et propre de chargement des leviers diminuant les volumes nécessaires. Mais, également, en appliquant la tension entre le liquide et le substrat de dépôt, nous modulons le volume et la surface déposés et ce même sur des surfaces très hydrophobes. Afin d'expliquer et confirmer les observations, un modèle théorique décrivant l'électromouillage sur les microleviers a été proposé et expérimentalement vérifié, faisant de cet actionnement électrique de fluide un candidat prédictif et fiable de manipulation de volumes finis de liquides. Dans un second temps, nous avons utilisé l'électroomose afin de concentrer des particules sur une micromembrane piezoélectrique résonante. Cet actionnement électrique de fluide permet d'augmenter sensiblement le nombre de particules à la surface du capteur et ainsi d'améliorer ses performances. Un modèle théorique complet est proposé pour décrire les effets du champ électrique sur le fluide et les objets en suspension et permet d'aboutir à de nombreuses données prédictives. Celles-ci ont ensuite été confirmées à l'aide de structures de tests spécifiquement fabriquées et une concentration 105 fois supérieure à celle obtenue par diffusion a été constatée. La dernière étape de ces travaux, l'utilisation des concentrateurs dans un milieu liquide biologique, a posé plus de problèmes que prévus et n'a pas permis d'obtenir les résultats escomptés. Néanmoins les observations d'ores et déjà réalisées nous laissent à penser que cet actionnement électrique de fluide demeure un excellent candidat pour diminuer fortement les temps de réponses des capteurs de tailles micrométriques.Recent advances in microfabrication, and the apparent necessity of miniaturizing detection and diagnostic systems has led to the emergence of a new research field called lab-on-chip, which are mostly working with fluids. Liquids actuations at the micrometer scale allow the development of many new methods based on electric field and potential. Electrowetting has been widely used to displace and manipulate droplets on a surface, whereas electroosmosis has been used for bulk fluid movement and mixing. In this thesis, we propose the integration of these two phenomena in existing microsystems. First, electrowetting is used in a liquid microspotting system based on a cantilevers array. A difference of electric potential between a fluid and a conducting substrate modify the energy of the liquid. Thus, the drop spreads on the surface and preferentially in existing grooves in the depositing tool. We thus obtain an original way to load the cantilevers decreasing the dead volumes in the loading droplet. Moreover, if the potential difference is applied between the liquid and the contacting surface, we can control the size and volume of deposited droplet, thus allowing the patterning of highly hydrophobic substrates. A theoretical model is proposed to describe and predict the fluid comportment ; Experimentations are in a very good agreement with the predicted comportment and values. Electrowetting reveals to be the best electric actuation for the manipulation of droplets and small volumes of liquids. Second, electroosmosis is used to concentrate particles on a piezoelectric micromembrane. This actuation allows us to increase the number of particles at the surface of the vibrating part therefore decreasing the response time of the sensor. A theoretical model is proposed to describe and understand the effect of the electric field on both the bulk fluid and the objects in solution. Experimental validations are proposed with the design and fabrication of dedicated structures. Observed concentration of particles was 105 higher that what is obtained with diffusion. The last step was to concentrate biological particles in adequate bulk fluid. Results were difficult to obtain and we do not observed the desired comportment. Nevertheless, the experimentations show clearly the huge potential of this electric actuation of fluid. Electroosmosis is therefore a real good phenomenon to decrease the response time of micrometric sensors