Development of microfluidic LED sensor platform

Persistent cells, representing a small fraction of a bacterial population, are refractory to antibiotic therapy. It is a phenotypic transient state. The underlying mechanisms of persistence remain largely unknown, but even divergent hypotheses agree on a fundamental component: persistence arises from phenotypic heterogeneity. Novel approaches are needed to elucidate which factors allow some bacteria to persist but not others. High-content screening (HCS), which uses microscopy as a screening tool, enables the visualization of single-cell phenotypes and is thus particularly suited for studying bacterial persistence. Traditionally performed with eukaryotic cells in micro-well plate format, executing HCS with prokaryotic cells required the development of a screening novel strategy due to their much smaller size. Microfluidics has proved to be a remarkable toolbox for bacterial studies. In combination with time-lapse microscopy, it permits the study of dynamic processes with single-cell resolution in a tightly controlled microenvironment. Though numerous microfluidic platforms for prokaryotes imaging have been developped, the current state of the art does not allow the imaging of thousands of genotypes in parallel, as is required for high-content screening. The focus of this thesis is to develop a high-throughput microfluidic screening platform enabling high-content imaging of prokaryotic cells. This platform allows 1152 bacterial strains to be imaged in parallel on both the single-cell and sub-cellular level with precise control over the growth environment. First, we demonstrate the ability of the platform to effectively screen for both altered cell division phenotypes through subcellular markers imaging in a static environment and for phenotypes emerging in complex medium profiles, such as bacterial persistence. Second, we employed our platform in a genetic screen, exposing a mutant library of Mycobacterium smegmatis to the antibiotic Isoniazid (INH), and identified a total of 31 mutants with altered persistence rates during antibiotic exposure as compared to the wild-type. Eight of these mutants had decreased persistence rates while 23 mutants displayed increased persistence. Third, using the same platform we performed in depth analysis of mutants with altered rates of persistence in order to identify the cell division and cell death dynamics underlying persistence. This single-cell analysis revealed unexpected dynamics where a mutant with decreased persistence displayed a lower killing rate compared to the wild-type. This high-throughput microfluidic screening platform is highly versatile and can be used in a wide range of bacterial studies, as it is capable of imaging not only single cells, but also subcellular markers, such as those based on the cell cycle or bacterial septa. This novel technology thus opens new doors for microbiologists.

Measuring the concentration of different compounds in aquatic ecosystem and understanding their impact on the environment are some of the main objectives in the environmental monitoring field. Trace metals, nutrients, volatile organic compounds (VOCs), biotoxins and algaes are among the most important targets of such assessment. Their quantification at an appropriate time scale is crucial in order to understand their dynamics in the marine biogeochemical processes. In situ continuous monitoring is the key to achieve these objectives. This thesis is focused on the development of new micro-engineered tools for improving the performance of electroanalytical techniques by exploiting microfabrication techniques. The developed devices were fabricated by using different techniques, such as thin-film technology, printing and additive manufacturing techniques. The quantification of nutrients is hampered by the high sodium chloride content present in the seawater. Literature is weak concerning systems which provide a selective removal of chloride with respect to other ions. One of the developed tools of this thesis is an environmentally-friendly microfluidic platform for selective sodium chloride removal prior to nutrients detection: chloride removal occurs upon bulk electrolysis in a thin-layer configuration and a ten times-fold reduction was achieved. Trace metals speciation may vary continuously reflecting changing in aquatic system physicochemical conditions. Their quantification is commonly achieved by voltammetric techniques. Voltammetric probes have been reported in literature for detection of trace metals such as lead, cadmium, copper and zinc. The core of these systems is a miniaturized sensor composed by an array of microelectrodes serving as WE and a CE. The reference electrode is a crucial component for the functioning of such sensors however its integration is still challenging. The second tool developed in the framework of this thesis is a three-electrode on-chip sensor for the detection of trace metals, where RE integration is achieved by using a PVC membrane plasticized with ionic liquids as diffusion barrier on top of a silver/silver chloride electrode. These sensors were capable to detect trace metals, but further improvement is required for their quantification. Marine biotoxins are compounds with toxic activity that accumulate in fish and shellfish. Among the various species of biotoxins, another target of this work is saxitoxin, a low molecular weight neurotoxin. Its detection is hindered by its low concentration, therefore a pre-concentration unit is required. Another herein developed tool is a simple microfluidic platform predisposed for pre-concentration of saxitoxin; its working principle is based on the binding of the saxitoxin to a specific aptamer and its release upon a temperature increase. VOCs, such as hydrocarbons and chlorinated hydrocarbons, are considered as significant indicators of urban and industrial pollution. Mid-infrared fiber-optic evanescent wave sensors constitute one of the most promising approaches for continuous VOCs pollution monitoring in seawater. In this thesis a microfluidic platform designed to optimize the enrichment of the polymer layer coating the fiber with the target analytes is developed. The optimization of the device design allowed the simultaneous detection of ten VOCs species. All the tools developed are intended to be integrated in submersible probes for in situ deployment.

The function of mammalian tissue relies greatly on the microscale tissue architecture into which specific types of cells are three-dimensionally arranged. To replicate these microscale tissue architectures and observe cell behaviors inside these architectures, techniques for handling, observing and stimulating the cells with microscale resolution are required; microfluidic technology—the technology that deals with the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small scale—is one of the most promising candidate technologies for the fabrication and modeling of three-dimensional (3D) tissues with microscale architectures. In this book chapter, we report the recent progresses of biofabrication and 3D tissue modeling utilizing microfluidic platforms. We cover the applications of microfluidic platforms in the following two aspects: (1) microfluidic biofabrication platforms to fabricate microtissues such as cell-laden beads, cell-laden fibers and cell-laden sheets with high throughput and precise patterning of cells; (2) tissue-on-a-chip and organ-on-a-chip platforms to perform on-chip housing/installation, sensing and stimulation of tissues for 3D tissue modeling.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

On-site bioaerosol sampling and detection in microfluidic platforms

Drug releasing microparticles play an important role in drug delivery as they can be used for site specific delivery as well as control over the release time of therapeutics. The use of microfluidic technologies for the fabrication of these particles is of increasing interest since they provide enhanced control over microparticle size and size distribution compared to bulk production methods. However, the use of microfluidic platforms in the production of drug releasing microparticles with therapeutically relevant cargo still requires optimization depending on their application, and the effect of the addition of cargo on the production process is still unexplored. Here we show the formation of therapeutically relevant (in terms of size and dose of cargo) polycaprolactone (PCL) microparticles using a microfluidic platform and analyze the effect of the addition of cargo in the microparticle size and size distribution. This microfluidic platform was designed with the aid of computational fluid dynamic simulations, allowing us to construct a polydimethylsiloxane (PDMS) microfluidic device capable of making microparticles in the range of 15 to 35 μm with low coefficient of variation (CV) both with and without cargo by varying the flow rate ratios of the phases used during droplet generation. Our data show the effect of the addition of cargo on the droplet and microparticle sizes and monodispersity. Our fabrication method allows the formation of spherical microparticles, optimal for biomedical applications. In addition, our microfluidic platform can maintain the generation of monodisperse droplets (with an average size of 52.5 μm) over extended periods of time, suggesting it has the capacity to be used for scaled-up production of PCL microparticles. This microfluidic device is a robust and reliable method for the fabrication of PCL microparticles with cargo, which can potentially be loaded with other relevant therapeutic molecules for biomedical applications.

Infectious diseases caused by bacteria, viruses, fungi, and parasites pose a significant societal challenge. In response, scientists are developing advanced technology to enhance the prevention, diagnosis, and treatment of such diseases. One such promising technology is microfluidic systems, which are utilized in organ‐on‐a‐chip systems to replicate the microenvironments of organs. These systems have potential applications in drug screening, disease modeling, and personalized medicine. This review provides an overview of recent advances in organ‐on‐a‐chip platforms and their potential for preventing and diagnosing various infections. After discussing traditional techniques employed in studying infectious diseases, the role of microfluidic platforms in detecting infections is delved in. It is expound on advanced microfluidic‐based strategies for infection diagnosis, such as the polymerase chain reaction‐based microfluidic devices, enzyme linked immunosorbent assay‐based microfluidic devices, hierarchical nanofluidic molecular enrichment systemand µWestern blotting‐based microfluidic devices, and smartphone‐based microfluidic devices. Additionally, future research challenges and perspectives are discussed on microfluidic systems in biomedical and regenerative medicine applications. Consequently, microfluidic platforms have the potential to serve as fundamental frameworks for understanding infectious diseases, thereby leading to personalized regenerative medicine. hierarchical nanofluidic molecular enrichment system

A novel and versatile optical reader for microfluidic platforms is presented. The reader includes a modular insertion port based on the lock and key concept for reproducible alignment with a miniaturized optical detection system comprising an interchangeable light emitting diode (LED) and a photodiode. The modular nature of the insertion port allows the use of microfluidic platforms in variable shapes and fluidic configurations. Three different analytical methodologies based on absorbance or fluorescence measurements were used to demonstrate the flexibility and reproducibility of the proposed experimental setup.

Early and timely detection of disease biomarkers can prevent the spread of infectious diseases, and drastically decrease the death rate of people suffering from different diseases such as cancer and infectious diseases. Because conventional diagnostic methods have limited application in low-resource settings due to the use of bulky and expensive instrumentation, simple and low-cost point-of-care diagnostic devices for timely and early biomarker diagnosis is the need of the hour, especially in rural areas and developing nations. The microfluidics technology possesses remarkable features for simple, low-cost, and rapid disease diagnosis. There have been significant advances in the development of microfluidic platforms for biomarker detection of diseases. This article reviews recent advances in biomarker detection using cost-effective microfluidic devices for disease diagnosis, with the emphasis on infectious disease and cancer diagnosis in low-resource settings. This review first introduces different microfluidic platforms (e.g. polymer and paper-based microfluidics) used for disease diagnosis, with a brief description of their common fabrication techniques. Then, it highlights various detection strategies for disease biomarker detection using microfluidic platforms, including colorimetric, fluorescence, chemiluminescence, electrochemiluminescence (ECL), and electrochemical detection. Finally, it discusses the current limitations of microfluidic devices for disease biomarker detection and future prospects.

This study describes a microfluidic thread-based analytical device (μTAD) capable of in situ mass spectrometric analysis for continuous flow reaction monitoring. Organic reaction screening is foundational to drug discovery. Microfluidic devices are of special interest here because they provide continuous reaction monitoring with advantages such as the use of smaller reagent volumes and short analysis times. We created a μTAD platform from cellulose thread and a microscope glass slide in a Y-shaped format, which enabled multiple reactants to be introduced through individual channels/reservoirs. The naturally corrugated and intertwined subfibers within cellulose thread substrates serve as serpentine mixing medium, requiring no special preparation. The capillary action behind the flow of the reactants in the thread substrate leads to controlled solvent evaporation, which is found to accelerate the chemical reactions. When performed in front of a mass spectrometer, the μTAD platform provides a facile approach to directly characterize the reaction products formed on the thread substrate. We have used the μTAD mass spectrometry platform to monitor (1) Katritzky chemistry for the synthesis of pyridinium species, (2) aza-Michael addition for uncatalyzed formation of C-N bonds, and (3) Mannich reaction, which is a three-component condensation reaction that affords β-amino carbonyl compounds. Similar reactions were screened using nanoelectrospray ionization, which suggested limited reactivity in charged microdroplets. Therefore, the main factors observed to influence reactivity on the μTAD platform were identified to include effective mixing and solvent evaporation, leading to reagent concentration in the resultant thin films. The μTAD proved to be a rapid, efficient, and low-cost platform for organic reaction monitoring, which has the potential to act as an alternative to the traditional batch screening method.

Microfluidic systems have enormous potential as investigational devices in biomedical research, in particular immunology. Microfluidic devices can be fabricated with the precise regulation of any number of parameters including controlled surface chemistries, geometrical dimensions, signal input and output and timing. The adjustable nature of microfluidic devices makes them an ideal platform to recreate cellular microenvironments for studying cell-cell interactions, migration, antibody and cytokine production, and differentiation in vitro. Investigations with customized microfluidic devices have the potential for elucidating the mechanism behind T lymphocyte commitment to either the αβ or γδ lineage. Both types of T cells arise from immature CD4-CD8- precursors in the thymus but diverge during development. Recent publications indicate that γδ T cell receptor (TCR) works in concert with ligand to direct progenitors to the γδ fate. We propose to develop a microfluidic device in which immature T lymphocytes can migrate through a thymus-like microenvironment allowing for αβ or γδ lineage commitment. As of yet, the microfluidic device fabrication protocol has been developed and preliminary cell culture experiments with the commonly used murine stromal cell line OP9 have success. Such a device would allow for analysis of specific developmental points in T cell development.

Electrophoresis in microfluidic devices is becoming a useful analytical platform for a variety of biological assays. In this report, we present a method that allows for an increased sensitivity of detection of fluorescent molecules in microfluidic electrophoresis devices. This capability is provided by the implementation of a particular buffer system that is designed to initially function in an isotachophoretic (ITP) mode and, then after a controlled amount of electric current has been applied to the system, to transition to a zone electrophoretic mode. In the initial ITP mode, analytes dissolved in a large volume of injected sample are concentrated into a single narrow zone. After application of a sufficient and adjustable amount of electric current, the system switches into a zone electrophoretic mode, where the concentrated analytes are separated according to their electrophoretic mobilities. Application of this tandem ITP-zone electrophoretic strategy to the concentration, separation, and detection of fluorescent reporter molecules in a standard microfluidic device results in an approximately 50-fold increase in detection sensitivity relative to equivalent separations that are obtained with zone electrophoresis alone. Even with very long initial sample plugs (up to 3000 microm), this strategy produces electrophoretic separations with high resolutions and peak efficiencies. This strategy can be implemented to increase detection sensitivity in any standard microfluidic electrophoresis platform and does not require any specialized hardware or microchannel configurations.

A new poly(vinyl alcohol) (PVA) sponge-supported nanocatalysis platform on a continuous-flow microfluidic multireactor device was constructed for high-efficiency and high-reusability catalytic degradation of environmental pollutants. PVA sponge-supported metal nanoparticle catalysts (MNPs/PVA) were prepared by a simple improved impregnation self-assembly method, without any complicated surface modification. The MNPs/PVA catalysts were further integrated on a multireactor microfluidic device to form a continuous flow (CF) reactor platform (MNPs/PVA/chip) for simultaneous catalytic degradation of pollutants with two different nanocatalysts. After condition and catalyst optimizations, the catalytic activities of AuNPs/PVA and CoNPs/PVA were evaluated on this continuous flow microfluidic multireactor platform, by using p-nitrophenol (4-NP) as a model organic pollutant. Both catalysts exhibited outstanding catalytic efficiency (e.g., 100% for fresh catalysts), and the strong interactions between MNPs and PVA ensured high reusability (e.g., >20 cycles). After 20 cycles of catalysis, the optimal catalyst AuNPs/PVA still maintained a high catalytic efficiency of 97.6%. Compared to AuNPs/PVA-5, the cost-effective CoNPs/PVA catalyst exhibited similar catalytic performance within the first 10 cycles, while AuNPs/PVA showed better stability for long-term use. Hence, this continuous flow catalytic platform that combines the advantages of porous material-supported nanocatalysts with microfluidic devices has tremendous potential for various cost-effective environmental research and practical applications.

This thesis covers devices exploring basic areas of physical and biological research including: surface plasmon enhanced InGaN light-emitting diode, analysis of using AlGaN emitters coupled with thin film heaters to cure onychomycosis infections, tunable organic transistors that use microfluidic source and drain electrodes, as well as an electrical microfluidic pressure gauge for PDMS MEMS. Also analyzed are devices created through the use of novel three-dimensional rapid prototyping techniques, such as: the replication of three-dimensional valves from printed wax molds, chemically robust three-dimensional monolithic SIFEL fluoropolymer microfluidics, microfluidic valves for customized radioactive positron emission dyes, reduction of microfluidic control inputs through the use of pressure multiplexing, bicuspid-inspired microfluidic check valves and microfluidic three-dimensional separation column. Devices created to analyze blood are also treated including: a microfluidic device to extract blood plasma from a fingerstick; inexpensive, portable immunoassay devices and their use in in small cell lung carcinoma and multiple sclerosis; as well as a device to screen metastasizing cancer cells. Devices created to perform polymerase chain reactions are also studied, including: an evaporative cooler for microfluidic channels, thermal management in microfluidics using micro-Peltier junctions in a microfluidic polymerase chain reaction system, and an accessible polymerase chain reaction system.

Development of pH sensitive biocompatible block copolymer polymersomes, which are stable in physiological conditions, is enabling the intracellular delivery of water soluble drugs and proteins. As a result, it is becoming increasingly important to develop robust production methods to enhance the polymersome encapsulation efficiency. One way that this could be achieved is through production in microfluidic devices that potentially offer more favourable conditions for encapsulation. Here a flow focussing microfluidic device is used to induce self-assembly of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacrylate) (PMPC-b-PDPA) block copolymer by changing the pH of the flows within the microchannels. The laminar flow conditions within the device result in a pH gradient at either interface of the central flow, where diffusion of hydrogen ions enables the deprotonation of the PDPA block copolymer and results in self-assembly of polymersomes. Dynamic light scattering reveals hydrodynamic diameters in the range of 75-275 nm and double membrane structures visualized using transmission electron microscopy indicate that polymersome nanostructures are being produced. The encapsulation efficiency for Bovine Serum Albumin (BSA) was calculated by measuring the spectroscopic absorbance at 279 nm and indicates that the encapsulation efficiency produced in the microfluidic device is equivalent to the standard in solution production method. Critically, the microfluidic system eliminates the use of organic solvents, which limit biological applications, through the pH induced self-assembly process and offers a continuous production method for intracellular delivery polymersomes.