Continuous Nanoparticle Sizing and Characterization via Microfluidics

High-throughput manufacturing of nanomaterial-based products demands robust online characterization and quality control tools capable of continuously probing the in suspension state. But existing analytical techniques are challenging to deploy in production settings because they are primarily geared toward small-batch ex-situ operation in research laboratory environments. Here we introduce an approach that overcomes these limitations by exploiting surface complexation interactions that emerge when a micron-scale chemical discontinuity is established between suspended nanoparticles and a molecular tracer. The resulting fluorescence signature is easily detectable and embeds surprisingly rich information about composition, quantity, size, and morphology of nanoparticles in suspension independent of their agglomeration state. We show how this method can be straightforwardly applied to enable continuous sizing of commercial ZnO nanoparticles, and to instantaneously quantify the anatase and rutile composition of multi-component TiO2 nanoparticle mixtures pertinent to photo catalysis and solar energy conversion. A transport model of the interfacial complexation process is formulated to qualitatively confirm the experimental discovery and to provide understanding of the transport and binding processes. Practical utility is demonstrated by combining our detection method with a cyclone sampler to enable continuous monitoring of airborne nanoparticles. Our method uniquely combines ultra-high flow rate sampling (up to thousands of liters per minute) with sensitive detection based on localized fluorescent complexation, permitting rapid quantitative measurement of airborne nanoparticle concentration. By coupling these components, we show initial results demonstrating detection of airborne ultrafine Al2O3 nanoparticles at environmental concentrations below 200 μg m^−3 in air sampled at 200 L min^−1. This capability suggests potential for online monitoring, making it possible to establish dynamic exposure profiles not readily obtainable using current-generation personal sampling instruments. The underlying fluorescent complexation interactions are inherently size and composition dependent, offering potential to straightforwardly obtain continuous detailed characterization. The increasing commercial prevalence of nanoparticle-based materials also introduces a new demand for robust online characterization tools amenable toward online monitoring in manufacturing settings. We address this need by showing how electrical conductivity measurements can be exploited to instantaneously obtain size and species information in oxide nanoparticle suspensions. This approach is readily implemented in an easy to build platform that can be employed either online to provide real-time feedback during continuous synthesis and processing, or offline for evaluation of test samples obtained from larger batches. Our implementation enables accurate results to be obtained using inexpensive digital multimeters, suggesting broad potential for on-site deployment in industrial settings

Optical Printing of Multiscale Hydrogel Structures

Hydrogel has been a promising candidate to recapitulate the chemical, physical and mechanical properties of natural extracellular matrix (ECM), and they have been widely used for tissue engineering, lab on a chip and biophotonics applications. A range of optical fabrication technologies such as photolithography, digital projection stereolithography and laser direct writing have been used to shape hydrogels into structurally complex functional devices and constructs. However, it is still greatly challenging for researchers to design and fabricate multiscale hydrogel structures using a single fabrication technology. To address this challenge, the goal of this work is the design and develop novel multimode optical 3D printing technology capable of printing hydrogels with multiscale features ranging from centimeter to micrometer sizes and in the process transforming simple hydrogels into functional devices for many biomedical applications. Chapter 2 presents a new multimode optical printing technology that synergistically combined large-scale additive manufacturing with small-scale additive/subtractive manufacturing. This multiscale fabrication capability was used to (i) align cells using laser induced densification in Chapter 3, (ii) develop diffractive optics based on changes in refractive indices in Chapter 4, (iii) print diffractive optical elements in Chapter 5, and (iv) digitally print complex microfluidic devices and other 3D constructs in Chapter 6. Overall, this work open doors to a new world of fabrication where multiscale functional hydrogel structures are possible for a range biomedical application

Establishment of a fully automatized microfluidic platform for the screening and characterization of novel Hepatitis B virus capsid assembly modulators

El procés de descobriment de fàrmacs s'enfronta a importants desafiaments a causa de la constant disminució dels guanys per medicament atesa la disminució en les noves aprovacions de la FDA combinada amb el constant augment dels costos i el temps de desenvolupament. Les plataformes integrades de detecció usant microfluídica van sorgir com a possibles solucions per accelerar el desenvolupament de molècules actives i reduir els requisits de temps i costos. El projecte VIRO-FLOW té com a objectiu identificar nous agents curatius per al virus de l'hepatitis B (VHB), integrant els avantatges de la química de flux continu amb tecnologies de bioassaigs in vitro en microfluídica. Durant aquesta tesi es va construir un sistema microfluídic aplicant dispositius modulars automatitzats. Es van redactar protocols d'avaluació per a les dades de fluorescència i reflexió, permetent el càlcul del factor Z, les desviacions estàndard, les corbes de dilució i els valors de concentracions efectives mitjanes màximes (EC50). La proteïna central del VHB (HBc) es va seleccionar com a objectiu principalEl proceso de descubrimiento de fármacos se enfrenta a importantes desafíos debido a la constante disminución de las ganancias por medicamento dada la disminución en las nuevas aprobaciones de la FDA combinada con el constante aumento de los costes y el tiempo de desarrollo. Las plataformas integradas de detección usando microfluídica surgieron como posibles soluciones para acelerar el desarrollo de moléculas activas y reducir los requisitos de tiempo y costes. El proyecto VIRO-FLOW tiene como objetivo la identificación de nuevos agentes curativos para el virus de la hepatitis B (VHB), integrando las ventajas de la química de flujo continuo con tecnologías de bioensayos in vitro en microfluídica. Durante la presente tesis se construyó un sistema microfluídico aplicando dispositivos modulares automatizados. Se redactaron protocolos de evaluación para los datos de fluorescencia y reflexión, permitiendo el cálculo del factor Z, desviaciones estándar, curvas de dilución y valores de concentraciones efectivas medias máximas (EC50). La proteína central del VHB (HBc) se seleccionó como objetivo principal.Drug Discovery as known today faces major challenges due to the constant decrease of earnings per drug given the decrease in new FDA approvements combined with the steadily rising development costs and time. Integrated microfluidic screening platforms emerged as possible solutions by accelerating the hit-to-lead development cycle and reducing time and cost requirements. The VIRO-FLOW project aims at the fast and efficient identification of novel curative agents for the Hepatitis B Virus (HBV), integrating the advantages of continuous flow chemistry with in vitro microfluidic bioassay technologies. During the present thesis a microfluidic system was built, applying automatized modular devices. Evaluation protocols were written for the fluorescence and reflection data, allowing the Z´-factor calculation, standard deviations, dilution curves, and half‐maximal effective concentrations (EC50) values. HBV core protein (HBc) was selected as primary target due to the ongoing demand for a functional cure to reduce the economic and social challenges imposed by the chronic diseas

Determination of epithelial growth factor receptor mutations in circulatory tumour cells from non-small cell lung cancer patients isolated using a novel microfluidic device

Patients with epidermal growth factor receptor (EGFR) sensitizing mutations in non small cell lung cancer (NSCLC) receive benefit from Tyrosine Kinase inhibitors. Accurate selection of patients before treatment is highly dependent on precise molecular diagnosis of EGFR mutations. Presently in the clinic, the diagnostic samples routinely used tumour biopsy and/or cell free DNA (cfDNA), are not sufficiently effective for precise diagnosis. Circulatory tumour cells (CTC) in blood have been explored successfully as alternative and complementary diagnostic markers to the current clinical tools. However, utility in the clinics has been hampered by the relatively low concentration of CTC in blood, and the lack of robust technologies that are adaptable for routine use. The present study describes the design and optimization of an immunomagnetic based microfluidic device (Lung card version II) that isolates CTC expressing the epithelial cell adhesion molecule (EpCAM) from blood with high capture efficiency and purity. The device is a 2-part system comprising a disposable chip that is simple in design and a reusable microfluidic unit that contains a mobile magnetic arm. The simple design and work-flow process of the device ensures cost efficiency for scalability and, ultimately, use in the clinic. The device was initially validated for its capability to isolate EpCAM positive cells. Results from spiking carboxylfluorescein succinimidyl ester stained EpCAM positive cells in media/blood showed a capture efficiency of ≥ 65% and a purity ≥ 97% from a 13ml sample in 50 minutes. The isolated CTC from NSCLC patients (n=38) were analysed for mRNA markers specific to malignant cells and were characterized for EGFR mutations following PCR and next generation sequencing. The mutational status of CTC was compared to that obtained from matched, tumour biopsy, samples. Significantly more mutations (P=0.0173) were detected in CTC enriched samples than the matched biopsy. Interestingly, mutations were detected in only 4 biopsy samples and the mutations detected in the biopsy were only concordant with results from CTC enriched samples for 1 patient. Exon 19 deletion was the most frequent mutation detected (86.7%) with rare mutations such as: L792P, C797S, H509R also been detected in CTC, and the present study reports the detection of K708R mutation in NSCLC for the first time. The clinical outcomes of patients who were positive for EGFR mutation from CTC, but had been placed on therapies based on mutation results from tissue biopsy were evaluated in this study. The results showed that no significant progression free survival (PFS) benefit was attained when comparing treatment response between patients whose CTC possessed an EGFR mutation and patients whose CTC possessed no EGFR mutation (10 months vs26 months p value-0.3420 HR- 0.76 95% CI- 0.2498-2.319). In summary the results from this study showed that the microfluidic device captured CTC with efficiency equal to other immuno-affinity based devices but had better purity rates and throughput and also that the device can be utilized for CTC processing for downstream analysis. Results from this current study further demonstrated the clinical potential of CTC+NGS matrix for the detection of EGFR mutations and the prospective impact it would have for precision oncology in NSCLC are discussed

Plasmonic Nanomaterials-Based Point-of-Care Biosensors

Point-of-care (POC) biosensors, although rapid and easy-to-use, are orders magnitude less sensitive than laboratory-based tests. Further they are plagued by poor stability of recognition element thus limiting its widespread applicability in resource-limited settings. Therefore, there is a critical need for realizing stable POC biosensors with sensitivity comparable to gold-standard laboratory-based tests. This challenge constitutes the fundamental basis of this dissertation work– to expand access to quality and accurate biodiagnostic tools. At the heart of these solutions lies plasmonic nanoparticles which exhibit unique optical properties which are attractive for label-free and labelled biosensors.Firstly, we improve the stability and applicability of label-free plasmonic biosensors for implementing biodiagnostics in POC and resource-limited settings. We demonstrate a cost-effective plasmonic paper-based biosensor for non-invasive detection of renal cancer. We also demonstrate a facile integration of plasmonic paper and microneedle patch to realize a POC biosensor which enables detection of target biomarkers present in interstitial fluid in an easy-to-use two-step process. We introduce a polymer encapsulation strategy to realize a stable and refreshable biosensor for long-term monitoring of protein biomarkers under harsh conditions. Next, we demonstrate dramatic improvement in bioanalytical parameters of POC biosensors by designing and realizing an ultrabright fluorescent nanolabel, plasmonic fluor. We discuss a novel approach for detection and quantification of inflammatory disease burden via plasmonically-active tissue analog which can undergo in vivo or ex vivo degradation in the presence of biological fluid associated with the tissue. We demonstrate a partition-free digital fluoroimmunoassay for ultrasensitive, multiplexed, and quantitative detection of protein biomarkers present in human biospecimens. Significantly, utilizing plasmonic-fluor, we overcome long-standing limitations associated with lateral flow immunoassays (LFA)– limited sensitivity, low accuracy and smaller analytical range compared to laboratory tests, and limited quantitation ability. Taken together, these advances are expected to overcome fundamental challenges associated with POC biosensors, and to bridge the gap between laboratory-based and at-home or point-of-care (POC) diagnosis. Through this dissertation work we demonstrate a complete workflow of a POC diagnostic platform that outperforms gold-standard laboratory tests in sensitivity, speed, dynamic range, ease of use, and cost

DEVELOPMENT OF SYNTHETIC CHEMOTAXIS BASED BACTERIAL BIOREPORTERS

With increasing industrialization and the increasing amount of chemical compounds produced every day, the need of a proper monitoring of the environment is crucial. The use of biosensors based on living microorganisms is an interesting alternative to common chemical analysis. Since microorganisms are easy and cheap to produce, and thanks to their small size, they can be implemented in miniaturized portable devices, which may be used directly in the field. Common whole-cell bacterial bioreporters produce an easy detectable signal by induction of expression of a reporter protein in presence of either a specific molecule or a general stress reaction. Whole-cell bioreporter analysis is robust, but requires a couple of hours to obtain a clear reporter signal. In this thesis, we envisioned to develop bacterial biosensors based on a different physiological response than de novo gene expression that could lead to a faster response time while keeping target sensitivity and specificity. In particular, we tried to exploit chemotaxis, the behaviour of motile bacteria to sense their environment and swim in the direction of or away from chemical compounds. Chemotaxis is rapid (sec–min scale) and some species show naturally chemotaxis towards compounds of environmental interest. In Chapter 2, we quantified bacterial chemotaxis from direct measurements of cellular motility. We developed a microfluidic chip, which generated a stable attractant gradient and into which motile bacteria could be added. The bacteria sensed the chemical gradient and accumulated where the concentration of attractant is highest. Accumulation of cells was quantified over time by epifluorescence microscopy. As a proof of concept, we used chemotaxis of Escherichia coli towards serine, aspartate and methylaspartate. Notably, E. coli accumulated to 10 µM serine within 10 minutes, but showed maximum accumulation to 100 µM serine after 20 minutes or longer. Secondly, we quantified chemotaxis of Cupriavidus pinatubonensis JMP134 to 2,4-dichlorophenoxyacetate, a commonly used herbicide. Unfortunately, accumulation of JMP134 was not very sensitive and could only be observed with 1 mM 2,4- dichlorophenoxyacetic acid or higher. Steady-state chemodynamic and chemotaxis modelling was used to support the observed cellular response in the microfluidic chip as a function of attractant concentration. In Chapter 3, we wanted to facilitate the manipulation of the microfluidic chip by the development of a different chip that integrates valves inside the structure. This could facilitate the control of the liquid flow inside the chip, as well as enable sample exchange. By focusing on individual cell movement, we expected we might achieve very short response times upon addition of attractants. The gradient was generated by alternating valve opening and motile E. coli were inserted in the middle of this pre-established gradient. Individual cell trajectories were monitored in the few first minutes of response. Contrary to our expectations, no significant difference in trajectory characteristics was measured in presence compared to absence of a gradient. Mathematical simulations of single cell chemotaxis response suggested that more time is required to observe cell accumulation, or that cells would have to be introduced closer to the source. In Chapter 4, I focused on chemotaxis responses at the molecular and single cell level by measuring CheY–CheZ interactions. I fused two non-fluorescent parts of the green fluorescent protein (Gfp) to CheY and CheZ, components of chemotaxis pathway. I could demonstrate that Gfp fluorescent foci appear in single cells as a genuine interaction between CheY and CheZ. By mutant analysis, I showed that foci form mostly at the motor complex and less frequently at the sites of chemoreceptors. Not completely unexpected, the reformed split-Gfp was relatively stable and little dynamics in position or fluorescent intensity of foci was detected. However, single cell analysis indicated that the turnover of split-Gfp is more important immediately after addition of 100 µM nickel as repellent. In Chapter 5, I attempted to measure chemotaxis response through pH changes at single cell levels. Notably, the flagellar motor is powered by an influx of protons through the cytoplasmic membrane. I deployed the pH-sensitive fluorescent protein pHluorin, either expressed inside the cytoplasm or in the periplasm of E. coli, to measure pH differences in chemotactically active cells. For this, I used a modified agarose-block test as attractant and recorded fluorescent changes in accumulating cells. Interestingly, a 100 µM serine source induced an increase of pH in the cytoplasm and a decrease in the periplasm in cells close to the source, but not in cells further away. This suggests an active export of protons from the cytoplasm to the periplasm during chemotaxis in order to compensate for the increased flux needed for the flagellar motors. Finally in chapter 6, I attempted to change E. coli chemotaxis specificity by introducing receptors coming from Pseudomonas putida. I focused on two P. putida receptors, one for benzoate and the other for toluene. I demonstrated expression of both receptors in E. coli – although we cannot be completely certain that the proteins inserted into the membrane. Agarose-block tests with serine, toluene and benzoate, in comparison to no attractants, showed that E. coli cell accumulation close to a source of toluene was significantly higher in strains expressing the toluene receptor. E. coli without benzoate receptor accumulated as well as those with benzoate receptor near sources with benzoate. In this work, we investigated different approaches to exploit chemotaxis in order to produce biosensing signals. Our results are promising and show that functional biosensors based on chemotaxis may be achieved in a variety of ways. -- En raison de l’avancée de l’industrialisation et de l’augmentation du nombre et de la quantité de composés chimiques produits chaque jour, la surveillance de la pollution touchant notre environnement est cruciale. L’utilisation de biosenseurs basés sur des micro-organismes vivants est une alternative intéressante aux analyses chimiques couramment utilisées. Ces micro-organismes sont faciles et peu coûteux à produire et, grâce à leur petite taille, ils peuvent être facilement implémentés dans des appareils miniaturisés et portables pouvant être utilisés directement sur le terrain. Les biorapporteurs bactériens usuels produisent un signal facilement détectable conséquence de l’induction de l’expression d’une protéine rapportrice en présence d’une molécule spécifique ou en réponse à un stress. Les biorapporteurs sont des outils d’analyse robustes mais requièrent quelques heures pour obtenir un signal clair. Dans cette thèse, nous avons eu pour but de développer des biosenseurs bactériens basés sur une autre réponse physiologique que l’expression de novo de gènes, ceci dans le but de produire une réponse plus rapide tout en conservant la sensibilité et la spécificité pour la molécule cible. En particulier, nous avons exploité le chimiotactisme, soit le comportement des bactéries mobiles qui peuvent sentir leur environnement et se déplacer pour s’approcher ou s’éloigner de composés chimiques. Le chimiotactisme présente l’avantage de fournir une réponse rapide, prenant de quelques secondes à quelques minutes. De plus, certaines espèces bactériennes sont connues pour montrer naturellement une attraction pour des composées d’intérêt environnemental comme des polluants. Dans le chapitre 2, nous avons quantifié le chimiotactisme bactérien par mesure directe de la mobilité cellulaire. Nous avons développé une puce micro-fluidique qui génère un gradient stable d’attractant dans lequel des bactéries mobiles peuvent être ajoutées. Les bactéries sentent le gradient chimique et s’accumulent là où la concentration d’attractant est la plus élevée. Cette accumulation de cellules est quantifiée au cours du temps par microscopie à épifluorescence. Comme preuve de concept, nous avons utilisé le chimiotactisme d’Escherichia coli vers la sérine, l’aspartate et le méthylaspartate. E. coli est attiré par 10 µM de sérine en 10 minutes, mais montre une accumulation maximale avec 100 µM de sérine après au moins 20 minutes. Nous avons également quantifié le chimiotactisme de Cupriavidus pinatubonensis JMP134 pour le 2,4- dichlorophenoxyacetate, un herbicide communément utilisé. Malheureusement JMP134 n’était pas très sensible et la réponse n’a pu être observée qu’avec un minimum de 1 mM de 2,4- dichlorophenoxyacetate. La modélisation mathématique du chimiotactisme en fonction de la concentration d’attractant a été utilisée pour appuyer les résultats obtenus expérimentalement en utilisant la puce micro-fluidique. Dans le chapitre 3, nous avons voulu faciliter la manipulation de la puce micro-fluidique en développant un autre type de puce qui intègre des valves à l’intérieure de leur structure. Cela facilite le contrôle du flux de liquide dans la puce, et permet ainsi un potentiel échange d’échantillons. En se focalisant sur le mouvement de cellules individuelles, nous nous attendions à obtenir un temps de réponse plus court après l’ajout d’attractant. Le gradient d’attractant est généré par des ouvertures alternées des valves et des cellules d’E. coli sont ensuite insérées au milieu du gradient préétabli. Les trajectoires de cellules individuelles sont enregistrées pendant les premières minutes de réponse. Contrairement à nos attentes, aucune différence significative dans les caractéristiques des trajectoires n’a été mesurée en présence ou en absence de gradient. Des simulations mathématiques de la réponse chimiotactique de cellules individuelles suggère la nécessitée d’un plus long temps d’observation ou encore d’introduire les cellules plus proche de la source d’attractant. Dans le chapitre 4, nous nous sommes focalisés sur le chimiotactisme aux niveaux cellulaire et moléculaire en mesurant l’interaction entre deux acteurs de la signalisation. Pour cela, deux parties non-fluorescentes de la protéine fluorescente verte (GFP) ont été fusionnées aux protéines CheY et CheZ, composants de la signalisation du chimiotactisme. J’ai pu démontré que les foci de fluorescence apparaissant dans les cellules individuelles montrent l’interaction entre CheY et CheZ. Par l’analyse de mutants, j’ai montré que les foci se forment principalement au niveau du moteur des flagelles et moins fréquemment au niveau des récepteurs. La GFP reformée lors de l’interaction de CheY et CheZ est relativement stable et se révèle peu dynamique dans la position ou dans l’intensité de fluorescence des foci. Néanmoins, l’analyse des cellules individuelles indique que le turnover de la GFP est plus important immédiatement après ajout de 100 µM de nickel, utilisé comme substance répulsive. Dans le chapitre 5, j’ai mesuré la réponse chimiotactique à travers les changements de pH au niveau des cellules individuelles. Le moteur des flagelles est énergisé par un influx de protons à travers la membrane cytoplasmique. J’ai donc exprimé une protéine fluorescente sensible au pH, la pHluorin, soit dans le cytoplasme ou le périplasme d’E. coli et mesuré les différences de pH dans des cellules actives pour le chimiotactisme. Pour cela, j’ai utilisé un bloc d’agarose contenant la source d’attractant et mesuré les changements de fluorescence des cellules attirées. Une source de 100 µM de serine induit une augmentation du pH dans le cytoplasme et inversement, une diminution dans le périplasme dans les cellules proches de la source mais pas dans les cellules plus éloignées. Cela suggère un export actif de protons depuis le cytoplasme dans le périplasme pendant le chimiotactisme afin de compenser l’augmentation de l’influx de protons nécessaire à la rotation des flagelles. Finalement dans le chapitre 6, j’ai changé la spécificité du chimiotactisme d’E. coli en y introduisant des récepteurs venant de Pseudomonas putida. Je me suis intéressée à deux récepteurs de P. putida, l’un liant le benzoate et l’autre le toluène. J’ai démontré que l’expression des deux récepteurs se fait de façon correcte chez E. coli, même si l’on ne peut pas être complétement certain que les protéines soient correctement repliées ou encore bien insérées dans la membrane. Les tests par bloc d’agarose contenant la sérine, le toluène ou le benzoate en comparant à l’absence d’attractant, montrent que l’accumulation d’E. coli proche de la source est significativement plus important pour la souche exprimant le récepteur pour le toluène. D’autre part, la souche n’exprimant pas le récepteur au benzoate s’accumule aussi bien que la souche avec récepteur autour d’une source de benzoate. Dans ce travail, nous avons investigué différentes approches pour exploiter le chimiotactisme dans le but de produire des signaux par des biosenseurs bactériens. Nos résultats sont prometteurs et montrent que des biosenseurs fonctionnels basés sur le chimiotactisme peuvent être obtenu par différentes approches