DEVELOPMENT OF MICROFLUIDIC PLATFORMS AS A TOOL FOR HIGH-THROUGHPUT BIOMARKER SCREENING

Droplet microfluidic platforms are in the early stages of revolutionizing high throughput and combinatorial sample screening for bioanalytical applications. However, many droplet platforms are incapable of addressing the needs of numerous applications, which require high degrees of multiplexing, as well as high-throughput analysis of multiple samples. Examples of applications include single nucleotide polymorphism (SNP) analysis for crop improvement and genotyping for the identification of genes associated with common diseases. My PhD thesis focused on developing microfluidic devices to extend their capabilities to meet the needs of a wide array of applications

Microfluidique en gouttes à l'échelle femtolitrique

Droplet-based microfluidics has demonstrated its multiple advantage over standard microtiter plates technologies by increasing analysis throughputs, decreasing costs and enabling the encapsulation of single cells into individual reservoirs. In the state-of-the-art, droplet volumes usually range from 2 pL to 4nL, one thousand to one million times smaller than microtiter plate wells. This PhD work focuses on the miniaturization of biological reservoirs down to the femtoliter scale which would enable an increase of throughputs of analysis and open up access to new studies, such as single-molecule studies or drug delivery. The first part of this manuscript concentrates on the miniaturization of elementary operations of droplet-based microfluidics down to the femtoliter scale. Production, mixing, electrocoalescence, DEP sorting, splitting, drop-on-demand, stability, biocompatibility were successfully demonstrated on droplets of a few micrometers diameter. The second part of this manuscript focuses on some biological applications that were developed with the LBC. A platform for the in situ encoding of droplets with DNA barcodes readable per sequencing was developped. Two other applications were envisioned: a project studying the conditions that prevailed the apparition of chromosomes in an early RNA world and a genotype-phenotype mapping project benefited from downscaling to the femtoliter scale.La microfluidique en gouttes permet l’analyse de systèmes biochimiques à grand débit, en utilisant de faibles volumes réactionnels, à faible coût. Dans l’état de l’art, le volume des gouttes varie entre 2 pL et 4 nL, un millier à million de fois inférieur au volume d’un puit de microplaque. Miniaturiser d’avantage les volumes réactionnels permettrait d’augmenter encore les débits d’analyse, de d’avantage réduire les coûts et ouvre également l’accès à de nouvelles études, telles que les études sur molécule unique ou la délivrance de médicaments. La première partie de ce travail de thèse concerne la miniaturisation des opérations classiques de la microfluidique en gouttes à l’échelle femtolitrique: production, stabilité, biocompatibilité, mélange en gouttes, coalescence, tri, division de gouttes, production à la demande ont été démontrés avec succès sur des gouttelettes femtolitriques. Le tout a été permis en restant dans les limites de la technologie PDMS et des standards classiques de la lithographie. La seconde partie s’intéresse à certaines applications biologiques issues du couplage de gouttelettes picolitriques et femtolitriques. Une plateforme permettant l’encodage in situ de gouttes à l’aide de codes barres d’ADN lisibles par séquençage a été construite. Deux autres applications ont été envisagées: un projet concernant l’émergence des chromosomes dans un monde prébiotique qui nécessitait des facteurs de dilution de l’ordre de 1:1000 et un projet de cartographie génotype-phénotype sur une enzyme qui nécessitait deux dilutions par 10 ont bénéficié de la miniaturisation à l’échelle femtolitrique

THERMOPLASTIC MICROFLUIDIC PCR TECHNOLOGIES FOR NEAR-PATIENT DIAGNOSTICS

Microfluidic technologies have great potential to help create portable, scalable, and cost-effective devices for rapid polymerase chain reaction (PCR) diagnostics in near patient settings. Unfortunately, current PCR diagnostics have not reached ubiquitous use in such settings because of instrumentation requirements, operational complexity, and high cost. This dissertation demonstrates a novel platform that can provide reduced assay time, simple workflow, scalability, and integration in order to better meet these challenges. First, a disposable microfluidic chip with integrated Au thin film heating and sensing elements is described herein. The system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. With extensive multiphysics modeling and empirical testing we were able to optimize the system and achieve cycle times of 14 seconds and completed 35 PCR cycles plus HRMA in a total of 15 minutes, for successful identification of a mutation in the G6PC gene indicative of von Gierke’s disease. Next, a scalable sample digitization method that exploits the controlled pinning of fluid at geometric discontinuities within an array of staggered microfluidic traps is described. A simple geometric model is developed to predict the impact of device geometry on sample filling and discretization, and validated experimentally using fabricated cyclic olefin polymer devices. Finally, a 768-element staggered trap array is demonstrated, with highly reliable passive loading and discretization achieved within 5 min. Finally, a technique for reagent integration by pin spotting affords simplified workflow, and the ability to perform multiplexed PCR. Reagent printing formulations were optimized for stability and volume consistency during spotting. Paraffin wax was demonstrated as a protective layer to prevent rehydration and reagent cross contamination during sample loading. Deposition was accomplished by a custom pin spotting tool. A staggered trap array device with integrated reagents successfully amplified and validated a 2-plex assay, showing the potential of the platform for a multiplexed antibiotic resistance screening panel

Enhancing Throughput of Combinatorial Droplet Devices via Droplet Bifurcation, Parallelized Droplet Fusion, and Parallelized Detection

Combinatorial droplet microfluidic devices with programmable microfluidic valves have recently emerged as a viable approach for performing multiplexed experiments in microfluidic droplets. However, the serial operation in these devices restricts their throughput. To address this limitation, we present a parallelized combinatorial droplet device that enhances device throughput via droplet bifurcation, parallelized droplet fusion, and parallelized droplet detection. In this device, sample droplets split evenly at bifurcating Y-junctions before multiple independent reagent droplets are injected directly into the split sample droplets for robust droplet fusion. Finally, the fused sample and reagent droplets can be imaged in parallel via microscopy. The combination of these approaches enabled us to improve the throughput over traditional, serially-operated combinatorial droplet devices by 16-fold—with ready potential for further enhancement. Given its current performance and prospect for future improvements, we believe the parallelized combinatorial droplet device has the potential to meet the demand as a flexible and cost-effective tool that can perform high throughput screening applications

Development of Microwave/Droplet-Microfluidics Integrated Heating and Sensing Platforms for Biomedical and Pharmaceutical Lab-on-a-Chip Applications

Interest in Lab-on-a-chip and droplet-based microfluidics has grown recently because of their promise to facilitate a broad range of scientific research and biological/chemical processes such as cell analysis, DNA hybridization, drug screening and diagnostics. Major advantages of droplet-based microfluidics versus traditional bioassays include its capability to provide highly monodispersed, well-isolated environment for reactions with magnitude higher throughput (i.e. kHz) than traditional high throughput systems, as well as its low reagent consumption and elimination of cross contamination. Major functions required for deploying droplet microfluidics include droplet generation, merging, sorting, splitting, trapping, sensing, heating and storing, among which sensing and heating of individual droplets remain great challenges and demand for new technology. This thesis focuses on developing novel microwave technology that can be integrated with droplet-based microfluidic platforms to address these challenges. This thesis is structured to consider both fundamentals and applications of microwave sensing and heating of individual droplets very broadly. It starts with developing a label-free, sensitive, inexpensive and portable microwave system that can be integrated with microfluidic platforms for detection and content sensing of individual droplets for high-throughput applications. This is, indeed, important since most droplet-based microfluidic studies rely on optical imaging, which usually requires expensive and bulky systems, the use of fluorescent dyes and exhaustive post-imaging analysis. Although electrical detection systems can be made inexpensive, label-free and portable, most of them usually work at low frequencies, which limits their applications to fast moving droplets. The developed microwave circuitry is inexpensive due to the use of off-the-shelf components, and is compact and capable of detecting droplet presence at kHz rates and droplet content sensing of biological materials such as penicillin antibiotic, fetal bovine serum solutions and variations in a drug compound concentration (e.g., for Alzheimer’s Disease). Subsequently, a numerical model is developed based on which parametrical analysis is performed in order to understand better the sensing and heating performance of the integrated platform. Specifically, the microwave resonator structure, which operates at GHz frequency affecting sensing performance significantly, and the dielectric properties of the microfluidic chip components that highly influence the internal electromagnetic field and energy dissipation, are studied systematically for their effects on sensing and heating efficiency. The results provide important findings and understanding on the integrated device operation and optimization strategies. Next, driven by the need for on-demand, rapid mixing inside droplets in many applications such as biochemical assays and material synthesis, a microwave-based microfluidic mixer is developed. Rapid mixing in droplets can be achieved within each half of the droplet, but not the entire droplet. Cross-center mixing is still dominated by diffusion. In this project, the microwave mixer, which works essentially as a resonator, accumulates an intensive, nonuniform electromagnetic field into a spiral capacitive gap (around 200 μm) over which a microchannel is aligned. As droplets pass by the gap region, they receive spatially non-uniform energy and thus have non-uniform temperature distribution, which induces non-uniform Marangoni stresses on the interface and thus three-dimensional (3D) chaotic motion inside the droplet. The 3D chaotic motion inside the droplet enables fast mixing within the entire droplet. The mixing efficiency is evaluated by varying the applied power, droplet length and fluid viscosity. In spite of various existing thermometry methods for microfluidic applications, it remains challenging to measure the temperature of individual fast moving droplets because they do not allow sufficient exposure time demanded by both fluorescence based techniques and resistance temperature detectors. A microwave thermometry method is thus developed here, which relies on correlating fluid temperature with the resonance frequency and the reflection coefficient of the microwave sensor, based on the fact that liquid permittivity is a function of temperature. It is demonstrated that the sensor can detect the temperature of individual droplets with ±1.2 °C accuracy. At the final part of the thesis, I extend my platform technology further to applications such as disease diagnosis and drug delivery. First, I develop a microfluidic chip for controlled synthesis of poly (acrylamide-co-sodium acrylate) copolymer hydrogel microparticles whose structure varies with temperature, chemical composition and pH values. This project investigates the effects of monomer compositions and cross-linker concentrations on the swelling ratio. The results are validated through the Fourier transform infrared spectra (FTIR), SEM and swelling test. Second, a preliminary study on DNA hybridization detection through microwave sensors for disease diagnosis is conducted. Gold sensors and biological protocols of DNA hybridization event are explored. The event of DNA hybridization with the immobilized thiol-modified ss-DNA oligos and complimentary DNA (c-DNA) are monitored. The results are promising, and suggests that microwave integrated Lab-on-a-chip platforms can perform disease diagnosis studies

Microdroplet reactors for high-throughput chemistry and biology

Droplet-based microfluidic systems have recently been developed to overcome the problems of slow mixing and dispersion associated with traditional microfluidic systems. By utilising flow instabilities between two immiscible phases, droplets can be generated using normal microfluidic formats. Further, aqueous solutions can be confined and mixed within droplets, resulting in rapid homogenisation and no dispersion. Accordingly, droplet-based microfluidic systems have been utilised in various applications in a high-throughput manner. However, the techniques and methods for droplet formation, manipulation and detection have been continuously studied and improved upon to develop, prepare, manipulate and implement droplet systems for real-world applications. Since droplets can be controllably produced with variable reagent compositions at high generation frequencies (1 kHz or above), on-line detection and characterisation of every high-speed droplet is one of the most important challenges associated with droplet analysis. The ability to extract information from each droplet microreactor is crucial for applications in high-throughput analysis and screening. An appropriate detection technique able to extract the vast amount of information produced in such systems is key in unlocking the full capabilities of droplet-based. In this work, a custom built confocal spectroscopic system was coupled with a droplet-based microfluidic system to conduct high-sensitivity and high-throughput biological experiments. The integration of a confocal system allows for online characterisation of individual droplets in terms of their size, formation frequency, fluorescence intensity and population. The combination of a droplet-based microfluidic system and the confocal detection setup has been successfully used to demonstrate a few high-throughput chemical and biological applications. For example, the droplet system was utilised to demonstrate high-throughput single cell encapsulation, characterisation and quantification for the first time. In addition, highthroughput binding assays and kinetic measurements using a well-known streptavidin-biotin binding model and a protein-protein interaction were performed. Furthermore, a novel approach for fluorescence lifetime imaging (FLIM) was developed and used to analyse mixing patterns within droplets. Specifically, data from FLIM measurements were extracted to determine spatially localised fluorescence lifetimes within droplets and thus a twodimensional map of droplet mixing. Finally, the droplet-based microfluidic approach was exploited to perform biological analysis at the single molecule level