Dissertations / Theses on the topic 'Acoustophoresi'

Cette thèse s’inscrit dans le contexte du développement des laboratoires sur puce (LOC, « Lab On a Chip », permettant de réaliser plusieurs opérations nécessaires à l’analyse d’un échantillon biologique à l'intérieur d'un seul microsystème. Dans ce type de dispositif, de nombreuses étapes sont nécessaires avant d’arriver au résultat d’une analyse donnée (introduction de l'échantillon, concentration, mélange, purification, séparation, etc.). L’équipe microsystèmes du laboratoire Ampère étudie depuis plusieurs années différentes techniques de manipulation sans contact de particules, pour le tri ou de manipulation de particules individuelles dans les laboratoires sur puce, telles que la diélectrophorèse ou la magnétophorèse. Dans cette thèse, nous nous intéressons à la manipulation acoustique de micro particules. Cette technique se révèle notamment avantageuse pour la manipulation d’objets biologiques comme des bactéries, car elle permet de s’affranchir de certaines contraintes de marquage ou de changement de milieu. Notre choix s’est porté sur l’emploi des ondes acoustiques de surface (SAW, « Surface Acoustic Waves »), compatibles avec la filière PDMS très utilisée dans la communauté des LOC. Outre la possibilité de simplifier l’intégration microfluidique de la pince acoustique, la technologie SAW offre une alternative aux dispositifs à pièges acoustiques fixes existant dans la littérature en permettant un contrôle en temps réel des particules piégées. C’est ce que nous avons réalisé expérimentalement : en jouant sur le déphasage entre les signaux d’alimentation électriques des transducteurs électromécaniques, nous pouvons modifier la position des noeuds et des ventres de l’onde acoustique résultante. Ainsi, nous avons pu contrôler en temps réel la position d’une bille en latex de 3 μm ou encore d’un faisceau de bactéries E.coli. Par ailleurs, nous avons réalisé une simulation par éléments finis de la puce acoustofluidique dans son ensemble permettant une meilleure compréhension de tous les phénomènes en jeu et l’optimisation du transfert énergétique entre la source électrique et la particule manipulée. Cette simulation nous indique notamment que l’amplitude de l’onde acoustique stationnaire sur le substrat piézoélectrique varie environ d’un facteur deux en fonction du déphasage imposé entre les deux sources électriques. Cela impacte donc dans la même proportion la force acoustique résultante. Cette variation semble être validée par nos dernières expériences
In lab-on-a-chip (LOC) technologies, many sample preparation steps are required before achieving a biological analysis on a single chip (sample introduction, concentration, mixing, purification, separation, etc.). The microsystem team of the Ampere Lab has studied for many years different contactless particle manipulation techniques, for sorting or manipulating bioparticles in LOC platforms, such as dielectrophoresis and magnetophoresis. In this thesis, we focus on acoustic manipulation of microparticles. This technique is advantageous for the manipulation of biological objects such as bacteria, because labelling and medium exchange can be avoided. We chose to work with surface acoustic waves (SAW), because this approach is consistent with the use of PDMS, widely used in microfluidics. Besides an easier microfluidic integration of the acoustic tweezers, the SAW technology provides an alternative to the existing devices with fixed acoustic traps, allowing a real time control of the trapped particles. This was experimentally achieved by playing on the phase shift between the two electrical signals driving the IDT, thereby modifying the position of nodes and antinodes of the resulting pressure wave. As a result, we could control in real time the position of a 3 μm latex bead or an E.coli bacteria alignment. We have also developed a finite-element model of the whole acoustofluidic chip allowing a better understanding of the physics and the optimization of the energy transfer between the electrical source and the trapped particle. Among different results, this model informs us that the magnitude of the acoustic radiation force varies by a factor of two with the phase shift between the electrical sources. This result seems to be validated by our last experiments

Acoustophoresis is a label free method where the acoustic radiation force is used to manipulate microparticles inside microfluidic channels. The magnitude of the force is dependent of several parameters, which include the density, speed of sound and size of the microparticles, as well as the amplitude of the pressure waves. Recently, acoustophoresis has been used in microfluidics to manipulate microparticles inside moving droplets. In this Master's thesis project, two microfluidic chip designs are used to enrich droplets with polystyrene beads (10 μm in diameter) using acoustophoresis. The microchips have been fabricated with two different fabrication methods; crystalline dependent wet etching and crystalline independent dry etching. In the microchips, water droplets in oil are generated with microparticles suspended in them. By using a channel width that is half a wavelength of the incoming acoustic waves, pressure nodal lines are created in the middle of the channel in which the microparticles align. The droplets then enters a droplet splitting feature, where they are divided into three daughter droplets. Since the majority of the incoming particles are recovered in the center daughter droplet while some of the droplet volume is removed, the center droplet is enriched with the microparticles. For the wet etched design stable droplet splitting was observed when the volumetric flow was 18 μL/min and the incoming droplets had a length-to-width ratio larger than 3. The maximum recovery for this design was 81.1% ± 13.8% with an applied voltage at 10 Vpp. Stable droplet splitting was observed for the dry etched chip at 10.5 μL/min and 18 μL/min at 10 and 20 Vpp, when the incoming droplet had a length-to-width ratio of 3. In this chip the maximum recovery was 93.2% ± 8.3% at the volumetric flow of 10.5 μL/min and an applied voltage of 20 Vpp.

Despite the many promising advances made in microfluidics, sample preparation remains the single largest challenge and bottleneck in the field of miniaturised diagnostics. This thesis is focused on the development of sample preparation methods using active and passive particle manipulation techniques for point of care diagnostic applications. The active technique is based on acoustophoresis (acoustic manipulation) while the passive method is based on inertial microfluidics (hydrodynamic manipulation). In paper I, acoustic capillary-based cavity resonator was used to study aggregation of silica and polystyrene particles. We found that silica particles show faster aggregation time (5.5 times) and larger average area of aggregates (3.4 times) in comparison to polystyrene particles under the same actuation procedure. The silica particles were then used for acoustic based bacteria up-concentration. In paper II, a microfluidic-based microbubbles activated acoustic cell sorting technique was developed for affinity based cell separation. As a proof of principle, separation of cancer cell line in a suspension with better than 75% efficiency is demonstrated. For the passive sample preparation, inertial and elasto-inertial microfluidic approach that uses geometry-induced hydrodynamic forces for continuous size-based sorting of particles in a flow-through fashion were studied and applied for blood processing (paper III-V). In paper III, a simple ushaped curved channel was used for inertial microfluidics based enrichment of white blood cells from diluted whole blood. A filtration efficiency of 78% was achieved at a flow rate of 2.2 ml/min. In paper IV, elasto-inertial microfluidics where viscoelastic flow enables size-based migration of cells into a non- Newtonian solution, was used to continuously separate bacteria from unprocessed whole blood for sepsis diagnostics. Bacteria were continuously separated at an efficiency of 76% from undiluted whole blood sample. Finally, in paper V, the inertial and elasto-inertial techniques were combined with a detection platform to demonstrate an integrated miniaturized flow cytometer. The all-optical-fiber technology based system allows for simultaneous measurements of fluorescent and scattering data at 2500 particles/s. The use of inertial and acoustic techniques for sample preparation and development of an integrated detection platform may allow for further development and realization of point of care testing (POCT) systems.

QC 20170124

This project aimed at exploring new hybrid materials to be used for acoustophoresis applications. Acoustophoresis can be used to manipulate particles inside a microfluidic channel by creating ultrasound standing waves within the channel [1]. This can be used for cell separation [2] or trapping of particles [3]. The intent of this project was to create materials for use in microfluidic channels that would be cheaper and easier to manufacture than those traditionally used, while still having adequate acoustic properties to allow for use in acoustopheresis. This was done by investigating whether the addition of glass-beads or glass-bubbles could increase the acoustic properties of an off-stoichiometry-thiol-enes (OSTE) based polymer. Hybrid samples with different volume fractions of glass-beads or glass-bubbles added to the OSTE polymer were manufactured and characterised according to their acoustic properties using the pulse-echo buffer-rod method. The acoustic properties measured were the density, attenuation, acoustic impedance and the reflection coefficient between water and the material. The addition of glass-beads was found to increase the acoustic impedance while the inverse was found for the addition of glass-bubbles. Both the addition of glass-beads and glass-bubbles were found to increase the attenuation. The hybrid material that was found to have the most suitable acoustic properties was OSTE/Glass-beads 40%, whose acoustic impedance had been increased ∼60% compared to pure OSTE. Consequently, the OSTE/Glass-beads 40% material was used to manufacture a microfluidic channel. A particle trapping experiment showed that the OSTE/Glass-beads 40% microfluidic channel was able to obtain bead trapping. This means that a standing wave was able to be generated within the channel and that it was strong enough to trap particles in the centre of the channel. However, evaluation of the particle trapping efficiency of the channel showed that it was not as effective as those using traditional materials. Therefore, future work is recommended to optimise a channel design for the OSTE/Glass-beads 40% material to increase the particle trapping efficiency.
I detta projekt undersöktes ett nytt hybridmaterial för användning i applikationer inom akustofores. Akustofores kan användas till att manipulera partiklar inuti mikrofluidkkanaler genom att generera ståendevågor i kanalen med hjälpav ultraljud [1]. Detta kan användas till cellseparation [2] eller till att fånga partiklar [3]. Målet i detta projekt var att skapa material som skulle bli billigare och möjliggöra enklare fabricering av kanalerna som används inom akustofores än de material som traditionellt används, med bibehållande av tillräckliga akustiskaegenskaper. Detta genomfördes genom att undersöka om tillsättning av glaspärlor eller glasbubblor kunde förbättra de akustiska egenskaperna av en off-stoichiometry-thiol-enes (OSTE) baserad polymer. Hybridprover gjorda på OSTE-polymeren med olika volymandelar av glaspärloroch glasbubblor tillverkades och kategoriserades med avseende på deras akustiska egenskaper med hjälp av pulseeko buffertstång metoden. De akustiska egenskaperna som uppmättes var densitet, attenuering, akustisk impedans och reflektions koefficienten mellan vatten och materialet. Resultatet av projektet visade att tillsättning av glaspärlor ökade den akustiska impedansen  i motsatts till glasbubblorna som visade sig minska den. Vidare visade det sig att både tillsättningen av glaspärlor och glasbubblor ökade attenueringen. Det hybridmaterial som visade sig ha de mest lämpliga akustiska egenskaperna var OSTE/glaspärlor med en 40% volymandel av glaspärlor. Den akustiska impedansen hade förhöjts med cirka 60% jämfört med vanlig OSTE. Därför valdes det hybrid-materialet till att tillverka en mikrofluidikkanal. Därefter genomfördes ett partikelfångstexperiment som visade att, OSTE/glaspärlor med en 40% volymandel av glaspärlor, kunde erhålla partikelfångst i kanalen. Detta innebär att en stående våg kunde genereras i kanalen och att den var tillräckligt stark för att kunna fånga partiklarna i mitten av kanalen. Däremot visade utvärdering av kanalens partikelfångsteffektivitet att den inte var lika effektiv som kanaler gjorda av traditionellt använda material. Därför rekommenderas framtida arbete till att designa en optimerad kanaldesign med OSTE/Glas-pärlor 40% materialets egenskaper i åtanke för att förhoppningsvis kunna öka partikelfångst effektivitet.

Analysis of blood samples is one of the major steps in diagnosing pathological conditions like cancer. The upstream sample preparation for the pathological cell analysis from complex biological fluid like blood, involves selective cell sorting. It can be achieved using fluorescently activated or magnetically activated cell sorters. Another way is to sort them using acoustophoresis which is cheaper, gives better spatial control and is also rapid apart from the fact that, it does not affect the cellular viability.6,9 In acoustophoresis, particles depending upon their density and compressibility relative to the suspended medium migrate to either pressure anti-nodes or nodes, when subjected to acoustic field. Poly vinyl alcohol-based microbubbles have a strong negative acoustic contrast factor and hence migrate to the anti-nodes in a standing ultrasonic wave. Previously, this property was utilized for cell separation by conjugating the bubbles to cells and subjecting them to ultrasonic waves in a silicon glass based microfluidic channel.55 A protocol for coating the microbubbles with avidin, so that these can readily attach to the cells has been developed in this work. However, microfluidic channel is obtained from a master mold which is developed in a clean room facility using photolithography. A cost-effective way has been developed for the production of a mold using a Computerized Numerical Control system (where the positive master for the microfluidic channel is drilled onto a PMMA sheet) for continuous separation of cancer cells. Alternate methods like a cutting plotter (which uses a double sided adhesive tape as a positive master) and a 3-D printer have been investigated, in order to be used as a mold for the microfluidic channel. As a proof, microbubbles-cell complex was focused in a PDMS based microfluidic channel, by utilizing standing Bulk acoustic waves. At flow rate of 10µl/min, efficiency greater than 80% has been achieved. This technique is low cost and can be implemented in places without a clean room facility for size independent cell sorting.

Diagnostic ultrasound (US) is safer, quicker and cheaper than other diagnostic imaging modalities. Over the past two decades, the applications of US imaging has been widened due to the development of injectable, compressible and encapsulated microbubbles (MBs) that provide an opportunity to improve conventional echocardiographic imaging, blood flow assessment and molecular imaging. The encapsulating material is manufactured by different biocompatible materials such as proteins, lipids or polymers. In current research, researchers modify the encapsulated shell with the help of advanced molecular chemistry techniques to load them with dyes (for fluorescent imaging), nanoparticles and radioisotopes (for multimodal imaging) or functional ligands or therapeutic gases (for local drug delivery). The echogenicity and the radial oscillation of MBs is the result of their compressibility, which undoubtedly varies with the encapsulated shell characteristics such as rigidity or elasticity. In this thesis, we present acoustic properties of novel type of polyvinyl alcohol (PVA)-shelled microbubble (PVA-MB) that was further modified with superparamagnetic iron oxide nanoparticles (SPIONs) to work as a dual-modal contrast agent for magnetic resonance (MR) imaging along with US imaging. Apparently, the shell modification changes their mechanical characteristics, which affects their acoustic properties. The overall objective of the thesis is to investigate the acoustic properties of modified and unmodified PVA-MBs at different ultrasound parameters. The acoustic and mechanical characterization of SPIONs modified PVA-MBs revealed that the acoustical response depends on the SPION inclusion strategy. However they retain the same structural characteristics after the modification. The modified MBs with SPIONs included on the surface of the PVA shell exhibit a soft-shelled behavior and produce a higher echogenicity than the MBs with the SPIONs inside the PVA shell. The fracturing mechanism of the unmodified PVA-MBs was identified to be different from the other fracturing mechanisms of conventional MBs. With the interaction of high-pressure bursts, the air gas core is squeezed out through small punctures in the PVA shell. During the fracturing, the PVA-MBs exhibit asymmetric (other modes) oscillations, resulting in sub- and ultra-harmonic generation. Exploiting the US imaging at the other modes of the oscillation of the PVA-MBs would provide an opportunity to visualize very low concentrations of (down to single) PVA-MBs. We further introduced the PVA-MBs along with particles mimicking red blood cells in an acoustic standing-wave field to observe the acoustic radiation force effect. We observed that the compressible PVA-MBs drawn toward pressure antinode while the solid blood phantoms moved toward the pressure node. This acoustic separation method (acoustophoresis) could be an efficient tool for studying the bioclearance of the PVA-MBs in the body, either by collecting blood samples (in-vitro) or by using the extracorporeal medical procedure (ex-vivo) at different organs. Overall, this work contributes significant feedback for chemists (to optimize the nanoparticle inclusion) and imaging groups (to develop new imaging sequences), and the positive findings pave new paths and provide triggers to engage in further research.

QC 20150827

3MiCRON

We investigate experimentally the effects of acoustic forces on submicron aerosol in a channel flow. This technique can potentially overcome some of the limitations of conventional separation systems and provide advanced manipulation capabilities such as sorting according to size or density. The theoretical framework for acoustophoresis at such small length scales where molecular effects are expected to be significant is still incomplete and in need of experimental validation. The main objectives of this thesis are to identify the physical limitations and capabilities of acoustophoretic manipulation for submicron aerosol particles. Two sets of experiments were carried out: first, qualitative results revealed that acoustic manipulation is possible for submicron particles in air and that the acoustic force follows the trend expected by theoretical models developed for particles in inviscid fluids. The acoustic force on submicron particles was estimated in a second set of measurements performed with quantitative diagnostic tools. Comparison of these results with available theoretical models for the acoustic radiation forces demonstrates that for such small particles additional forces have to be considered. At submicron length scales, the magnitude of the forces observed is orders of magnitude higher than the predictions from the inviscid theory. One potential application for acoustophoresis is specifically investigated in this thesis: assist electrostatic precipitation (ESP) samplers to target very small aerosols, such as those carrying airborne viruses. To identify the shortcomings of ESP samplers that acoustophoresis should overcome, two ESP designs have been investigated to quantify capture efficiency as a function of the particle size and of the air velocity in a wind tunnel. The results reveal that both designs have limitations when it comes to sampling submicron aerosol particles. When exposed to polydispersed suspensions they behave as low-pass filters.

QC 20161125

In both, cell secretome analysis and bacteria evolution, controlled handling of particles with a few to sub-micrometers in size and media exchange are inevitable in order to investigate body fluid’s proteins or change the surrounding culture conditions for pivoted evolution. Typically, nanofiltration and ultra-centrifugation are employed which can lead to cell damage, need large sample volumes and have a high sample loss. Using contactless and label-free acoustic cell manipulation, disadvantages of other magnetic, dielectric or hydrodynamic methods can be avoided. Here, a novel design using acoustic forces for small particle trapping and media exchange is thoroughly numerically investigated including first- and second-order acoustic effects. The device comprises parallel aligned medium and air channels separated by a thin wall. Particle trapping occurs at this thin wall. The medium channel dimensions (height and width) and thin wall thickness are optimized with respect to trapping forces. Thinnest walls are preferable and an aspect ratio of 0.8. First preliminary experimental variation with polystyrene particles showed good agreement with the simulations. Thereby the particle trapping efficiency is evaluated under quiescent flow conditions. For particle trapping, a device with a channel height of 290μm and an aspect ratio of 0.7 is superior which supports the numerical results. Finally, medium exchange of E. coli bacteria is demonstrated with best results for a device with a channel height of 450μm and an aspect ratio of 0.8 showing that 13.4% of the initial bacteria were released after medium exchange which can be used for further processing.

Dropp-genereringstekniker är viktiga för industrier som läkemedelsindustrin, livsmedelsindustrin, kosmetikindustrin etc. Traditionella droppgenereringstekniker är dock begränsade i mängden av material som kan processas till droppform. Ett exempel inkjet som är en väletablerad teknik för att generera droppar med hög hastighet (1-10 kHz) och precision (10-20 μm), men kan bara stöta ut vätskor med låga viskositet, ungefär 10-100 gånger viskositeten av vattnet. Akustophoretisk utskrift motiv är att övervinna denna materialbegränsning och har framgångsrikt avkopplat dropputstötning från bläckviskositet. Metoden utnyttjar ickelinjära akustiska krafter för att skriva ut en stor mängd av material med hög kontroll, med viskositet som sträcker sig över fyra storleksordningar (0,5 mPa · s till 25 000 mPa · s). Emellertid är utstötningen baserad på bildandet av en hängande droppe, och i den aktuella prototypen begränsas materialpaletten av akustophoretisk utskrift genom sprider sig över munstycket, vilket begränsar den minsta tillåtnas ytspänningen till ungefär 60 mN / m. I detta arbete införs en munstycksbeläggningsteknik för att expandera mängden av utskrivbara material, med tillåtna ytspänningar så låga som 25 mN / m. Genom att utnyttja generera nanostrukturer med låg ytenergi på munstyckspetsen, tillverkas superavstötande beläggning. Grunden för nanostrukturerna genererades med hjälp av sot från ett paraffin-vaxljus. Ett robust tillverkningsprotokoll har etablerats, och beläggningen fysikaliska egenskaper och prestanda har karaktäriserats. Tre nya tillämpningsområden undersöktes, vilket demonstrerade noviteten hos denna nya metod. Detta arbete banar vägen för en ny uppsättning material som ska behandlas i en droppe-per droppe metodik.
Droplet generation techniques are essential for industries such as the pharmaceutical, food industry, cosmetic industry, etc. However, traditional droplet generation techniques are limited in the palette of materials that can processed in a droplet form. For example, inkjet which is a well-established technology to generate droplets of high speed (1-10 kHz) and precision (10-20 μm), but can only eject fluids with low viscosities, roughly 10-100 folds the one of water. Acoustophoretic printing aims to overcome this material limitation and have successfully decoupled droplet ejection from ink viscosity. The method harnesses nonlinear acoustic forces to print a wide range of materials on demand, spanning over four orders of magnitudes (0.5 mPa·sto 25,000 mPa·s). However, the ejection is based on the formation of a pendant drop, and in the current prototype, the material palette of acoustophoretic printing is limited by nozzle wetting, limiting the allowable minimum surface tension to about 60 mN/m. In this work, a nozzle coating technique is introduced in order to expand the material window by processing fluid with a surface tension as low as 25 mN/m. By leveraging self-assembling of nanostructures on the nozzle tip, superamphiphobic coating is successfully manufactured by using a candle soot template.A robust manufacturing protocol has been established, and the coating characterized in its physics and performance.

Dans ce travail nous decrivons l'effet de force ionique dans l'acoustophorese et la conductivite de melanges d'electrolytes. Cet effet est considere comme une perturbation lineaire de l'etat d'equilibre, etat decrit a l'aide de l'approximation spherique moyenne, et les resultats obtenus sont analytiques. L'influence de la frequence est prise en compte dans les equations de l'acoustophorese et la reproduction des experiences d'acoustophorese est etendue a 1m au lieu de 0,01m. Une description de la conductivite des sels simples en terme de rayons moyens est aussi proposee et donne des resultats tres satisfaisants. Enfin, la description, en rayons individuels, de la conductivite des melanges d'electrolytes simples et de systemes micellaires avant et apres la concentration micellaire critique (cmc) concorde avec l'experience jusqu'a des concentrations de 1m au lieu de 0,1m. Ces ameliorations theoriques permettent une meilleure comprehension et une quantification de l'effet de force ionique. Ainsi, le nombre d'hydratation des cations devient accessible par l'acoustophorese et la quantification plus coherente de l'effet des ions simples est proposee pour les suspensions colloidales. Concernant la conductivite des melanges d'electrolytes, une fois la description validee pour les melanges d'ions simples, on determine, de facon plus coherente pour les systemes micellaires, les coefficients de diffusion a dilution infinie et a la cmc. Ce travail devrait aussi permettre de mieux prendre en compte des proprietes physico-chimiques telles que l'association ou la condensation des contre-ions a des concentrations relativement elevees

Microfluidics is a field of research that enables the manipulation of fluids in the submillimetre length scale. The technology allows the development of lab-on-a-chip devices, which are miniaturized systems for chemical and biological analysis. Currently, the conventional manufacturing methods for these systems require multiple time-consuming steps. Therefore, focus has shifted towards laser micromachining as an alternative method. Direct laser writing would circumvent many of the steps required for the conventional methods, drastically reducing the process time. In this Master thesis project, it was shown that microfluidic chips can be manufactured using a Nd:YVO4 (532 nm) nanosecond laser system. The process was optimized for silicon and borosilicate glass substrates. Acoustic focusing of polystyrene beads was demonstrated for a system etched in silicon. The optimized process used a power of 50%, a frequency of 10 kHz, a scan speed of 60 mm/s with triple lines as fill type and it had an etch rate of 4.3 μm/pass. Processed wafers were cleaned in buffered HF and bonded using anodic bonding as well as adhesive bonding. Processing of glass proved unpredictable, resulting in cracks and chippings. However, in- and outlets were successfully etched through thin glass wafers. It was found that crucial factors for the process were to control the focus, positioning of structures, structure orientation and the pulse separation for a uniform distribution of pulses. Based on the results, it is estimated that the manufacturing process could be done in two to three days using the laser micromachining process.

Changes in the biomechanical properties of cells accompanying the development of various pathological conditions have been increasingly reported as biomarkers for various diseases and as a predictor of disease progression stages. For instance, cancer cells have been found to be less stiff compared to their healthy counterparts due to the proteomic and lipidomic dysregulations conferred by the underlying pathology. The separation and selective recovery of cells or extracellular vesicles secreted from such cells that have undergone these changes have been suggested to be of diagnostic and prognostic value. This dissertation first describes the implementation of a stiffness-based separation of phosphatidylcholine-based vesicles using a method first introduced based on the research in this work and was dubbed thermally-assisted acoustophoresis, or thermo-acoustophoresis. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to change in the compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature (Tϕ), corresponding to the temperature at which the contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop this separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. We have further explored the functionality of this method by experimenting with cholesterol-containing vesicles. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. We evaluated the Tϕ of vesicles at different cholesterol molar ratios (Xchol) and developed a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we obtained efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells affected by diseases that affect the stiffness and extracellular vesicles such as exosomes and microvesicles.

Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening. The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.

QC 20160926

Que ce soit pour la transfusion mais aussi pour le comptage et l’examen d’un type cellulaire en particulier, des techniques de fractionnement du sang fiables sont d’une nécessité absolue pour la médecine. Parmi les techniques de fractionnement les plus récentes, l'acoustophorèse permet de séparer des cellules en exploitant à l’échelle microfluidique la force de radiation ultrasonore. Lors d'un fractionnement par acoustophorèse, le maintien du débit est une tâche essentielle. Néanmoins, les bas débits utilisés rendent difficile le développement de débitmètres adaptés. Également, l’acoustophorèse et les techniques de fractionnement microfluidiques en général sont demandeuses de nouveaux outils de caractérisation des propriétés physiologiques du sang. L’hématocrite est en particulier une quantité intéressante dont la mesure permet de quantifier le procédé de fractionnement via les globules rouge. Dans cette thèse, nous présentons un système Doppler ultrasonore conçu pour caractériser un écoulement de sang dans un dispositif d’acoustophorèse. Dans un premier temps, nous montrerons comment ce système peut être simplement exploité à l’aide d’un algorithme d’optimisation pour mesurer un débit très faible de sang. Puis, nous appliquerons quelques éléments théoriques pour évaluer la capacité du système à mesurer l’hématocrite d’un écoulement de sang dilué via la puissance ultrasonore rétrodiffusée. Enfin, nous montrerons que le comportement non-Newtonien de l’écoulement de sang nous permet de mesurer l’hématocrite dans une gamme physiologique, en sondant de légères variations du profil des vitesses
Blood transfusion is a major issue for medicine. To ensure the quality of blood products, reliable and safe blood fractionation techniques are required. Acoustophoresis separation is a recent microfluidic technique that exploits the ultrasonic radiation force to fractionate blood. During acoustophoresis, the stability of blood flow rate is essential. However, the development of a flowmeter adapted to blood acoustophoresis is not an easy task due to very low microfluidic flow rates. In addition, there is a need for new blood characterization techniques that would allow to monitor acoustophoresis. In particular, the blood hematocrit is an interesting parameter to measure that could help to assess red blood cells fractionation. In this PhD thesis, we present a simple Doppler ultrasound system that has been conceived to characterize the blood flow in an acoustostophoresis channel. We will first demonstrate how the system can be simply exploited with an optimization algorithm to measure blood flow rate in a range relevant to acoustophoresis. Then, we will apply some theoretical elements to evaluate the system capacity to measure the hematocrit of a diluted blood flow using ultrasonic backscattered power. Lastly, we will show how blood non-Newtonian behavior can be used to measure hematocrit in a physiological range through small variations of blood maximum velocity

Cette thèse de doctorat porte sur la description analytique des phénomènes acoustophorétiques, en solutions et suspensions. L'acoustophorèse est la création d'un champ électrique par une onde acoustique.La première partie porte sur les solutions d'électrolytes, et est basée sur l'analyse critique de la littérature. A partir des différents articles, un modèle original, basé sur la résolution des équations de la dynamique pour les ions est trouvé, lequel permet la prédiction, sans paramètres ajustables, de l'acoustophorèse des sels simples pour des solutions allant de très diluées à assez concentrées. Ce modèle est ensuite étendu aux cas de solutions avec trois espèces ioniques différentes, et un programme informatique calculant l'acoustophorèse en fonction de la concentration est en annexe. Une seconde extension est faite pour les liquides ioniques, et permet de déduire le volume des ions. Des tentatives d'extension du modèle sont faites pour les micelles et les colloïdes, en précisant les écueils. Une deuxième approche, basée sur la thermodynamique irréversible et les relations de réciprocité d'Onsager, est faite dans le cas des suspensions colloïdales. Les principaux résultats sont la proportionnalité entre l'acoustophorèse et la mobilité électrique des colloïdes, et donc l'applicabilité de cette technique à la caractérisation des suspensions, y compris concentrées ; le lien rigoureux entre l'acoustophorèse et son corollaire, la création d'une onde acoustique et son champ électrique ; enfin une procédure pour séparer le signal des colloïdes du signal de l'électrolyte support dans l'acoustophorèse des suspensions réelles
This Ph.D. thesis is on the analytical description of acoustophoretic phenomena, in solutions and suspensions. Acoustophoresis is the creation of an electric field by an acoustic wave.First part is on electrolytic solutions, and it begins by a critical review of literature, from Debye first paper to a recent Ph.D. thesis on the same subject. Hypotheses are carefully selected, and a new model is deduced. This model, using pressure, friction, electric, inertia and corrective force, allows the prediction of acoustophoresis up to 0,3 molar for a simple salt, without any fitting parameter. An extension to solutions with three ionic species is done, and a Fortran program to compute the acoustophoresis as a function of the concentration is given in annex. Extension of the model, in the case of ionic liquid, allows the measurement of the volume of ions. A brief point is done on micellar and colloidal suspensions. A second part is on the application of non-equilibrium thermodynamic, especially Onsager reciprocal relation, to the acoustophoresis of suspensions. Acoustophoresis is shown to be proportional to the electric mobility, which allows the measurement of the latter in dark and concentrated suspensions. A link between acoustophoresis and the creation of acoustic wave by an electric field is also found, and a process to isolate contributions of colloids in real suspensions, with a supporting electrolyte, is proposed

Dans le domaine du diagnostic in vitro, l'étape d'extraction de micro-organismes à partir d'un échantillon complexe est une étape clé pour permettre l'identification du pathogène responsable d'une infection. Pour les septicémies, cette étape d'extraction est généralement précédée d'une étape de culture, ce qui conduit à une obtention des résultats au bout de plusieurs jours. Un résultat plus rapide (typiquement inférieur à 24h) permettrait d'augmenter le taux de survie des patients, et aurait ainsi une forte valeur ajoutée pour le corps médical. Le but de ces travaux est donc de développer une nouvelle méthode d'extraction et de concentration de pathogènes directement à partir d'un échantillon sanguin, sans étape de culture. Une stratégie en deux modules microfluidiques associés en série est proposée : elle repose sur la modification de la conductivité et de l'osmolarité de l'échantillon dans un premier module, puis sur la capture des micro-organismes par diélectrophorèse dans un second module. L'étude du premier module a permis de déterminer l'impact de la conductivité et de l'osmolarité du milieu sur les propriétés diélectriques des cellules. Deux voies ont ainsi été abordées, afin de diriger les cellules du sang et les micro-organismes vers un milieu de conductivité et d'osmolarité contrôlées : la dilution, et l'utilisation de forces acoustiques. L'étude du deuxième module a ensuite permis de démontrer la possibilité de capturer et concentrer des micro-organismes à partir d'un échantillon hypotonique et faiblement conducteur dans un écoulement microfluidique par diélectrophorèse. L'architecture d'un microsystème dédié a été définie grâce à un modèle numérique, puis validé expérimentalement avec des échantillons sanguins et différents micro-organismes (E. coli, S. epidermidis et C. albicans). La capture générique des micro-organismes est démontrée, et un taux de capture de 97% a été obtenu pour la séparation de \EC, avec une vitesse moyenne de l'échantillon dans le microsystème de 100 à 200 µm.s-1. Enfin, des perspectives d'amélioration sont présentées pour permettre d'effectuer cette étape de séparation sur un gros volume d'échantillon (1 à 10mL) en quelques heures, afin de répondre aux exigences imposées par l'urgence des tests de diagnostic des septicémies
Extraction of pathogens from a biological sample is a key step for efficient diagnostic tests of infectious diseases. For bloodstream infections, current diagnostic methods are usually based on bacterial growth and take several days to provide valuable information. An accelerated result would have a high medical value to adjust therapeutic strategies. The aim of this study is to design a new approach for separation and concentration of microorganisms directly from a blood sample, to avoid time-consuming growth stages. We report a method based on two different microsystems connected in series: it combines modification of conductivity and osmolarity of the sample with generic capture of microorganisms by dielectrophoresis. First we explore the impact of conductivity and osmolarity on the dielectric properties of blood cells and microorganisms. Dilution and acoustic forces are both analyzed to transfer blood cells and microorganisms to the optimized buffer. Then we demonstrate the feasibility of achieving the dielectrophoretic separation of microorganisms from blood cells in a low conductivity and low osmolarity medium inside a fluidic device. The structure of the device is optimized with numerical simulations and experiments performed on blood samples and various microorganisms (E. coli, S. epidermidis and C. albicans).The generic capture of microorganisms is validated, and we achieved a separation of 97% efficiency with E. coli, with an optimal inlet velocity around 100-200 µm.s-1. Finally, we propose an improved microsystem to perform the sample preparation step on a larger volume (1-10mL) in a few hours, in order to fit the medical need

Recent advances and interest in ultrasound particle manipulation calls for theoretical understanding of acoustic radiation force and torque exerted on a configuration of multiple particles. In this thesis we theoretically study the acoustic radiation force and torque exerted by an arbitrary acoustic beam on a cluster of spherical particles in an inviscid fluid. The method is based on the partial-wave expansion (PWE) and the translational addition theorem for spherical wave functions. The combination of (PWE) and addition theorem Method enable us to solve the associated multiple scattering problem by numerically computing the (PWE) coefficients in a system of linear equations. On the other hand, when we consider the radiation force and torque exerted on a single sphere, the addition theorem has the advantage to solve this problem in a closed form. After obtaining the PWE coefficients, the acoustic radiation force and torque is computed through the farfield series solution. To illustrate the method, the acoustic radiation force and torque exerted on a single or multiple spheres are analyzed. In the case of a single sphere, the force is generated by a spherically focused ultrasound beam, where as the torque is generated by a Bessel vortex beam. For the multiple spheres configuration, the radiation force is induced by a traveling and a standing plane wave. In a specific configuration of three olive oil droplets suspended in water, with radii of the order of the wavelength, we found that rescattering events produce an acoustic interaction force, which significantly changes the radiation force on each droplet depending on the inter-droplet distance. In addition, we have found for the first time that an acoustic interaction torque due to the nonsymmetric spatial distribution of the acoustic energy density to the droplets. Further more, our study does not have restrictions on the spheres size compared to the wave length, nor on their composition material, which includes rigid, void, compressional liquid, elastic and viscoelastic solids, and layered material. Finally, this study has direct applications on methods for noncontact object handling by acoustic waves such as acoustic levitation, acoustical tweezers, and acoustophoresis in lab-on-a-chip devices.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Recentes avanços e interesse em manipulação de partículas necessitam de uma maior compreensão teórica da força de radiação e torque acústico exercidos sobre uma configuração de múltiplas partículas. Nesta tese, nós estudamos teoricamente a força de radiação e torque acústico exercido por um feixe acústico arbitrário em um conjunto de partículas esféricas suspensas em um fluido não viscoso. O método baseia-se na expansão de ondas parciais (EOP) e no teorema translacional da adição para funções de onda esférica. A combinação do método de ondas parciais com o teorema da adição nos permitir resolver o problema de espalhamento mútiplo computando numericamente os coeficientes da expansão em um sistema de equações lineares. Por outro lado, quando consideramos a força e torque de radiação exercidos sobe uma única esfera, o teorema da adição tem a vantagem para resolver este problema exatamente. Após a obtenção dos coeficientes, a força e o torque de radiação são calculados usando um método em séries no campo distante. Para ilustrar o método, a força e o torque exercidos sobre uma ou multiplas esferas são analisados. Para o de uma única esfera, a força de radiação é gerada por um feixe de ultrassom focalizado. Para uma configuração de multiplas esferas, a força de radiação é induzida por ondas planas e estacionarias. Numa configuração específica de três gotas de azeite suspensas em água, com raios da ordem do comprimento de onda, verificou-se que as ondas reespalhadas produzem uma força de interação acústica, o que altera significativamente a força de radiação em cada gota em função da distância inter-gota. Além disso, verificou-se, pela primeira vez que um torque de interação acústico devido a uma distribuição espacial não simétrica da densidade de energia acústica para as gotas. Além disso, nosso estudo não tem restrições quanto ao tamanho esferas em comparação com o comprimento de onda, nem sobre a sua composição, que inclui rígida, líquida, elástica e sólidos viscoelásticos. Por fim, este estudo tem aplicações diretas sobre os métodos de manipulação de objetos sem contato por ondas acústicas, tais como a levitação acústica, pinças acústicas e acoustophoresis em dispositivos lab-on-a-chip.

Dans le domaine du diagnostic in vitro, l'étape d'extraction de micro-organismes à partir d'un échantillon complexe est une étape clé pour permettre l'identification du pathogène responsable d'une infection. Pour les septicémies, cette étape d'extraction est généralement précédée d'une étape de culture, ce qui conduit à une obtention des résultats au bout de plusieurs jours. Un résultat plus rapide (typiquement inférieur à 24h) permettrait d'augmenter le taux de survie des patients, et aurait ainsi une forte valeur ajoutée pour le corps médical. Le but de ces travaux est donc de développer une nouvelle méthode d'extraction et de concentration de pathogènes directement à partir d'un échantillon sanguin, sans étape de culture. Une stratégie en deux modules microfluidiques associés en série est proposée : elle repose sur la modification de la conductivité et de l'osmolarité de l'échantillon dans un premier module, puis sur la capture des micro-organismes par diélectrophorèse dans un second module. L'étude du premier module a permis de déterminer l'impact de la conductivité et de l'osmolarité du milieu sur les propriétés diélectriques des cellules. Deux voies ont ainsi été abordées, afin de diriger les cellules du sang et les micro-organismes vers un milieu de conductivité et d'osmolarité contrôlées : la dilution, et l'utilisation de forces acoustiques. L'étude du deuxième module a ensuite permis de démontrer la possibilité de capturer et concentrer des micro-organismes à partir d'un échantillon hypotonique et faiblement conducteur dans un écoulement microfluidique par diélectrophorèse. L'architecture d'un microsystème dédié a été définie grâce à un modèle numérique, puis validé expérimentalement avec des échantillons sanguins et différents micro-organismes (E. coli, S. epidermidis et C. albicans). La capture générique des micro-organismes est démontrée, et un taux de capture de 97% a été obtenu pour la séparation de \EC, avec une vitesse moyenne de l'échantillon dans le microsystème de 100 à 200 µm.s-1. Enfin, des perspectives d'amélioration sont présentées pour permettre d'effectuer cette étape de séparation sur un gros volume d'échantillon (1 à 10mL) en quelques heures, afin de répondre aux exigences imposées par l'urgence des tests de diagnostic des septicémies.

碩士
國立雲林科技大學
機械工程系
107
Abstract Nowadays, using the simulation software is more frequent. The physical quantity can be selected and also combination of many physical quantities that are different from each type. In order to emerge more complex and more diversified in accordance with actual situations. By analyzing of the experimental circuit is feasible or not? After a lot of data is sorted out, adjust and integration the data one by one. So that can clearly distinguish the difference in details. After the subsequent processing, the experimental steps can be started. After all, in the simulation, the detailed condition can be simulated from the 2D plane, and then extended to the 3D model, although it is more complicated but more perfect. The wave pattern selected this time is: "plate wave". The single particle in the substrate makes an elliptical motion. Each single particle must affect the thickness of the half plate to achieve the simplest characteristics of the plate wave, because if the wave energy is only transmitted on the surface, you can use the surface wave directly. Since the energy needs to be transmitted to the entire substrate, the paper explains the influence of energy transfer on different designs, and the difference is reflected in the data comparison. Therefore, the finger electrodes are set on both sides of the substrate as a function of stable energy transfer. The dispersion curves measured according to the substrate width of different widths are used for different wavelengths, and are used on the basis of subsequent designs, and then other physical quantities are added to simulate in layers to achieve experimentally consistent geometric settings. To compare between the physical quantities of the sound pressure frequency domain and the thermal sound field, the reason for selecting the thermal sound field is also because the results of the comparison between the use conditions and the consideration factors in the two physical quantities are performed, and the screening is adjusted by adjusting different items or It is to reduce the boundary conditions used, and the obtained data is adjusted to be the parameters required for the experiment. Keyword: Plate wave、Electromechanical coupling coefficient、Perturb method、microfluid particle trapping

碩士
國立成功大學
奈米科技暨微系統工程研究所
100
Previous studies on particle focusing using acoustic radiation force have mainly focused on separation within a single fluid that needs a subsequent procedure to re-dilute separated particles into other media for cellular analysis. In this study, a twin fluid micro-flow system is proposed for separating particles from its original solvent and rediluting them into another solvent simultaneously. In this micro-flow system, two different miscible solvents flow parallel to each other through a 2-inlet-2-outlet micro-channel, where an acoustic standing wave is set up. Due to the differences in acoustic properties of these solvents, the pressure node of the acoustic wave is shifted from the middle line of the channel. Under the action of the acoustic radiation force, particles with positive A -factors are extracted from their original solvent and re-suspended into the other solvent, wherein the pressure node resides.