Microfluidic system for cell separation and deformation assessment by using passive methods

Tese de doutoramento em Biomedical EngineeringOs sistemas microfluídicos têm sido usados com sucesso em muitas aplicações biomédicas. As principais vantagens destes sistemas consistem na utilização de volumes de amostras reduzidos e com tempos de ensaios curtos. Além disso, os sistemas microfluídicos possibilitam a execução de várias tarefas em paralelo numa única plataforma microfluídica, como por exemplo a separação e medição da deformabilidade de células/partículas. Em dispositivos microfluídicos, existem dois métodos principais para separar células: métodos passivos, baseados em microestruturas e escoamentos laminares, e métodos ativos, baseados em campos de forças externos. Muitos estudos têm sido realizados com métodos passivos, pois estes não necessitam de forças externas. Nesta tese serão apresentadas diferentes geometrias passivas para um dispositivo microfluídico, constituído por vários filtros de fluxo cruzado e multiníveis com o intuito de separar células/partículas em função do seu tamanho. Outra característica importante é a implementação de microcanais hiperbólicos a montante das saídas por forma a criar um escoamento extensional homogéneo e consequentemente medir a deformabilidade das células de forma controlada. Após a separação e avaliação da deformação, a quantidade de glóbulos vermelhos será quantificada por um método de espectrofotometria. Os resultados indicam que várias geometrias mostraram uma boa taxa de separação, confirmada pelas medidas de camada livre de células e pela espectrofotometria. Verificou-se também que os sistemas microfluídicos testados são capazes de separar amostras patológicas de sangue, demostrando assim o seu potencial em realizar simultaneamente a separação e deformação de células patológicas, como por exemplo células provenientes de pacientes diagnosticados com malária e/ou diabetes.Microfluidic systems have been successfully used at many biomedical applications. Their great advantages allow working with minimal sample volumes and with short assays times. Additionally, microfluidic systems allow parallel operations in a single microfluidic platform such as separation and measurement of single cell/particles deformability. In microfluidic devices, there are two main methods for cells separation: passive methods, based on microstructures and laminar flow, and active methods, based on external force fields. Many studies have been made using passive methods because they do not require external forces. In this thesis it will be presented different geometrical passive approaches for a microfluidic device, that will have crossflow filters and multilevel steps that will separate the cells/particles by their size. Another important feature is the implementation of hyperbolic microchannels upstream the outlets in order to create a homogeneous extensional flow and consequently to measure the cells deformability in a controlled way. After the separation and deformation assessment, the amount of RBCs will be quantified by a spectrophotometry method. The results indicate that several geometries have shown a good separation rate, confirmed by the cell free layer and spectrophotometry measurements. It was also verified that the tested microfluidic systems are able to separate pathological blood samples, showing its potential to perform simultaneously separation and deformation assessments of blood diseases, such as malaria and diabetes.I want to acknowledge the financial support provided by scholarship SFRH/BD/99696/2014 from FCT (Science and Technology Foundation), COMPETE 2020, Portugal 2020 and POCH, that allow the successful development of this PhD project

Biophysical properties of blood-stage Plasmodium falciparum malaria: from single-cell host-pathogen interactions to human protective polymorphisms

Malaria is a mosquito-borne infectious disease responsible for half a million deaths every year and long-term economic stagnation in many countries where it is endemic. All symptoms and pathology of malaria are caused by Plasmodium falciparum parasite, and are initiated when parasites invade human red blood cells, then mature and multiply inside them in approximately 48 hours. The invasion process is completed in less than a minute and is one of the most crucial, yet least understood, phases of malaria infection. It also represents a brief window in which the parasites are extracellular and hence exposed to the host immune system, therefore representing a potential target for vaccines and treatments. The work described in this thesis firstly includes the optimisation of a real-time live microscopy platform for recording parasite egress-invasion sequences under controlled conditions, and to investigate their morphology and kinetics. This set-up was employed to address the role of calcium in mediating successful invasion by observing the invasion process simultaneously in bright-field and fluorescence. Elevated calcium signal was found to be absent during the early steps of the process, implying that calcium does not trigger invasion, and an alternative invasion mechanism was suggested. To investigate whether parasite and host cell physical parameters were actively involved in invasion, adhesion forces between parasites and red cells were measured with optical tweezers, while the biophysical properties of the red blood cells such as bending modulus, tension, radius, and viscosity were assessed by analysing their plasma membrane fluctuations. In particular, cells from the Dantu blood group, a rare blood variant found mainly in East Africa that provides up to 70% protection against malaria, and from Beta-thalassaemia individuals, were studied. A general correlation between red blood cell membrane tension, invasion efficiency and dynamics was established, determining a protective tension threshold above which cells are less likely to be invaded. Finally, mature parasites have the ability to bind to the endothelium of peripheral blood vessels, causing impair flow that can lead to a range of fatal conditions. To study malaria cytoadherence to endothelial cells, a microfluidic device was designed to produce an in vitro physiologically relevant model of human circulation. Increasing cytoadhesion was experimentally associated with endothelial glycocalyx disruption as initial factor for malaria pathogenesis. Live imaging methods and techniques adopted in this study highlight mechanisms crucial for malaria infection, and represent an innovative and complementary study of this disease with respect to purely biological approaches. These findings show how changes in red blood cell biophysics can be linked to human evolutionary response against malaria with tangible effects on the population

Micro/Nano Devices for Blood Analysis, Volume II

The development of micro- and nanodevices for blood analysis continues to be a growing interdisciplinary subject that demands the careful integration of different research fields. Following the success of the book “Micro/Nano Devices for Blood Analysis”, we invited more authors from the scientific community to participate in and submit their research for a second volume. Researchers from different areas and backgrounds cooperated actively and submitted high-quality research, focusing on the latest advances and challenges in micro- and nanodevices for diagnostics and blood analysis; micro- and nanofluidics; technologies for flow visualization and diagnosis; biochips, organ-on-a-chip and lab-on-a-chip devices; and their applications to research and industry

Micro/nano devices for blood analysis

[Excerpt] The development of microdevices for blood analysis is an interdisciplinary subject that demandsan integration of several research fields such as biotechnology, medicine, chemistry, informatics, optics,electronics, mechanics, and micro/nanotechnologies.Over the last few decades, there has been a notably fast development in the miniaturization ofmechanical microdevices, later known as microelectromechanical systems (MEMS), which combineelectrical and mechanical components at a microscale level. The integration of microflow and opticalcomponents in MEMS microdevices, as well as the development of micropumps and microvalves,have promoted the interest of several research fields dealing with fluid flow and transport phenomenahappening at microscale devices. [...

Development of a linear diattenuation sensitive semi-autonomous and portable digital lensless holographic microscope

Este documento presenta el desarrollo de un microscopio holográfico digital sin lentes (DLHM) sensible a la diatenuación lineal, semiautónomo y portátil. Con el desarrollo de este proyecto, pretendemos mejorar las prestaciones de esta técnica de microscopía y ampliar las posibilidades de un dispositivo DLHM portátil. Creemos que nuestro microscopio puede utilizarse potencialmente en el diagnóstico de enfermedades. Para abordar este objetivo, se investigaron los siguientes aspectos en el ámbito de este proyecto. i) El diseño de fuentes de iluminación especializadas para la técnica DLHM que sean eficientes, mecánicamente estables y fáciles de alinear. ii) La implementación de un DLHM sensible a la respuesta de diatenuación lineal de las muestras. iii) El estudio de métodos de autoenfoque basados en el aprendizaje automático para aplicarlos a los hologramas DLHM. iv) El diseño de software especializado para adquirir y reconstruir hologramas DLHM y iiv) el diseño y la fabricación de acoplamientos mecánicos adecuados que integren el montaje DLHM con el sensor digital del teléfono móvil. Cinco artículos y cinco actas son los productos resultantes de este proyecto de tesis. Constituyen parte del núcleo de esta tesis y se adjuntan al final del documento.This document presents the development of a linear diattenuation sensitive, semi-autonomous and portable digital lensless holographic microscope (DLHM). With the development of this project, we aim to improve the performance of this microscopy technique and to broaden the possibilities for a portable DLHM device. We believe our microscope can potentially be used in the diagnosis of diseases. In order to address this aim, the following aspects were investigated within the scope of this project. i) The design of specialized illumination sources for DLHM that are efficient, mechanically stable, and easy to align. ii) The implementation of a DLHM sensitive to the linear diattenuation response of samples. iii) The study of machine-learning based autofocusing methods to be applied to DLHM holograms. iv) The design of specialized software to acquire and reconstruct DLHM holograms and iiv) the design and manufacture of proper mechanical couplings that integrate the DLHM setup with the cellphone’s digital sensor. Five papers and five proceedings are the resultant products of this thesis project. They constitute part of the core of this thesis and are attached by the end of the document

Improvements in Digital Holographic Microscopy

The Ph.D. dissertation consists of developing a series of innovative computational methods for improving digital holographic microscopy (DHM). DHM systems are widely used in quantitative phase imaging for studying micrometer-size biological and non-biological samples. As any imaging technique, DHM systems have limitations that reduce their applicability. Current limitations in DHM systems are: i) the number of holograms (more than three holograms) required in slightly off-axis DHM systems to reconstruct the object phase information without applying complex computational algorithms; ii) the lack of an automatic and robust computation algorithm to compensate for the interference angle and reconstruct the object phase information without phase distortions in off-axis DHM systems operating in telecentric and image plane conditions; iii) the necessity of an automatic computational algorithm to simultaneously compensate for the interference angle and numerically focus out-of-focus holograms on reconstructing the object phase information without phase distortions in off-axis DHM systems operating in telecentric regime; iv) the deficiency of reconstructing phase images without phase distortions at video-rate speed in off-axis DHM operating in telecentric regime, and image plane conditions; v) the lack of an open-source library for any DHM optical configuration; and, finally, vi) the tradeoff between speckle contrast and spatial resolution existing in current computational strategies to reduce the speckle contrast. This Ph.D. dissertation is motivated to overcome or at least reduce the six limitations mentioned above. Each chapter of this dissertation presents and discusses a novel computational method from the theoretical and experimental point of view to address each of these limitations

Microfluidics for assessing the behaviour of deformable biological objects

Biological fluids, composed of polymeric solutions or suspensions of deformable particles, commonly present complex rheological behaviour. It is well known that particle-fluid interactions at the microscale dictate the macroscopic flow behaviour of these fluids, however the exact link in numerous situations is still missing. Recently, microfluidic techniques have been widely employed to study the dynamics of microscopic particles under flow.;Even though such techniques present a range of advantages, including the precise control of the flow conditions, as well as the consumption of a small amount of sample, the design of the microfluidic geometries still mostly relies on a trial-and-error approach. In this thesis, we experimentally test a set of microfluidic geometries, the design of which was previously optimised based on theoretical considerations or by means of numerical simulations in order to achieve specific flow conditions.;In addition, we have used complex observation techniques to study the dynamics of solutions and suspensions under flow, identifying microscopic dynamics as well as the major limitations of the microfluidic devices. Biological fluids such as solutions of DNA molecules and red blood cells suspensions were investigated in shear-dominated and extension-dominated flows and the performance of the optimised flow geometries for the study of such biological fluids was demonstrated.Biological fluids, composed of polymeric solutions or suspensions of deformable particles, commonly present complex rheological behaviour. It is well known that particle-fluid interactions at the microscale dictate the macroscopic flow behaviour of these fluids, however the exact link in numerous situations is still missing. Recently, microfluidic techniques have been widely employed to study the dynamics of microscopic particles under flow.;Even though such techniques present a range of advantages, including the precise control of the flow conditions, as well as the consumption of a small amount of sample, the design of the microfluidic geometries still mostly relies on a trial-and-error approach. In this thesis, we experimentally test a set of microfluidic geometries, the design of which was previously optimised based on theoretical considerations or by means of numerical simulations in order to achieve specific flow conditions.;In addition, we have used complex observation techniques to study the dynamics of solutions and suspensions under flow, identifying microscopic dynamics as well as the major limitations of the microfluidic devices. Biological fluids such as solutions of DNA molecules and red blood cells suspensions were investigated in shear-dominated and extension-dominated flows and the performance of the optimised flow geometries for the study of such biological fluids was demonstrated

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

In vitro modelling of cellular haemozoin and inhibition by β-haematin inhibitors and their derivatives

The discovery of new β-haematin inhibitors has become one focus for researches in response to the resistance of P. falciparum malaria parasites that emerged towards well-known antimalarials. While hundreds of new β-haematin inhibitors have been discovered using detergent mediated high-throughput screening methods, a crucial aspect is understanding exactly how these β-haematin inhibitors behave in the malaria parasite and inhibit the formation of haemozoin. What is known, is that well-known β-haematin inhibitors like chloroquine cause increased amounts of exchangeable haem in the parasite digestive vacuole and form a Fe(III)PPIX-inhibitor complex by accumulating at high concentrations which consequently inhibits parasite growth. Another important focus is on understanding the digestion of haemoglobin and its role in haemozoin formation. This research investigates the in vitro modelling of cellular haemozoin and inhibition by various β-haematin inhibitors across different scaffolds and the role of haemoglobin degradation in P. falciparum malaria parasites. The investigated β-haematin inhibitors resulted in micromolar IC50 (NF54) values and decreased parasite growth with increases in concentration. Using a pyridine-based parasite haem fractionation plate method, these β-haematin inhibitors were shown to target haemozoin formation by causing increased amounts of exchangeable haem that corresponded to decreasing amounts of haemozoin in chloroquine-sensitive parasites. The amounts of exchangeable haem were shown to be inversely proportional to the percentage of parasite growth in the presence of these β-haematin inhibitors. It was apparent that there was a tendency for parasite growth inhibition activity to decrease as the amount of exchangeable haem present in chloroquine sensitive parasites increased, although, the trend was not statistically significant. Moreover, it was observed that experimental cellular accumulation ratio values were low in comparison to chloroquine and amodiaquine. Based on the experimental cellular accumulation ratio values, it was deduced that the accumulation of these β-haematin inhibitors was not primarily due to pH trapping and more complex than previously proposed. Further investigations into the exchangeable haem amounts as a function of intracellular test compound amounts at the IC50 values of these β-haematin inhibitors highlighted that there was an apparent 1:1 relationship with the amount of intracellular exchangeable haem, indicative of complex formation. Transmission electron microscopy images were obtained for untreated parasites that showed intact parasites inside red blood cells with clearly visible haemozoin crystals dispersed throughout the parasite digestive vacuole, whilst, treated parasites showed less defined haemozoin crystals as a result of inhibition. Moreover, electron energy-loss spectroscopy revealed that untreated parasites exhibited a strong iron signal which was associated with haemozoin in the parasite digestive vacuole with a weaker signal attributed to the red blood cell cytoplasm. Similarly, a strong iron signal was shown in the digestive vacuole of treated parasites which was associated with less defined haemozoin crystals. A halo around these haemozoin crystals was observed and was suggested to be indicative of the build-up of exchangeable haem. Additionally, a strong bromine signal attributed to a bromine-containing β-haematin inhibitor, test compound 1, was also observed in the same region as the haemozoin crystals. Overlaid signal distribution maps for iron and bromine showed direct evidence of Fe(III)PPIX and test compound 1, suggesting complexation. High-quality Raman spectra were obtained for the Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetically prepared samples for the Fe(III)PPIX porphyrin dominated spectral region of 1700-500 cm-1 at an excitation wavelength of 532 nm. From the spectra, a putative Fe(III)PPIX-test compound 1 complex was identified and shown to be similar to the synthetically prepared counterpart, haematin-test compound 1 mixture. It was highlighted that a unique peak at 1080 cm-1 indicated π- π interactions between the pyrrole-imidazole ring and thus confirming that the formation of this putative Fe(III)PPIX-inhibitor complex occurs. The confocal Raman true mapping technique proved to be efficient and reliable for imaging the signal distribution of haemozoin at the Raman peak of 754 cm-1 and 1080 cm-1 for the Fe(III)PPIX-test compound 1 complex which co-localized in the digestive vacuole of chloroquine sensitive parasites. Moreover, oxy- and deoxy-haemoglobin was observed to be localized to the red blood cell, where, deoxy-haemoglobin was located on the outer parts of the parasite. Principle component analysis, based on the Raman peak positions, exhibited significant differences in the spectra for Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetic samples where clusters were observed to separate mainly along principle component 1. These data proved that the spectra of the Fe(III)PPIX-test compound 1 complex was the same as its synthetically prepared counterpart but different from the remaining Fe(III)PPIX species. In comparison to the Fe(III)PPIX-test compound 1 complex, the cluster separations were observed to be significant, where, no significant separation was observed for the Fe(III)PPIX-test compound 1 complex and the haematin-test compound 1 mixture. Based on this, it was evident that a Fe(III)PPIX-test compound 1 complex existed in the digestive vacuole of treated chloroquine sensitive parasites. To fully understand the inhibition of haemozoin, the development of a haem pathway model is necessary, but, requires certain prerequisites. Bioinformatics data from PAXdb and ExPASy revealed that chloroquine resistance (Dd2) parasites, containing 1337 previously identified proteins with an average abundance-weighted molecular weight of 40,483 ± 77 g/mol. With this, the protein mass per cell for red blood cells, chloroquine-sensitive and - resistant parasites were consistent across three protein quantification methods was measured and revealed that chloroquine resistant parasites had a significantly higher protein mass per cell than chloroquine sensitive parasites and in turn a higher total number of protein molecules per cell. Aspartic proteases are 4-fold higher in concentration than cysteine proteases with histo-aspartic protease having the highest concentration in chloroquine resistant parasites. Along with these data, a time point quantification for chloroquine sensitive parasites throughout the blood-stage showed that the amount of haemoglobin decreased in a sigmoidal manner and corresponded to a linear increase in the amount of haemozoin and relatively constant exchangeable haem amount. This was consistent with Giemsa smears that showed that for early time points, large initial decreases in the amount of haemoglobin were observed between the early trophozoite to late trophozoite stage