Numerical investigation of cell encapsulation for multiplexing diagnostic assays using novel centrifugal microfluidic emulsification and separation platform

In the present paper, we report a novel centrifugal microfluidic platform for emulsification and separation. Our design enables encapsulation and incubation of multiple types of cells by droplets, which can be generated at controlled high rotation speed modifying the transition between dripping-to-jetting regimes. The droplets can be separated from continuous phase using facile bifurcated junction design. A three dimensional (3D) model was established to investigate the formation and sedimentation of droplets using the centrifugal microfluidic platform by computational fluid dynamics (CFD). The simulation results were compared to the reported experiments in terms of droplet shape and size to validate the accuracy of the model. The influence of the grid resolution was investigated and quantified. The physics associated with droplet formation and sedimentation is governed by the Bond number and Rossby number, respectively. Our investigation provides insight into the design criteria that can be used to establish centrifugal microfluidic platforms tailored to potential applications, such as multiplexing diagnostic assays, due to the unique capabilities of the device in handling multiple types of cells and biosamples with high throughput. This work can inspire new development of cell encapsulation and separation applications by centrifugal microfluidic technolog

Non-conventional solutions to physical and engineering problems facing microfluidics

Microfluidics is a vital tool for scientific research utilising micrometre scale features to provide unparalleled control of micro-, nano- and pico-litres of fluid. Planar lithographic design and fabrication techniques have become more versatile and refined over time. However, stagnation of novel designs, fabrication methodologies and experimental conditions is increasing due to the current limitations. 3D printing is approaching the resolution required in microfluidics, whilst also providing greater freedom of design, materials, and fabrication techniques. This thesis seeks to overcome the traditional limitations using 3D printing to innovate design and production, enabling rapid prototyping methodologies and truly 3D structures, which are typically expensive and labour intensive. The first system discussed within this work generates cells-laden gelatin microdroplets and featured heating and cooling water channels, with a performance comparative to commercial devices. Secondly, a flow cell for the screening of extracellular lectins via glycomimetic liposomes was produced. Additionally, stereolithographic printing was used to produce a bioinspired monolithic droplet generator which featured intertwined channels. Finally, a Van de Graaff generator-based electrophoresis system was developed in order to generate record breaking separation resolutions whilst extended capillary lifespan compared to other experimental systems.Die Mikrofluidik ist ein wichtiges Werkzeug für die wissenschaftliche Forschung, das Merkmale im Mikrometerbereich nutzt, um eine beispiellose Kontrolle von Mikro-, Nano- und Pikolitern von Flüssigkeiten zu ermöglichen. Planare lithografische Konstruktions- und Herstellungstechniken sind im Laufe der Zeit vielseitiger und verfeinert worden. Aufgrund der derzeitigen Einschränkungen nimmt jedoch die Stagnation neuartiger Designs, Herstellungsmethoden und experimenteller Bedingungen zu. Der 3D-Druck nähert sich der in der Mikrofluidik erforderlichen Auflösung und bietet gleichzeitig eine größere Freiheit bei Design, Materialien und Herstellungstechniken. Diese Dissertation versucht, die traditionellen Einschränkungen bei der Verwendung von 3D-Druck zu überwinden, um Design und Produktion zu erneuern und schnelle Prototyping-Methoden und echte 3D-Strukturen zu ermöglichen, die normalerweise teuer und arbeitsintensiv sind. Das erste in dieser Arbeit diskutierte System erzeugt mit Zellen beladene Gelatine-Mikrotröpfchen und verfügt über Heiz- und Kühlwasserkanäle mit einer Leistung, die mit kommerziellen Geräten vergleichbar ist. Zweitens wurde eine Durchflusszelle für das Screening von extrazellulären Lektinen über glykomimetische Liposomen hergestellt. Darüber hinaus wurde stereolithografischer Druck verwendet, um einen bioinspirierten monolithischen Tröpfchengenerator herzustellen, der ineinander verschlungene Kanäle aufweist. Schließlich wurde ein auf einem Van-de-Graaff-Generator basierendes Elektrophoresesystem entwickelt, um rekordverdächtige Trennauflösungen zu erzeugen und gleichzeitig die Kapillarlebensdauer im Vergleich zu anderen experimentellen Systemen zu verlängern

3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis

The emergence of microfluidic technologies has enabled the miniaturization of cell analysis processes, including nucleic acid analysis, single cell phenotypic analysis, single cell DNA and RNA sequencing, etc. Traditional chip fabrication via soft lithography cost thousands of dollars just in personnel training and capital cost. The design of these systems is also confined to two dimensions limited by their fabrication. To address the needs of smooth transition from technology to adoption by end-users, less complexity is urgently needed for microfluidics to be applied in pathogen detection under low-resource settings and more powerful integration of analyses to understand single cells. This dissertation presents my explorations in 3D microfluidics involving simulation-aided design of pretreatment devices for pathogen detection, fabrication through 3D printing, utilization of alternative commercial parts, and the combination with hydrogel material to link phenotypic analysis with in situ molecular detection for single cells. The main outputs of this dissertation are as follows: 1) COMSOL Multiphysics® was used to aid the design and understanding of microfluidic systems for environmental pathogen detection. In the development of an asymmetric membrane for concentration and digital detection of bacteria, the quantification requires Poisson distribution of cells into membrane pores; the flow field and particle trajectories were simulated to validate the cell distribution in capturing pores. In electrochemical bacterial DNA extraction, the hydroxide ion generation, species diffusion, and cation exchange were modeled to understand the pH gradient within the chamber. To address the overestimated risk by polymerase chain reactions (PCR) that detects all target nucleic acids regardless of cell viability, we developed a microfluidic device to carry out on-chip propidium monoazide (PMA) pretreatment. The design utilizes split-and-recombine (SAR) mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. Ten SAR mixers were employed based on fluid flow and diffusion simulation. High-resolution 3D printing was used for prototyping. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was experimentally demonstrated. 2) Water-in-oil droplet-based microfluidic platforms for digital nucleic acid analysis eliminates the need for calibration that is required for qPCR-based environmental pathogen detection. However, utilizing droplet microfluidics generally requires fabrication of sub-100 µm channels and complicated operation of multiple syringe pumps, thus hindering the wide adoption of this powerful tool. We designed a disposable centrifugal droplet generation device made simply from needles and microcentrifuge tubes. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil at the bottom of the tube. The average droplet size was tunable from 96 μm to 334 μm and the coefficient of variance (CV) was minimized to 5%. For droplets of a diameter of 175 μm, each standard 20 μL reaction could produce ~10⁴ droplets. Based on this calculated compartmentalization, the dynamic range is theoretically from 0.5 to 3×10³ target copies or cells per μL, and the detection limit is 0.1 copies or cells per μL. 3) Based on the disposable droplet generation device, we further developed a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol (PEG) hydrogel beads. The crosslinked hydrogel network in aqueous phase adds additional robustness to droplet microfluidics by allowing reagent exchange. The hydrogel beads demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital PCR, digital loop-mediated isothermal amplification (dLAMP), and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. This platform has the potential to advance the understanding of single cell genotype-to-phenotype correlations. 4) For effective sorting of the hydrogel beads after single cell phenotyping, a gravity-driven acoustic fluorescence-based hydrogel beads sorter was developed. The design involves a 3D-printed microfluidic tube, two sequential photodetectors, acoustic actuator, and a control system. Instead of bulky syringe pumps used in traditional cell or droplet sorting, this invention drives beads suspended in heavier fluorinated oil simply by buoyancy force to have the beads float through a vertical channel. Along the channel, sequential photodetectors quantify the bead acceleration and inform the action of downstream acoustic actuator. Hydrogel beads with different fluorescence intensity level were led into different collection chambers. The developed sorter promises cheap instrumentation, easy operation, and low contamination for beads sorting, and thus the full establishment of the single cell phenotype-genotype link. In summary, the work in this dissertation established a) the simulation-aided design and 3D printing to reduce the complexity of microfluidics, and thus lowered its barrier for environmental applications, b) a simple and disposable device using cheap commercial components to produce monodispersed water-in-oil droplets to enable easy adoption of droplet microfluidics by non-specialized labs, c) a hydrogel bead-based analysis platform that links single-cell phenotype and genotype to open new research avenues, and d) a gravity-driven portable bead sorting system that may extend to a broader application of hydrogel microfluidics to point of care and point of sample collection. These simple-for-end-user solutions are envisioned to open new research avenues to tackle problems in antibiotic heteroresistance, environmental microbial ecology, and other related fundamental problems.</p

Self-powered mobile sensor for in-pipe potable water quality monitoring

Traditional stationary sensors for potable-water quality monitoring in a wireless sensor network format allow for continuous data collection and transfer. These stationary sensors have played a key role in reporting contamination events in order to secure public health. We are developing a self-powered mobile sensor that can move with the water flow, allowing real-time detection of contamination in water distribution pipes, with a higher temporal resolution. Functionality of the mobile sensor was tested for detecting and monitoring pH, Ca2+, Mg2+, HCO3-/CO32-, NH4+, and Clions. Moreover, energy harvest and wireless data transmission capabilities are being designed for the mobile sensor

A novel, planar, microfluidic junction for multiphase flow, exemplified through the production of fusion energy targets, encapsulated mouse neuron stem cells and multi-compartmental capsules

Droplet microfluidics has been extensively studied in last two decades and found various applications in diverse research fields. In this thesis, we focused on the development of planar microfluidic devices, and explored their utility for the formation of multiple emulsions and microparticle fabrication. Here, a geometry variant flow-focusing junction was exhibited to control the location position of droplet breakup, and eliminate the satellite droplets. Then, this junction was tested to form monodispersed, multi-cored, double-emulsion droplets with controllable core numbers (up to 35) in a stepwise emulsification mechanism. Based on the above device, three diverse applications of droplet microfluidics have been conducted: (1) the mass fabrication of polymeric microcapsules with high sphericity and concentricity for inertial confinement fusion (ICF) target fabrication; (2) the production of microgels to encapsulate mouse neuron stem cells for stem cell therapy in the treatment of spinal cord injury; and (3) the formation of squalene droplets with motility and encapsulated droplet interface bilayers for the ultimate creation of artificial cells. Through the above, the following was achieved: (1) Single-core water/polymer/oil double emulsion droplets, as ICF target shells (for which sphericity and concentricity are paramount), were produced at tunable rates up to 20Hz. The polymeric microcapsules were solidified by using photopolymerization (minimum ultra violet exposure duration is 30ms) with an average 98.43±0.68% sphericity (best 99.69%), an average 98.44±0.62% concentricity (best 99.72%) and 100% yield rate; (2) Mouse neuron stem cells were encapsulated in alginate microspheres at 1 million cells/mL alginate. MTT assays were conducted to provide evidence that the cells survived the encapsulation process with continuous proliferation in vitro; and (3) xxviii Various arrangements of encapsulated droplet interface bilayer network were observed, and the motility of double emulsion droplets was realized by continuous interfacial reactions, through which the expulsion and capture actions of squalene droplets were identified

A novel, planar, microfluidic junction for multiphase flow, exemplified through the production of fusion energy targets, encapsulated mouse neuron stem cells and multi-compartmental capsules

Droplet microfluidics has been extensively studied in last two decades and found various applications in diverse research fields. In this thesis, we focused on the development of planar microfluidic devices, and explored their utility for the formation of multiple emulsions and microparticle fabrication. Here, a geometry variant flow-focusing junction was exhibited to control the location position of droplet breakup, and eliminate the satellite droplets. Then, this junction was tested to form monodispersed, multi-cored, double-emulsion droplets with controllable core numbers (up to 35) in a stepwise emulsification mechanism. Based on the above device, three diverse applications of droplet microfluidics have been conducted: (1) the mass fabrication of polymeric microcapsules with high sphericity and concentricity for inertial confinement fusion (ICF) target fabrication; (2) the production of microgels to encapsulate mouse neuron stem cells for stem cell therapy in the treatment of spinal cord injury; and (3) the formation of squalene droplets with motility and encapsulated droplet interface bilayers for the ultimate creation of artificial cells. Through the above, the following was achieved: (1) Single-core water/polymer/oil double emulsion droplets, as ICF target shells (for which sphericity and concentricity are paramount), were produced at tunable rates up to 20Hz. The polymeric microcapsules were solidified by using photopolymerization (minimum ultra violet exposure duration is 30ms) with an average 98.43±0.68% sphericity (best 99.69%), an average 98.44±0.62% concentricity (best 99.72%) and 100% yield rate; (2) Mouse neuron stem cells were encapsulated in alginate microspheres at 1 million cells/mL alginate. MTT assays were conducted to provide evidence that the cells survived the encapsulation process with continuous proliferation in vitro; and (3) xxviii Various arrangements of encapsulated droplet interface bilayer network were observed, and the motility of double emulsion droplets was realized by continuous interfacial reactions, through which the expulsion and capture actions of squalene droplets were identified

REVERSE INSULATOR DIELECTROPHORESIS: UTILIZING DROPLET MICROENVIRONMENTS FOR DISCERNING MOLECULAR EXPRESSIONS ON CELL SURFACES

Lab-on-a-chip (LOC) technologies enable the development of portable analysis devices that use small sample and reagent volumes, allow for multiple unit operations, and couple with detectors to achieve high resolution and sensitivity, while having small footprints, low cost, short analysis times, and portability. Droplet microfluidics is a subset of LOCs with the unique benefit of enabling parallel analysis since each droplet can be utilized as an isolated microenvironment. This work explored adaptation of droplet microfluidics into a unique, previously unexplored application where the water/oil interface was harnessed to bend electric field lines within individual droplets for insulator dielectrophoretic (iDEP) characterizations. iDEP polarizes particles/cells within non-uniform electric fields shaped by insulating geometries. We termed this unique combination of droplet microfluidics and iDEP reverse insulator dielectrophoresis (riDEP). This riDEP approach has the potential to protect cell samples from unwanted sample-electrode interactions and decrease the number of required experiments for dielectrophoretic characterization by ~80% by harnessing the parallelization power of droplet microfluidics. Future research opportunities are discussed that could improve this reduction further to 93%. A microfluidic device was designed where aqueous-in-oil droplets were generated in a microchannel T-junction and packed into a microchamber. Reproducible droplets were achieved at the T-junction and were stable over long time periods in the microchamber using Krytox FSH 157 surfactant in the continuous oil FC-40 phase and isotonic salts and dextrose solutions as the dispersed aqueous phase. Surfactant, salts, and dextrose interact at the droplet interface influencing interfacial tension and droplet stability. Results provide foundational knowledge for engineering stable bio- and electro-compatible droplet microfluidic platforms. Electrodes were added to the microdevice to apply an electric field across the droplet packed chamber and explore riDEP responses. Operating windows for droplet stability were shown to depend on surfactant concentration in the oil phase and aqueous phase conductivity, where different voltage/frequency combinations resulted in either stable droplets or electrocoalescence. Experimental results provided a stability map for strategical applied electric field selection to avoid adverse droplet morphological changes while inducing riDEP. Within the microdevice, both polystyrene beads and red blood cells demonstrated weak dielectrophoretic responses, as evidenced by pearl-chain formation, confirming the preliminary feasibility of riDEP as a potential characterization technique. Two additional side projects included an alternative approach to isolate electrode surface reactions from the cell suspension via a hafnium oxide film over the electrodes. In addition, a commercially prevalent water-based polymer emulsion was found to adequately duplicate microchannel and microchamber features such that it could be used for microdevice replication

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

A single-cell atlas of breast cancer cell lines to study tumour heterogeneity and drug response

Breast Cancer (BC) patient stratification is driven by receptor status and histological grading and subtyping, with about 20% of patients for which absence of any actionable biomarkers results in no clear therapeutic intervention. Clinical decision for breast cancer patients still relies primarily on the expression status of three biomarkers of therapeutic agents: the estrogen and progesterone receptors (ESR1 and PgR, respectively), and the aberrant expression/amplification of the epidermal growth factor receptor 2 (HER2/ERBB2). However, current clinical approaches for the diagnosis of such biomarkers do not account for the whole transcriptional landscape of the cell and the intrapopulation gene expression heterogeneity of tumors, that may be responsible for drug resistance in cancer patients. It is therefore necessary to discover and establish new predictive and prognostic biomarkers for patient stratification and personalized medicine that take into account tumor heterogeneity. Here, I evaluated the potentiality of single-cell RNA-sequencing (scRNA-seq) for automated diagnosis and drug treatment of BC. To this end, I implemented Drop-seq in the lab, a droplet-based microfluidic platform that enables to measure the gene expression profile in single-cell for thousands of cells. By means of Drop-seq, I transcriptionally profiled 35,276 individual cells from 32 cell lines covering all BC subtypes, showing that with scRNA-seq we successfully measured the expression of clinically relevant receptors. This breast cancer single-cell atlas can be used to computationally map single cell transcriptional profiles of patients' tumor biopsies to the atlas to determine their composition in terms of cell lines. By this approach, I found that each tumor is heterogeneous and composed of multiple cell lines mostly, but not exclusively, of the same subtype. I observed that in most cell lines there is a high degree of heterogeneity in the expression of BC receptors. I focused on whether such heterogeneity impacts a cell line's overall drug sensitivity. By correlating the percentage of cells expressing a given drug target (e.g. HER2, etc.) to the known toxicity of the relevant drug across the 33 cell lines, I observed a significant negative correlation (the higher the % of cells, the higher the toxicity). I then focused on the MDA-MB-361 cell-line of the luminal B subtype with a gain in genomic copy number of the locus containing the ERRB2 gene coding for HER2. Despite HER2 amplification, scRNA-seq showed that only about 70% of cells express its mRNA. To investigate the origin of this heterogeneity, I performed fluorescence-activating cell sorting (FACS) to isolate HER2 expressing cells (HER2+) from non-expressing cells (HER2-) in the MDA-MB-361 cell population. After approximately three weeks, both subpopulations re-established the original heterogeneity, thus showing that heterogeneity in HER2 expression in these cells is dynamic and not regulated by genetic mechanisms. This observation led us to the development of a bioinformatic approach named DREEP (DRug Estimation from Expression Profiles) to automatically predict responses to more than 450 anticancer agents starting from scRNA-seq and confirmed the validity of the approach using published large-scale studies on drug sensitivity. Application of DREEP to the MDA-MB-361 cell line identified drugs able to selectively inhibit the growth of the HER2- subpopulation. Etoposide was predicted to selectively inhibit the growth of the HER2- cells but not HER2+ cells. I experimentally validated the DREEP prediction of the effect of etoposide on the HER2- subpopulation. However, DREEP predicted afatinib, a specific and selective HER2 inhibitor, to be equally effective on both subpopulations, even though HER2- cells do not express the target of afatinib. Surprisingly, the experimental validation that I performed confirmed this counter-intuitive prediction. We thus developed a mathematical model to explain this counterintuitive result, in which we show that the afatinib treatment has the same effect on both subpopulations if the interconversion time between the two HER2 states is comparable to the cell cycle duration. Finally, I experimentally validated the model prediction by testing the interconversion dynamics of the HER2 state upon afatinib perturbation in MDA-MB-361 cell line

Development of novel nanomedicines for the treatment of non-small cell lung cancer

Lung cancer stands as one of the deadliest diseases, responsible for the most cancer related deaths worldwide. The UK 5-year survival rate of non-small-cell lung cancer (NSCLC), the predominant subtype of lung cancer, stands at 9.5%, highlighting an unmet need for therapeutic intervention. A key issue is the lack of efficacy current chemotherapy regimens have in the clinic. These therapies often suffer from poor tumour targeting, resulting in dissemination throughout the body and inadequate concentrations in the tumour. This causes deleterious side effects contributing to a reduced patient quality of life and ultimately survival. Nanomedicine may serve to overcome the current therapeutic hurdles in treating NSCLC; the use of nanoparticles (NPs) for the delivery of drugs can improve drug targeting to tumours, increasing efficacy and attenuating off-target side effects. NPs can be used to deliver multiple drugs and be made from varying materials such as gold (AuNPs) or polymers. Furthermore, the discovery of oncogenic mutations in genes like EGFR present druggable targets in patients harbouring the appropriate mutations. This can also be taken advantage of using NPs to more directly target tumours and increase therapeutic response. Therefore, the aim of this thesis was to develop novel NP formulations comprised of a chemically modified variant of the tyrosine kinase inhibitor afatinib and gold (Afb-AuNPs) or in combination with vinorelbine as a polymeric dual chemotherapy formulation (Dual-NPs). Drug-bearing NPs were synthesised using a combination of organic chemistry and hydrophobic ion pairing, after which the NPs were extensively characterised to discern their physicochemical properties. We then sought to investigate the in vitro efficacy of NPs. Cell viability studies revealed Afb-AuNPs and Dual-NPs were significantly cytotoxic to various NSCLC cell lines and comparatively nontoxic to noncancerous cells. Moreover, NP formulations were found to significantly inhibit proliferation of A549, H226 and PC-9 cells 3 compared to clinical formulations as determined by electric cell-substrate impedance sensing. The mechanism of uptake in cancer cells was elucidated using fluorescent NPs as a model system and quantified using confocal microscopy. Finally, the in vivo activity of biocompatibility of Dual-NPs was investigated in a physiologically relevant murine model of NSCLC. Taken together, these results highlight the therapeutic potential for NP formulations of chemotherapy.Open Acces