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Many methods of single-cell analysis are currently used, and each method provides different characteristics of the cell, such as dielectric and physical properties. In this thesis we investigate the use of optical analysis in tandem with dielectrophoresis analysis in a high throughput flow cytometer system. To examine the viability of combining optical and dielectrophoretic analysis methods, this thesis proposes an optical model using Mie scattering theory, and a data extraction and particle classification algorithm that employs both analysis modalities. The optical model presented here examines the effects of different experimental variables such as particle size and refractive index, the refractive index of the medium, and cytometer components like LED wavelength. The data extraction and particle classification algorithm presented extracts both optical and DEP data and is used to identify and sort particles in mixed population experiments. Combining the information gathered from examining the optical data from particles as well as the dielectric properties from dielectrophoretic measurements provides a more robust and confident classification of particles. In this thesis, the combination of the two methods was applied to both polystyrene polymer sphere (PSS) beads and Chinese hamster ovary (CHO) cell experiments. Particle size data was successfully extracted using optical analysis and used to provide more confidence and robustness to the particle classification conducted using DEP analysis. The findings of this thesis provide a starting point for further use of optical analysis in conjunction with DEP analysis in a high through put flow cytometer system.

High-pressure chemistry can be used to determine the contents of blood or water samples and to discover new chemistries. However, working with chemistry at pressures of many tens, or even hundreds, of bars often requires expensive and stationary equipment, such as autoclaves or chromatographic systems like high-performance liquid chromatography (HPLC). Since the introduction of microfluidics in the '90s, researchers have attempted to develop microfluidic chips as microreactors to speed up synthesis with faster mass and heat transfer. Other researchers have made efforts to create microfluidic chip-based HPLC to reduce the cost, increase the separation quality, speed up the analysis, and even enable portable systems for on-site medical or environmental analysis. Still, fully integrated systems have not yet been realized due to a lack of fluidic control components. This thesis presents novel methods for on-chip regulation and monitoring of pressure, flow, and temperature. Papers I and II specifically suggest a method for regulating backpressure and stabilizing pressure and flows using thermally controlled restrictors. Furthermore, a collaboration was made where a pressure-regulating chip was connected to an on-chip HPLC. The purpose of this was to activate sample plugs and therefore reduce the requirement for expensive surrounding equipment and enable portability, Paper III. Paper IV explores the use of a pressurized capsule to generate high-pressure flows that are coupled to a pressure-regulating chip to stabilize and regulate the pressure. Finally, an approach for integrating pressure sensors into high-pressure tolerant microchannels has been proposed, Paper V. The work conducted has provided new insights into fluid dynamics. The regulating method employed in Paper I-IV utilizes a restrictor that alters the pressure drop as temperature changes, hence changing the viscosity of the fluid. Although this technology has been known since before, new understandings have eme

The last years have witnessed a link between the COVID-19 pandemic with increasing numbers of vulnerable patients and globally emerging incidences of severe drug-resistant fungal infections, thus, calling for rapid, reliable, and sensitive diagnostic tools for fungal infections. However, despite strong warnings from health authorities, such as the World Health Organization, concerning the fatal consequences of the global spread of drug-resistant pathogenic fungi, progress in fungal infection diagnosis and therapy is still limited. Today, gold standard methods for revealing resistance and susceptibility in pathogenic fungi, namely antifungal susceptibility testing (AFST), require several days for completion, and thus this lengthy process can adversely affect antifungal therapy and further promote the spread of resistance. In this work, the use of photonic silicon chips consisting of micropatterned diffraction gratings as sensitive sensors for rapid AFST of clinically relevant fungal pathogens is investigated. These photonic chips provide a surface for the colonization of microbial pathogens at a liquid-solid interface and serve as the optical transducer element for label-free monitoring of fungal growth by detecting real-time changes in the white light reflectance. These sensor elements are used to track morphological changes of fungi in the presence of clinically relevant antifungals at varying concentrations to rapidly determine the minimum inhibitory concentration (MIC) values that help to classify pathogens as resistant or susceptible. We show that by careful design of the chip dimensions, this optical method can extend from bacteria, through yeasts, to filamentous fungi for accelerated AFST, which is at least three times faster than current gold standard methods and can provide same-day results. Moreover, a 3D-printed microfluidic gradient generator was designed to complement the assay and provide an integrated system, which can potentially be employed in point

Organs-on-chips (OoCs) hold promise to engineer progressively more human-relevant in vitro models for pharmaceutical purposes. Recent developments have delivered increasingly sophisticated designs, yet OoCs still lack in reproducing the inner tissue physiology required to fully resemble the native human body. This review emphasizes the need to include microarchitectural and microstructural features, and discusses promising avenues to incorporate well-defined microarchitectures down to the single-cell level. We highlight how their integration will significantly contribute to the advancement of the field towards highly organized structural and hierarchical tissues-on-chip. We discuss the combination of state-of-the-art micropatterning technologies to achieve OoCs resembling human-intrinsic complexity. It is anticipated that these innovations will yield significant advances in realization of the next generation of OoC models.

The global population is growing, rapidly increasing the demand for sustainable, novel, and safe food proteins with minimal risks of food allergy. In vitro testing of allergy-sensitizing capacity is predominantly based on 2D assays. However, these lack the 3D environment and crosstalk between the gut, skin, and immune cells essential for allergy prediction. Organ-on-a-chip (OoC) technologies are promising to study type 2 immune activation required for sensitization, initiated in the small intestine or skin, in interlinked systems. Increasing the mechanistic understanding and, moreover, finding new strategies to study interorgan communication is of importance to recapitulate food allergen sensitization in vitro. Here, we outline recently developed OoC platforms and discuss the features needed for reliable prediction of sensitizing allergenicity of proteins.

Current methods to detect PFOA in drinking water require expensive, specialized equipment and trained operators. This research builds on previous research with the goal of creating a low-cost flow rate based microfluidic assay to test water for PFOA. The aims of this research include creating a platform for repeatable capillary flow rate-based testing and to determine the optimal reagents that interact with PFOA. Utilizing System Automated Loading for Sample Analysis (SALSA), the platform achieved greater consistency within individual chip channels and across multiple chips, evidenced by reduced standard deviation when compared to traditional pipetting techniques. Additionally, the platform demonstrated improved k-value consistency over successive uses relative to manual pipetting. The research also evaluated seven reagents for their effectiveness in detecting perfluorooctanoic acid (PFOA) in deionized water samples. Of these reagents, glutamine and L-lysine showed no statistically significant differences between PFOA-spiked samples and controls, indicating they may not significantly alter k-values for further assay development. In contrast, larger proteins such as bovine serum albumin (BSA) and lysozyme yielded promising results for PFOA detection. BSA displayed linear detection within the range of 100 ag/µL to 1 pg/µL PFOA, enabling approximate concentration measurement. This study identifies five reagents—BSA, lysozyme, myoglobin, glycine, and L-aspartic acid—as suitable for PFOA detection in water samples using a flow rate-based assay. The potential for further refinement of the assay towards cost-effectiveness and specificity, as well as the application of machine learning for enhanced data analysis, is highlighted. In initial ML trails using XGBoost, samples spiked with PFOA were differentiated from a DI control with 95% accuracy. Notably, the SALSA platform provides a viable solution for consistent chip loading in various flow rate-based assays, offering rapi

The study investigated fluid dynamics in curved microchannels, exploring 3D printing parameters, channel geometry, and fluid properties, crucial for applications in medicine and energy. It highlighted the importance of microfluidics in handling small samples and enabling rapid analysis, stressing the need for precise measurement techniques to validate fluid velocity. Using 3D printing for microchannel design illustrated their utility, with microscopy aiding flow behavior comprehension. The research aimed to validate fluid velocity, covering technology analysis, microdevice design, fab rication, and measurement methodologies. It s uccessfully fabricated microdevices confirming fluid movement via capillarity, rev ealing the relationship between ch annel radius and flow velocity. Distinct flow velocity patterns were observed, vital for design optimization. The study affirmed capillary flow as a spontaneous phenomenon, with fluid velocity variations along curved microchannels co nsistent with mass conservation principles in incompressible flows., El estudio investigó el movimiento de fluidos en microcanales curvados, centrándose en la fabricación mediante impresión 3D (SLA). El artículo analiza la geometría del canal, las propiedades del fluido y la microfluídica en el dispositivo. El objetivo principal fue determinar la velocidad del fluido en estos microcanales curvos mediante manufactura aditiva (SLA). Los resultados del estudio permitieron la fabricación de microdispositivos que validaron el movimiento del fluido por capilaridad, resaltando la relación entre el radio del canal y la velocidad del flujo. Se observaron patrones característicos en la velocidad del flujo, fundamentales para la optimización de diseños en diversas aplicaciones. En resumen, se confirmó el flujo capilar como un fenómeno de absorción espontánea de líquidos, evidenciando la variación de la velocidad del fluido a lo largo de los microcanales curvados, en consonancia con el principio de conservación de masa en flujos incompresibles.

The study investigated fluid dynamics in curved microchannels, exploring 3D printing parameters, channel geometry, and fluid properties, crucial for applications in medicine and energy. It highlighted the importance of microfluidics in handling small samples and enabling rapid analysis, stressing the need for precise measurement techniques to validate fluid velocity. Using 3D printing for microchannel design illustrated their utility, with microscopy aiding flow behavior comprehension. The research aimed to validate fluid velocity, covering technology analysis, microdevice design, fab rication, and measurement methodologies. It s uccessfully fabricated microdevices confirming fluid movement via capillarity, rev ealing the relationship between ch annel radius and flow velocity. Distinct flow velocity patterns were observed, vital for design optimization. The study affirmed capillary flow as a spontaneous phenomenon, with fluid velocity variations along curved microchannels co nsistent with mass conservation principles in incompressible flows., El estudio investigó el movimiento de fluidos en microcanales curvados, centrándose en la fabricación mediante impresión 3D (SLA). El artículo analiza la geometría del canal, las propiedades del fluido y la microfluídica en el dispositivo. El objetivo principal fue determinar la velocidad del fluido en estos microcanales curvos mediante manufactura aditiva (SLA). Los resultados del estudio permitieron la fabricación de microdispositivos que validaron el movimiento del fluido por capilaridad, resaltando la relación entre el radio del canal y la velocidad del flujo. Se observaron patrones característicos en la velocidad del flujo, fundamentales para la optimización de diseños en diversas aplicaciones. En resumen, se confirmó el flujo capilar como un fenómeno de absorción espontánea de líquidos, evidenciando la variación de la velocidad del fluido a lo largo de los microcanales curvados, en consonancia con el principio de conservación de masa en flujos incompresibles.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, what you put is what you get (WYPIWYG) - that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.

The Human Genome Project ultimately aimed to translate DNA sequence into drugs. With the draft in hand, the Molecular Libraries Program set out to prosecute all genome-encoded proteins for drug discovery with automated high-throughput screening (HTS). This ambitious vision remains unfulfilled, even while innovations in sequencing technology have fully democratized access to genome-scale sequencing. Why? While the central dogma of biology allows us to chart the entirety of cellular metabolism through sequencing, there is no direct coding for chemistry. The rules of base pairing that relate DNA gene to RNA transcript and amino acid sequence do not exist for relating small molecule structure with macromolecular binding partner and subsequently cellular function. Obtaining such relationships genome-wide is unapproachable via state-of-the-art HTS, akin to attempting genome-wide association studies using turn-of-the-millennium Sanger DNA sequencing. Our laboratory has been engaged in a multi-pronged technology development campaign to revolutionize molecular screening through miniaturization in pursuit of genome-scale drug discovery capabilities. We employed DNA-encoded library (DEL) synthesis principles in the development of solid-phase DELs prepared on microscopic beads, each harboring 100 fmol of a single library member and a DNA tag whose sequence describes the structure of the library member. Loading these DEL beads into 100-pL microfluidic droplets followed by on-line photocleavage, incubation, fluorescence-activated droplet sorting, and DNA sequencing of the sorted DEL beads reveals the chemical structures of bioactive compounds. This scalable library synthesis and screening platform has proven useful in several proof-of-concept projects involving current clinical targets. Moving forward, we face the problem of druggability and proteome-scale assay development. Developing biochemical or cellular assays for all genome-encoded targets is not scalable and likely imposs

Analysis of mitochondrial structure and function is increasingly being recognized as central to understanding human health and disease. Mitochondria ultrastructure has been extensively characterized with transmission electron microscopy (TEM) and CryoTEM in fixed cells. Yet mitochondria within tissue cells can have markedly different structures and functions. Only through imaging functional, intact mitochondria can one ascertain information about the electrophysiology of the organelle. Hence, there is a critical need to be able to characterize the structure and function of the individual isolated mitochondrion.Our work aims to delve into procedures for characterizing the mitochondrial structure and function of isolated, functional mitochondria using super-resolution microscopy. This revealed that we can use super-resolution quantification of mitochondrial membrane potential using lipophilic cationic dye fluorescence to characterize the respiratory function of individual isolated, functional mitochondria in a way not possible in whole cells, potentially permitting elucidation of differences between individual mitochondria. Finally, by careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane-bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This quantitative voltage-dependent membrane binding model explains why super-resolution images of lipophilic cationic dyes show strong intensity near the cristae and not in the matrix. This model enables quantitative imaging of super-resolution voltages inside functional, intact mitochondria with super-resolution.

Biomarkers are measurable indicators of disease states or health conditions of an individual. Cellular and subcellular biomarkers offer insights for disease progression and therapeutic response. Traditional methods for biomarker detection, such as reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, are proven to be effective but often require complex procedures and extensive sample preparation.To overcome such limitations, this dissertation focuses on two innovative biosensing approaches: deformation-based microfluidic devices for cellular-level diagnosis and photonic sensors for subcellular-level diagnosis. Deformation-based microfluidic devices leverage principles of mechanical filtration, inertial microfluidics, and deterministic lateral displacement to sort cells based on their deformability. A novel microfluidic device featuring a flow-focusing channel with a cylindrical post is proposed to address challenges in current deformation-based sorting methods. On the other hand, photonic sensors offer label-free detection with high sensitivity and multiplexing capabilities. Specifically, ring resonator biosensors are highlighted for their compact size and sensitivity comparable to conventional methods. This dissertation presents design, fabrication, and characterization of integrated ring-assisted Mach-Zehnder interferometer (RA-MZI) biosensor with microfluidic channel. Such RA-MZI sensor offers high signal-to-noise ratio, sensitivity, and capability to detect optical changes in a larger dynamic range. Additionally, by integrating microfluidics, sample volume can be minimized. This dissertation research lays the groundwork for the development of point-of-care platform. By sequentially connecting the deformation-based droplet separation microfluidic device with the RA-MZI photonic sensor, a single system can be created which can be used for purification and detection of desired subcellular biomark

Microfluidic techniques have emerged as powerful tools to unveil dynamic processes occurring during foam and emulsion production, and instabilities during their lifetime (coalescence and digestion), and to gain detailed insights in interfacial effects at (sub)millisecond time scales, including but not limited to, interfacial adsorption (i.e. dynamic interfacial/surface tension) and interfacial rheology. These insights are pivotal in connecting the interfacial effects to dynamic processes as they would occur during emulsion/foam production. We highlight the importance of conducting research at relevant time scales.

The interaction of perfluorinated molecules, also known as “forever chemicals” due to their pervasiveness, with their environment remains an important yet poorly understood topic. In this work, the self-assembly of perfluorinated molecules with multivalent hosts, pillar-[5]-arenes, is investigated. It is found that perfluoroalkyl diacids and pillar-[5]-arenes rapidly and strongly complex with each other at aqueous interfaces, forming solid interfacially templated films. Their complexation is shown to be driven primarily by fluorophilic aggregation and assisted by electrostatic interactions, as supported by the crystal structure of the complexes, and leads to the formation of quasi-2D phase-separated films. This self-assembly process can be further manipulated using aqueous two-phase system microdroplets, enabling the controlled formation of 3D micro-scaffolds.

Ever since the implementation of microfluidics in the biomedical field, in vitro models have experienced unprecedented progress that has led to a new generation of highly complex miniaturized cell culture platforms, known as Organs-on-a-Chip (OoC). These devices aim to emulate biologically relevant environments, encompassing perfusion and other mechanical and/or biochemical stimuli, to recapitulate key physiological events. While OoCs excel in simulating diverse organ functions, the integration of the immune organs and immune cells, though recent and challenging, is pivotal for a more comprehensive representation of human physiology. This comprehensive review covers the state of the art in the intricate landscape of immune OoC models, shedding light on the pivotal role of biofabrication technologies in bridging the gap between conceptual design and physiological relevance. The multifaceted aspects of immune cell behavior, crosstalk, and immune responses that are aimed to be replicated within microfluidic environments, emphasizing the need for precise biomimicry are explored. Furthermore, the latest breakthroughs and challenges of biofabrication technologies in immune OoC platforms are described, guiding researchers toward a deeper understanding of immune physiology and the development of more accurate and human predictive models for a.o., immune-related disorders, immune development, immune programming, and immune regulation.

# Introduction In the current study, we expand the use of our EDGE (Edge-based Droplet GEneration) device, and validate the findings with ADT (automated drop tensiometer) results at complementary timescales. Furthermore, we explore if the EDGE tensiometer could distinguish effects occurring at high whey protein concentrations (2.5 – 10 wt%). In this dataset, you can find an excel file with an overview of all dataset named "EDGEvsADT_WPI_20240516_v09_final.xlxs" (in the Analyzed_Data folder). You can also find the raw data in the Raw_Data folder with .txt files. Finally, in the Plotted_Data_Scripts folder you can find all processed data from the excel file in .txt extension together with a Matlab code to plot the data. # Abstract Hypothesis Our hypothesis is that dynamic interfacial tension values as measured by the partitioned-Edge-based Droplet GEneration (EDGE) tensiometry can be connected to those obtained with classical techniques, such as the automated drop tensiometer (ADT), expanding the range of timescales towards very short ones. Experiments Oil-water and air-water interfaces are studied, with whey protein isolate solutions (WPI, 2.5 – 10 wt%) as the continuous phase. The dispersed phase consists of pure n-hexadecane or air. The EDGE tensiometer and ADT are used to measure the interfacial (surface) tension at various timescales. A comparative assessment is carried out to identify differences between protein concentrations as well as between oil-water and air-water interfaces. Findings The EDGE tensiometer can measure at timescales down to a few milliseconds and up to around 10 seconds, while the ADT provides dynamic interfacial tension values after at least one second from droplet injection and typically is used to also cover hours. The interfacial tension values measured with both techniques exhibit overlap, implying that the techniques provide consistent and complementary information. Unlike the ADT, the EDGE tensiometer distinguishes differences in prote

Microfluidics is an ever-expanding field featuring experiments and research on everdecreasing sample sizes. Though microfluidic thread-based experiments are not new, the idea of coupling them with optical techniques is. This work aims to demonstrate a method of detecting and quantifying fluorophores in an aqueous sample while wicking through a nylon thread and using a low-cost, yet surprisingly robust microscope setup. The technique is exceptionally sensitive, and relative detection limits were found to be < 10 ppm for riboflavin and < 0.1 ppm for quinine. Absolute detection limits were calculated to be 17.1 and 0.171 ng respectively. Individual quantification of these fluorophores in a mixture was also demonstrated while maintaining sensitivity by altering the pH of the solution.

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishingmore robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, wedemonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximaloxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters oftwo or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their abilityto adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covarianceof size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covarieslognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to theirtissue- or organ-level metabolism and describing its size-related variability through scaling laws.

This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics. The system integrates the extraction and purification of nucleic acids, followed by amplification via either reverse transcription-quantitative polymerase chain reaction (RT–qPCR) or reverse transcription loop-mediated isothermal amplification (RT–LAMP). By meeting diverse diagnostic and reagent needs, the platform yields testing results that closely align with those of commercial RT-LAMP and RTqPCR systems. Notable advantages of our system include its speed and cost-effectiveness. The assay is completed within 28min, including sample loading (5min), ribonucleic acid (RNA) extraction (3min), and RT-LAMP (20min). The cost of each assay is $9.5, and this pricing is competitive against that of Food and Drug Administration (FDA)-approved commercial alternatives. Although some RNA loss during on-chip extraction is observed, the platform maintains a potential limit of detection lower than 297 copies. Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located. Another key feature is the platform’s versatility, allowing users to choose between RTqPCR or RTLAMP tests based on specific requirements.

This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics. The system integrates the extraction and purification of nucleic acids, followed by amplification via either reverse transcription-quantitative polymerase chain reaction (RT–qPCR) or reverse transcription loop-mediated isothermal amplification (RT–LAMP). By meeting diverse diagnostic and reagent needs, the platform yields testing results that closely align with those of commercial RT-LAMP and RTqPCR systems. Notable advantages of our system include its speed and cost-effectiveness. The assay is completed within 28min, including sample loading (5min), ribonucleic acid (RNA) extraction (3min), and RT-LAMP (20min). The cost of each assay is $9.5, and this pricing is competitive against that of Food and Drug Administration (FDA)-approved commercial alternatives. Although some RNA loss during on-chip extraction is observed, the platform maintains a potential limit of detection lower than 297 copies. Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located. Another key feature is the platform’s versatility, allowing users to choose between RTqPCR or RTLAMP tests based on specific requirements.

Type I diabetes (T1D) is an autoimmune disorder affecting the insulin-producing beta cells of the islets of Langerhans, disrupting the glucose homeostasis regulatory system. Nowadays, islet transplantation is one of the anticipated treatments to revive the endocrinal function by injecting isolated pancreatic islets from a deceased donor into the patient’s liver’s portal vein. Regardless of the promising aspect, the main issue prior to transplantation is the inconsistent quality and low percentage of functioning islets post-transplantation. Therefore, a rapid islet functionality test with minimal complicated operation becomes necessary to tackle the pre-transplantation issue. This project revolves around the end-to-end development of an electrophysiology recording platform to monitor extracellular activities in murine pancreatic islets. A microfluidic perfusion system with hydrodynamic trapping is integrated with planar gold microelectrode arrays (MEA) as the preliminary device directly interfacing the islets. The design and fabrication of both the microfluidics and electrode devices, as well as in-house front-end electronics with analog filters and amplifiers tailored to capture the microvolt-scale signals, covered most of the project. Offline digital processing was performed in Python to analyse the recorded signals further. As a result, the complete platform and recording setup have been fully integrated, with successful islet trapping on top of electrodes and front-end electronics with 220x voltage gain and 0.1-3000 Hz bandwidth to record extracellular electrophysiology signals from intact pancreatic islets. While the current preliminary electrophysiology recordings are still quite inconclusive and require further validation, the project serves as a starting point in developing devices for extracellular electrophysiology measurement, which has not commonly been investigated specifically in pancreatic islets, and enables further exploration in the field., Typ I-diabetes (T1D) är en autoimmun sjukdom som påverkar de insulinproducerande betacellerna på de Langerhanska öarna och stör det reglerande systemet för glukoshomeostas. Nuförtiden är ötransplantation en av de förväntade behandlingarna för att återuppliva den endokrina funktionen genom att injicera isolerade pankreasöar från en avliden donator i patientens levers portven. Oavsett den lovande aspekten är huvudfrågan före transplantation den inkonsekventa kvaliteten och låga andelen fungerande öar efter transplantationen. Därför blir ett snabbt funktionstest av öar med minimalt komplicerad operation nödvändigt för att ta itu med problemet före transplantation. Detta projekt kretsar kring end-to-end utveckling av en elektrofysiologisk inspelningsplattform för att övervaka extracellulära aktiviteter i murina pankreatiska öar. Ett mikrofluidiskt perfusionssystem med hydrodynamisk infångning är integrerat med plana guldmikroelektrodarrayer (MEA) som den preliminära enheten som direkt gränsar till öarna. Designen och tillverkningen av både mikrofluidik och elektrodenheter, såväl som intern front-end-elektronik med analoga filter och förstärkare skräddarsydda för att fånga signalerna i mikrovoltskala, täckte större delen av projektet. Offline digital bearbetning utfördes i Python för att analysera de inspelade signalerna ytterligare. Som ett resultat har den kompletta plattformen och inspelningsuppsättningen integrerats helt, med lyckad ö-infångning ovanpå elektroder och front-end-elektronik med 220x spänningsförstärkning och 0,1-3000 Hz för att registrera extracellulära elektrofysiologiska signaler från intakta pankreatiska öar. Medan de nuvarande preliminära elektrofysiologiska inspelningarna fortfarande är ganska ofullständiga och kräver ytterligare validering, fungerar projektet som en utgångspunkt för att utveckla enheter för extracellulär elektrofysiologisk mätning, som inte vanligtvis har undersökts specifikt i pankreasöar, och möjliggör ytterligare utforskning in

Understanding how organisms respond to oxygen (O2) fluctuations in their aquatic microenvironments and studying O2 consumption as an indicator of metabolism is crucial for advancing ecology, toxicology, tissue engineering and environmental sciences. However, despite their importance across scientific disciplines, traditional O2 measurements often lack the sensitivity to capture rapid or localized changes occurring at the microscale. This PhD thesis addresses this challenge by developing novel microfluidic techniques and systems to sense O2. To investigate advective and diffusive O2 fluxes at the microscale, this thesis developed chemical sensing particle image velocimetry (sensPIV), a technique that combines optode microparticles and rapid microscope imaging. Specifically, I contributed by testing the performance of sensPIV particles within O2-permeable polydimethylsiloxane (PDMS) devices. This demonstrated the effectiveness of sensPIV in visualizing microscale O2 fluxes, with potential applications around complex biological structures such as coral segments. To characterize single-cell O2 respiration rates this thesis developed a micro-respiration chamber device. This device consists of gastight microwells that isolate single-cells, allowing for measuring their O2 consumption via immobilized O2-sensitive optodes. When applied to human hepatic cells, this device revealed size-related respiration kinetics in single-cells and adaptively changing respiration rates due to O2 limitations. The micro-respiration device was then refined into SlipO2Chip, a microfluidic platform that quantifies single-cell O2 respiration rates before and after chemical exposures. This was achieved by adding a mechanism for opening and closing microwells via slipping a dedicated channel that introduces chemical solutions. SlipO2Chip demonstrated a dose-dependent decrease in diatom respiration when exposed to the bacterial infochemical 2-Heptyl-4-Quinolone (HHQ), thus enhancing our understandin

Infertility is a global challenge, often remedied with In vitro fertilization (IVF) and Intra cytoplasmic sperm injection (ICSI). Sperm quality is crucial, prompting ICSI when compromised. Routine sperm preparation via gradient centrifugation raises concerns about sperm stress and DNA fragmentation. Zymot, a new device that utilizes microfluidic technology, emerges as a promising alternative. It minimizes sperm stress and DNA damage, potentially enhancing fertilization rates and embryo quality. The aim of this study was to compare the outcome of ICSI samples treated with Zymot against gradient centrifuged samples. Focus was on fertilization rates, embryo quality, and pregnancy outcomes. The results of 104 Zymot treated samples from men with compromised sperm quality were compared with 144 gradient centrifugations retrospective. Results revealed a significant difference between methods in the number of pronulear (PN), specifically in the Good Quality Embryo (GQE) where 62% with Zymot were 2PN compared to 59% with gradient (p=0.017). No significant difference in pregnancy rates or embryo utilization rate were observed. A tendency towards higher proportion (54.6%) of Zymot-treated embryos were cryopreserved compared to gradient (49.4%, p=0,27). In conclusion, a significant difference between methods in the GQE proportion of 2PN embryos favored Zymot. Closer examination revealed a higher proportion of embryos cryopreserved with Zymot, suggesting a potential for increased treatment success in future cycles. Zymot, requiring less time, yielded equivalent results to gradient centrifugation, with higher GQE proportions and more embryos cryopreserved. This merits consideration as a high-quality alternative to sperm preparation for ICSI in cases of poor sperm quality.

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishingmore robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, wedemonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximaloxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters oftwo or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their abilityto adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covarianceof size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covarieslognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to theirtissue- or organ-level metabolism and describing its size-related variability through scaling laws.

ZIF-8/PVA microspheres with an even size were synthesized with the assistance of a simple microfluidic device. The ZIF-8/PVA microdroplets were first generated in a simple co-flow microfluidic device using a PVA aqueous solution dispersed with ZIF-8 nanoparticles as the dispersed phase and fatty acid methyl ester (FAME) as the continuous phase. Subsequently, the ZIF-8/PVA microdroplets were further extracted to form ZIF-8/PVA microspheres. The influence of the type of continuous phase on the sphericity of microspheres was investigated and the effect of ZIF-8 content was studied. Further studies showed that decreasing the extraction rate and using additives of NaCl in the dispersed phase could improve the sphericity of the ZIF-8/PVA microspheres. Finally, the synthesized ZIF-8/PVA microspheres were used to study the loading and release of tetracycline. The results showed that the loading of ZIF-8/PVA microspheres was more than 5 times higher than that of pure PVA microspheres. The drug release time of ZIF-8/PVA microspheres was much longer than that of the PVA microspheres and the release rate was significantly affected by the pH of the environment., Validerad;2024;Nivå 2;2024-06-26 (joosat);Funder: Natural Science Basic Research project of Changzhou (CJ20235065);Full text license: CC BY

Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager (‘PACMan’), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv/Fm). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv/Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.

The analysis of many environmental and clinical samples requires the modification of the original pH, which is conventionally carried out by manual/automatic addition of acid, base, or buffering reagents. In the case of decentralized measurements, often, this approach is not plausible. Instead, reagentless alternatives, such as electrochemically activated in-situ pH adjustments, are suitable. Herein, we present a method for electrochemical, reversible pH modulation of thin-layer samples (<100 µm thickness) using the co-polymer poly(aniline-co-o-aminophenol) (PANOA). The PANOA's electropolymerization strategy was optimized considering the proton exchange properties in the final material. Thus, limiting the maximum anodic potential to 0.85 V and with the number of cyclic scans being ≤150), the optimal pH modulation capabilities were observed. The reversible proton exchange properties of PANOA were quantified by monitoring the pH inside the thin-layer sample (volume of 0.6 µL), which was defined by a 3D-printed microfluidic cell and a pH-sensor placed in a face planar configuration to the PANOA film. A pH value in the range of 2–4 can repeatably be reached in the samples in 3 min, purely by an electrochemical means and without the addition of external reagents. The concept has been demonstrated to acidify samples at environmental pH (artificial samples and Seawater). The outcomes suggest that the family of polyaniline-co-polymers are interesting to be explored and utilized for electrochemically based pH modulation strategies, if careful considerations are taken regarding their electropolymerization process. Overall, such materials could contribute to the development of continuous, decentralized measuring devices requiring acidification for the formal detection of environmental markers, such as nutrients, carbon species speciation and alkalinity, among others., QC 20240815

The elimination of micropollutants such as highly fluorinated substances, including per- and polyfluoroalkyl substances (PFAS), in wastewater treatment plants has been receiving growing attention due to the urgent need to minimize their adverse effects on natural water and associated ecosystems. Conventional treatment methods often fall short in effectively removing PFAS. In this study, the Hydrodynamic Cavitation on a Chip concept (HCOC) was utilized to degrade 11 common PFAS variants (PFAS11) for the first time in three different hydrodynamic cavitation reactor set-ups, each enhanced with surface modifications involving roughness elements. Stockholm municipal wastewater treated by a Membrane BioReactor (MBR) process was subjected to fully developed cavitating flow treatment using the three distinct microscale hydrodynamic cavitation (HC) reactors. The obtained results indicate that the chemical-free HCOC technique employed in this study has a significant potential in the degradation of nearly all investigated PFAS11 compounds at a notable rate of 36.1 % while the combination with MBR process can prevent blockage within the fluidic channels, enabling continuous operation with high throughput processing rates. Our proposed methodology demonstrated promising results in eliminating PFAS and could contribute to advancements in the use of microscale HC to treat micropollutants in wastewater. These findings could be a major leap in water treatment technologies addressing the global burden of resource-efficient micropollutant water treatment., QC 20240722

Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context., QC 20240605

Understanding and controlling fluid entrapment during forced imbibition in porous media is crucial for many natural and industrial applications. However, the microscale physics and macroscopic consequences of fluid entrapment in these geometric-confined porous media remain poorly understood. Here, we introduce a novel multidepth microfluidic chip, which can mitigate the depth confinement of traditional two-dimensional (2-D) microfluidic chips and mimic the wide pore size distribution as natural-occurring three-dimensional (3-D) porous media. Based on microfluidic experiments and direct numerical simulations, we observe the fluid-entrapment scenarios and elucidate the underlying complex interaction between geometric confinement, capillary number and wettability. Increasing depth variation can promote fluid entrapment, whereas increasing capillary number and contact angle yield the opposite effect, which seemingly contradicts conventional expectations in traditional 2-D microfluidic chips. The fluid-entrapment scenario in depth-variable microfluidic chips stems from microscopic interfacial phenomena, classified as snap-off and bypass events. We provide theoretical analyses of these pore-scale events and validate corresponding phase diagrams numerically. It is shown that increasing depth variation triggers snap-off and bypass events. Conversely, a higher capillary number suppresses snap-off events under strong imbibition, and an increased contact angle inhibits bypass events under imbibition. These macroscopic imbibition patterns in microfluidic porous media can be linked with these pore-scale events by improved dynamic pore-network models. Our findings bridge the understanding of forced imbibition between 2-D and 3-D porous media and provide design principles for newly engineered porous media with respect to their desired imbibition behaviours., QC 20240524

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishingmore robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, wedemonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximaloxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters oftwo or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their abilityto adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covarianceof size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covarieslognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to theirtissue- or organ-level metabolism and describing its size-related variability through scaling laws.

This thesis explores flow control and sensing in microfluidic chips for high-pressure applications. Sub-cm glass chips have been designed and fabricated with the aim of miniaturizing chemical analysis systems. Today, chemical analyses are performed worldwide for medical and environmental purposes. As more tests become available for a wider audience, the demand further increases. The large instruments that are typically used are expensive and have high chemical and power consumption. Miniaturizing components has, on the contrary, the ability to decrease volumes, costs, and environmental impacts. In addition to lower consumption, miniaturization carries several features: quick heat distribution, laminar flow, and higher pressure tolerances, to name a few. In this thesis, microfluidic chips are developed aiming to replace larger-scale instruments. Applications are centered around high-performance chromatography, which is a separation method used to separate and detect compounds in a sample. Different flow phenomena are also investigated, including fluid compressibility and capacitance, which become interesting when working at elevated pressures. Experiments have been made showing how this impacts the regulation of microfluidic flow. Thermal regulation of viscosity has been a centerpiece of this work. Controlling flow rate and pressure in a system by changing the viscosity of a fluid has proven effective for several applications. This was utilized to maintain back pressure at the end of a system as well as to control and stabilize flow at the beginning. It was also used to regulate composition and adjust parallel flows during experiments. Multiple chips were also connected to utilize several features and to get close to fully miniaturized and portable analysis systems. Apart from flow actuation, the microfluidic chips were also equipped with sensors for accurate sensing in close proximity.

Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager (‘PACMan’), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv/Fm). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv/Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishingmore robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, wedemonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximaloxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters oftwo or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their abilityto adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covarianceof size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covarieslognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to theirtissue- or organ-level metabolism and describing its size-related variability through scaling laws.

ZIF-8/PVA microspheres with an even size were synthesized with the assistance of a simple microfluidic device. The ZIF-8/PVA microdroplets were first generated in a simple co-flow microfluidic device using a PVA aqueous solution dispersed with ZIF-8 nanoparticles as the dispersed phase and fatty acid methyl ester (FAME) as the continuous phase. Subsequently, the ZIF-8/PVA microdroplets were further extracted to form ZIF-8/PVA microspheres. The influence of the type of continuous phase on the sphericity of microspheres was investigated and the effect of ZIF-8 content was studied. Further studies showed that decreasing the extraction rate and using additives of NaCl in the dispersed phase could improve the sphericity of the ZIF-8/PVA microspheres. Finally, the synthesized ZIF-8/PVA microspheres were used to study the loading and release of tetracycline. The results showed that the loading of ZIF-8/PVA microspheres was more than 5 times higher than that of pure PVA microspheres. The drug release time of ZIF-8/PVA microspheres was much longer than that of the PVA microspheres and the release rate was significantly affected by the pH of the environment., Validerad;2024;Nivå 2;2024-06-26 (joosat);Funder: Natural Science Basic Research project of Changzhou (CJ20235065);Full text license: CC BY

Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager (‘PACMan’), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv/Fm). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv/Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.

Archived patient-derived tissue specimens play a central role in understanding disease and developing therapies. To address specificity and sensitivity shortcomings of existing single-cell resolution proteoform analysis tools, we introduce a hybrid microfluidic platform (DropBlot) designed for proteoform analyses in chemically fixed single cells. DropBlot serially integrates droplet-based encapsulation and lysis of single fixed cells, with on-chip microwell-based antigen retrieval, with single-cell western blotting of target antigens. A water-in-oil droplet formulation withstands the harsh chemical (SDS, 6 M urea) and thermal conditions (98 °C, 1-2 hr) required for effective antigen retrieval, and supports analysis of retrieved protein targets by single-cell electrophoresis. We demonstrate protein-target retrieval from unfixed, paraformaldehyde-fixed (PFA), and methanol-fixed cells. Key protein targets (HER2, GAPDH, EpCAM, Vimentin) retrieved from PFA-fixed cells were resolved and immunoreactive. Relevant to biorepositories, DropBlot profiled targets retrieved from human-derived breast tumor specimens archived for six years, offering a workflow for single-cell protein-biomarker analysis of sparing biospecimens.

Small particles exhibit a wide range of dynamic behaviours in the microscopic acoustic environment due to the interplay of acoustic radiation and viscous streaming. These dynamics, including migration, trapping, propulsion and steady rotation, have been studied extensively by considering the hydrodynamic forces and torques these particles experience. This thesis investigates the flow fields generated by small particles in acoustic fields due to acoustic radiation and steady streaming phenomena. The series of studies starts from investigating a solid sphere with a misalignment between its centre of mass and geometric centre, where its dynamic behaviour becomes significantly more intricate when subjected to a single standing wave in a viscous fluid. We identify and compute a series of combined effects on the particle when it deviates from the equilibrium positions of the standing wave, encompassing the levitation and trapping due to acoustic radiation, propulsion due to acoustic streaming force and rotational motion due to torque, which are all arising from the coupled linear oscillations of the rigid body. Due to these combined forces and torques, we determine the stable configurations of spheres with various density asymmetries and flow conditions. We then break down the problem of acoustic motions and study a range of streaming flow fields that derive from and correspond to each component of the complex dynamics exhibited by the asymmetric solid particle, incorporating detailed studies of the flow fields listed below: First, we perform a detailed study of a solid body in a viscous fluid undergoing oscillatory motion, which naturally produces a steady secondary flow due to convective inertia. This phenomenon is embodied in the streaming flow generated by a sphere in an unbounded fluid executing rectilinear oscillations. We review the literature on this canonical problem and summarise exact and asymptotic formulas in the small-amplitude limit. These analytical formul

Emulsion drops in formulated products, such as paints and personal care products, can show attractive or adhesive interactions if they are stabilized by surface-active coatings. Such adhesive interactions between drops provide a pathway to help impart product functions such as gelling, flocculation and deposition. Therefore, understanding the adhesion behaviour between drops is important to formulation design, yet the soft, deformable nature of drops often makes this difficult to study and quantify. The aim of this thesis is to study the adhesion behaviour between drops in formulated emulsions where the synergistic interactions between multiple surfactants and polymers mediate or control the adhesion. The multicomponent nature of the formulations and the need to study a large parameters space, in both surfactant composition and solution conditions, required developing an experimental framework using a novel microfluidic platform to screen for systems that warrant more detailed study. A novel microfluidic method was developed to study drop adhesion in various mixtures of surface-active species, with my focus being polymer-surfactant (PS) mixtures. This method is initially linked with atomic force microscopy (AFM) methods to measure pairwise drop interactions – a more detailed and laborious method, by studying the effect of dilution on the drop adhesion where the adhesion was mainly induced by the bridging attraction between the adsorbed PS complexes on drops. Agreement between the results from the two methods suggests that the microfluidic method is reliable and can be used to screen through systems quickly and rank different PS systems through an empirical number – “adhesion number”, even from a semi-quantitative perspective. For systems of particular interest, the AFM method can then be used to gain more quantitative information of the adhesive force such as force magnitude, force range and work of adhesion. Using the microfluidic method under the dilution mode and

Infertility is a global challenge, often remedied with In vitro fertilization (IVF) and Intra cytoplasmic sperm injection (ICSI). Sperm quality is crucial, prompting ICSI when compromised. Routine sperm preparation via gradient centrifugation raises concerns about sperm stress and DNA fragmentation. Zymot, a new device that utilizes microfluidic technology, emerges as a promising alternative. It minimizes sperm stress and DNA damage, potentially enhancing fertilization rates and embryo quality. The aim of this study was to compare the outcome of ICSI samples treated with Zymot against gradient centrifuged samples. Focus was on fertilization rates, embryo quality, and pregnancy outcomes. The results of 104 Zymot treated samples from men with compromised sperm quality were compared with 144 gradient centrifugations retrospective. Results revealed a significant difference between methods in the number of pronulear (PN), specifically in the Good Quality Embryo (GQE) where 62% with Zymot were 2PN compared to 59% with gradient (p=0.017). No significant difference in pregnancy rates or embryo utilization rate were observed. A tendency towards higher proportion (54.6%) of Zymot-treated embryos were cryopreserved compared to gradient (49.4%, p=0,27). In conclusion, a significant difference between methods in the GQE proportion of 2PN embryos favored Zymot. Closer examination revealed a higher proportion of embryos cryopreserved with Zymot, suggesting a potential for increased treatment success in future cycles. Zymot, requiring less time, yielded equivalent results to gradient centrifugation, with higher GQE proportions and more embryos cryopreserved. This merits consideration as a high-quality alternative to sperm preparation for ICSI in cases of poor sperm quality.

Type I diabetes (T1D) is an autoimmune disorder affecting the insulin-producing beta cells of the islets of Langerhans, disrupting the glucose homeostasis regulatory system. Nowadays, islet transplantation is one of the anticipated treatments to revive the endocrinal function by injecting isolated pancreatic islets from a deceased donor into the patient’s liver’s portal vein. Regardless of the promising aspect, the main issue prior to transplantation is the inconsistent quality and low percentage of functioning islets post-transplantation. Therefore, a rapid islet functionality test with minimal complicated operation becomes necessary to tackle the pre-transplantation issue. This project revolves around the end-to-end development of an electrophysiology recording platform to monitor extracellular activities in murine pancreatic islets. A microfluidic perfusion system with hydrodynamic trapping is integrated with planar gold microelectrode arrays (MEA) as the preliminary device directly interfacing the islets. The design and fabrication of both the microfluidics and electrode devices, as well as in-house front-end electronics with analog filters and amplifiers tailored to capture the microvolt-scale signals, covered most of the project. Offline digital processing was performed in Python to analyse the recorded signals further. As a result, the complete platform and recording setup have been fully integrated, with successful islet trapping on top of electrodes and front-end electronics with 220x voltage gain and 0.1-3000 Hz bandwidth to record extracellular electrophysiology signals from intact pancreatic islets. While the current preliminary electrophysiology recordings are still quite inconclusive and require further validation, the project serves as a starting point in developing devices for extracellular electrophysiology measurement, which has not commonly been investigated specifically in pancreatic islets, and enables further exploration in the field., Typ I-diabetes (T1D) är en autoimmun sjukdom som påverkar de insulinproducerande betacellerna på de Langerhanska öarna och stör det reglerande systemet för glukoshomeostas. Nuförtiden är ötransplantation en av de förväntade behandlingarna för att återuppliva den endokrina funktionen genom att injicera isolerade pankreasöar från en avliden donator i patientens levers portven. Oavsett den lovande aspekten är huvudfrågan före transplantation den inkonsekventa kvaliteten och låga andelen fungerande öar efter transplantationen. Därför blir ett snabbt funktionstest av öar med minimalt komplicerad operation nödvändigt för att ta itu med problemet före transplantation. Detta projekt kretsar kring end-to-end utveckling av en elektrofysiologisk inspelningsplattform för att övervaka extracellulära aktiviteter i murina pankreatiska öar. Ett mikrofluidiskt perfusionssystem med hydrodynamisk infångning är integrerat med plana guldmikroelektrodarrayer (MEA) som den preliminära enheten som direkt gränsar till öarna. Designen och tillverkningen av både mikrofluidik och elektrodenheter, såväl som intern front-end-elektronik med analoga filter och förstärkare skräddarsydda för att fånga signalerna i mikrovoltskala, täckte större delen av projektet. Offline digital bearbetning utfördes i Python för att analysera de inspelade signalerna ytterligare. Som ett resultat har den kompletta plattformen och inspelningsuppsättningen integrerats helt, med lyckad ö-infångning ovanpå elektroder och front-end-elektronik med 220x spänningsförstärkning och 0,1-3000 Hz för att registrera extracellulära elektrofysiologiska signaler från intakta pankreatiska öar. Medan de nuvarande preliminära elektrofysiologiska inspelningarna fortfarande är ganska ofullständiga och kräver ytterligare validering, fungerar projektet som en utgångspunkt för att utveckla enheter för extracellulär elektrofysiologisk mätning, som inte vanligtvis har undersökts specifikt i pankreasöar, och möjliggör ytterligare utforskning in

Understanding how organisms respond to oxygen (O2) fluctuations in their aquatic microenvironments and studying O2 consumption as an indicator of metabolism is crucial for advancing ecology, toxicology, tissue engineering and environmental sciences. However, despite their importance across scientific disciplines, traditional O2 measurements often lack the sensitivity to capture rapid or localized changes occurring at the microscale. This PhD thesis addresses this challenge by developing novel microfluidic techniques and systems to sense O2. To investigate advective and diffusive O2 fluxes at the microscale, this thesis developed chemical sensing particle image velocimetry (sensPIV), a technique that combines optode microparticles and rapid microscope imaging. Specifically, I contributed by testing the performance of sensPIV particles within O2-permeable polydimethylsiloxane (PDMS) devices. This demonstrated the effectiveness of sensPIV in visualizing microscale O2 fluxes, with potential applications around complex biological structures such as coral segments. To characterize single-cell O2 respiration rates this thesis developed a micro-respiration chamber device. This device consists of gastight microwells that isolate single-cells, allowing for measuring their O2 consumption via immobilized O2-sensitive optodes. When applied to human hepatic cells, this device revealed size-related respiration kinetics in single-cells and adaptively changing respiration rates due to O2 limitations. The micro-respiration device was then refined into SlipO2Chip, a microfluidic platform that quantifies single-cell O2 respiration rates before and after chemical exposures. This was achieved by adding a mechanism for opening and closing microwells via slipping a dedicated channel that introduces chemical solutions. SlipO2Chip demonstrated a dose-dependent decrease in diatom respiration when exposed to the bacterial infochemical 2-Heptyl-4-Quinolone (HHQ), thus enhancing our understandin

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishingmore robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, wedemonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximaloxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters oftwo or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their abilityto adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covarianceof size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covarieslognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to theirtissue- or organ-level metabolism and describing its size-related variability through scaling laws.

Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager (‘PACMan’), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv/Fm). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv/Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.

High-pressure chemistry can be used to determine the contents of blood or water samples and to discover new chemistries. However, working with chemistry at pressures of many tens, or even hundreds, of bars often requires expensive and stationary equipment, such as autoclaves or chromatographic systems like high-performance liquid chromatography (HPLC). Since the introduction of microfluidics in the '90s, researchers have attempted to develop microfluidic chips as microreactors to speed up synthesis with faster mass and heat transfer. Other researchers have made efforts to create microfluidic chip-based HPLC to reduce the cost, increase the separation quality, speed up the analysis, and even enable portable systems for on-site medical or environmental analysis. Still, fully integrated systems have not yet been realized due to a lack of fluidic control components. This thesis presents novel methods for on-chip regulation and monitoring of pressure, flow, and temperature. Papers I and II specifically suggest a method for regulating backpressure and stabilizing pressure and flows using thermally controlled restrictors. Furthermore, a collaboration was made where a pressure-regulating chip was connected to an on-chip HPLC. The purpose of this was to activate sample plugs and therefore reduce the requirement for expensive surrounding equipment and enable portability, Paper III. Paper IV explores the use of a pressurized capsule to generate high-pressure flows that are coupled to a pressure-regulating chip to stabilize and regulate the pressure. Finally, an approach for integrating pressure sensors into high-pressure tolerant microchannels has been proposed, Paper V. The work conducted has provided new insights into fluid dynamics. The regulating method employed in Paper I-IV utilizes a restrictor that alters the pressure drop as temperature changes, hence changing the viscosity of the fluid. Although this technology has been known since before, new understandings have eme

We demonstrated resonance-based detection of magnetic nanoparticles employing novel designs based upon planar (on-chip) microresonators that may serve as alternatives to conventional magnetoresistive magnetic nanoparticle detectors. We detected 130 nm sized magnetic nanoparticle clusters immobilized on sensor surfaces after flowing through PDMS microfluidic channels molded using a 3D printed mold. Two detection schemes were investigated: (i) indirect detection incorporating ferromagnetic antidot nanostructures within microresonators, and (ii) direct detection of nanoparticles without an antidot lattice. Using scheme (i), magnetic nanoparticles noticeably downshifted the resonance fields of an antidot nanostructure by up to 207 G. In a similar antidot device in which nanoparticles were introduced via droplets rather than a microfluidic channel, the largest shift was only 44 G with a sensitivity of 7.57 G/ng. This indicated that introduction of the nanoparticles via microfluidics results in stronger responses from the ferromagnetic resonances. The results for both devices demonstrated that ferromagnetic antidot nanostructures incorporated within planar microresonators can detect nanoparticles captured from dispersions. Using detection scheme (ii), without the antidot array, we observed a strong resonance within the nanoparticles. The resonance’s strength suggests that direct detection is more sensitive to magnetic nanoparticles than indirect detection using a nanostructure, in addition to being much simpler.

The data consists of LSC measurements of HTO for different sample volumes as well as HTO and scinitllation cocktail concentrations prepared using a microfluidic chip.

Microduct flows are known for their inherent laminar regimes resulting from the characteristic small dimensions and low velocities. In this regard, direct numerical simulations are employed to investigate an innovative approach that harnesses the unique thermophysical properties of high-pressure transcritical fluids to achieve significantly higher rates of mixing and heat transfer in microduct geometries. The strategy is based on the sizeable changes in properties that supercritical fluids, at pressures and temperatures exceeding their critical value, undergo across the pseudo-boiling region. To this end, four different cases are considered, and systematically analyzed, in which the bulk pressure and temperature difference between walls are varied. The results obtained indicate that laminar flow prevails at low-pressure conditions, while flow regimes with turbulent characteristics can be achieved when operating at high-pressure conditions with a transversal temperature difference. The transition to the turbulence-like regime is assessed by quantifying variations in velocity and temperature profiles, accompanied by the observation of secondary flow motions. As a result, substantial increases in the Nusselt number of roughly 20×, indicative of enhanced heat transfer, are obtained at the hot wall in comparison to cases with same temperature differences at low pressure., This work is funded by the European Union (ERC, SCRAMBLE, 101040379). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them., Peer Reviewed, Postprint (published version)

Blood-brain-barrier (BBB) serves as a fatal guard of the central nervous system as well as a formidable obstacle for the treatment of brain diseases such as brain tumors. Cell membrane-derived nanomedicines are promising drug carriers to achieve BBB-penetrating and brain lesion targeting. However, the challenge of precise size control of such nanomedicines has severely limited their therapeutic effect and clinical application in brain diseases. To address this problem, this work develops a microfluidic mixing platform that enables the fabrication of cell membrane-derived nanovesicles with precise controllability and tunability in particle size and component. Sub-100 nm macrophage plasma membrane-derived vesicles as small as 51 nm (nanoscale macrophage vesicles, NMVs), with a narrow size distribution (polydispersity index, PDI: 0.27) and a high drug loading rate (up to 89% for indocyanine green-loaded NMVs, NMVs@ICG (ICG is indocyanine green)), are achieved through a one-step process. Compared to beyond-100 nm macrophage cell membrane vesicles (general macrophage vesicles, GMVs) prepared via the traditional methods, the new NMVs exhibits rapid (within 1 h post-injection) and enhanced orthotopic glioma targeting (up to 78% enhancement), with no extra surface modification. This work demonstrates the great potential of such real-nanoscale cell membrane-derived nanomedicines in targeted brain tumor theranostics. A new microfluidic platform is designed, enabling flexible and precise control of cell membrane nanomedicine preparation. The produced full-range-nanoscaled macrophage vesicles (NMVs) as small as 50 nm can achieve a high drug loading rate (89%) as well as rapid and enhanced orthotopic glioma targeting, without any extra surface modification.image

The global population is growing, rapidly increasing the demand for sustainable, novel, and safe food proteins with minimal risks of food allergy. In vitro testing of allergy-sensitizing capacity is predominantly based on 2D assays. However, these lack the 3D environment and crosstalk between the gut, skin, and immune cells essential for allergy prediction. Organ-on-a-chip (OoC) technologies are promising to study type 2 immune activation required for sensitization, initiated in the small intestine or skin, in interlinked systems. Increasing the mechanistic understanding and, moreover, finding new strategies to study interorgan communication is of importance to recapitulate food allergen sensitization in vitro. Here, we outline recently developed OoC platforms and discuss the features needed for reliable prediction of sensitizing allergenicity of proteins.