Magnetic components and microfluidics optimization on a Lab-on-a-chip platform

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Sinais e Imagens Médicas), Universidade de Lisboa, Faculdade de Ciências, 2017Desde 1934, quando Moldovan criou o primeiro instrumento que poderia ser descrito como um citómetro de fluxo, este equipamento tornou-se um importante componente em várias especialidades dentro do laboratório clínico para o diagnóstico, prognóstico e monitorização de um número incontável de doenças. Esta tecnologia biofísica suspende entidades biológicas num fluxo de fluido, sinalizando-as usando reconhecimento biomolecular, para depois as detetar através de um aparelho de detecção eletrónica. Com o crescimento das técnicas de fabricação de semicondutores e microfluídos, foram e continuam a ser feitas muitas tentativas de criar citómetros de fluxo do tipo Lab-on-a-Chip (LOC), o que certamente irá afastar os equipamentos usados hoje me dia nos laboratórios por equipamentos usados in situ de custo e tamanho reduzidos, portáteis e sem necessidade de pessoal especializado. Após uma revisão bibliográfica das técnicas e princípios de funcionamento dos equipamentos já existentes foi possível perceber que a utilização de partículas magnéticas (PM) pode ter várias vantagens quando comparadas com o uso convencional de deteção por fluorescência, removendo assim a necessidade de integrar e alinhar componentes ópticos, permitindo uma medição direta e a construção de um citómetro de fluxo LOC com preparação, separação e deteção de amostras totalmente magnético. No INESC-MN foi feito um protótipo que permite a deteção de um tipo de PMs em tempo real a velocidades da ordem de cm/s usando sensores magnetoresistivos integrados em canais microfluídicos mas a primeira demonstração desta técnica para aplicações de citómetro foi realizada através da detecção de células Kg1-a marcadas com PMs de 50 nm que passaram, através de um canal microfluídico, sobre 3 sensores magnetoresistivos demonstrando que, para amostras de elevada concentração, pode ter a mesma eficiência que um hemocitómetro, mas com menor erro. Tendo como ambição um dispositivo LOC capaz de contar várias entidades biológicas na mesma amostra, um módulo de contagem com vários canais paralelos é necessário. Nesse sentido, foi projetado um novo chip com 4 colunas separadas por 3 mm, cada uma com 7 sensores do tipo válvula de spin (SV) com uma área de deteção de 100x4 μm2 distanciados 150 μm uns dos outros. Os sensores são abordados individualmente por uma linha de corrente de alumínio de 300 nm e passivados com 300 nm de nitreto de silicio. Alinhados com as colunas de sensores, 4 canais de polydimethylsiloxane (PDMS) com uma secção de 20 μm de altura e 100 μm de largura foram irreversivelmente colados ao chip por ultravioleta-ozono (UVO) criando o canal onde a amostra irá fluir. Para que as PMs sinalizem a sua passagem é necessário colocá-las sob um campo magnético forte o suficiente para induzir a sua magnetização e para que, consequentemente, as PMs emanem um campo marginal significativo. Aproveitando a insensibilidade das SVs às componentes perpendiculares ao seu plano (xy), aplica-se um campo magnético nesse sentido (z) para magnetizar as partículas. As PMs ao passarem sobre o sensor geraram um sinal bipolar devido ao campo marginal criado pela sua magnetização perpendicular. Como é apresentado na simulação do sinal, a amplitude do mesmo depende apenas da altura da partícula em relação ao sensor e da magnetização das mesmas, idealmente, uma saturação da magnetização das partículas e o máximo de proximidade aos sensores geraria a maior amplitude possível. O campo magnético perpendicular foi criado usando um magnete de neodímio posicionado sob a placa de circuito impresso (PCB), onde o chip do citómetro é colado e as ligações entre o chip e a PCB soldadas por ultrassons com fio de alumínio. Na abordagem usada em Loureiro et al., 2011, um magnete de 20 mm x 10 mm x 1 mm foi simplesmente colado sob a PCB, mas devido aos campos magnéticos serem sempre fechados as componentes x e y criam desvios nas curvas de transferência dos sensores deixando apenas 1 ou 2 sensores de uma coluna do chip operacionais. Numa abordagem seguinte foi usado um magnete 20 mm x 20 mm x 3 mm distanciado 2 cm abaixo da PCB, isto tornou as curvas de transferência dos sensores adequadas para medição, mas fez com que a componente z do campo magnético não fosse grande o suficiente para que as PMs emanassem um campo magnético suficientemente forte. Percebendo as falhas de cada uma das configurações anteriores, foram feitas simulações do campo magnético que iria influenciar o chip originado por magnetes de vários tamanhos a várias distancias para perceber qual conseguiria fornecer uma maior área em que as componentes x e y fossem menores que 10 Oe e em que a componente z fosse de pelo menos 1 kOe. Através das simulações foi concluído que o magnete de 20 mm x 10 mm x 1 mm o mais próximo possível do chip seria a melhor solução, mas que um alinhamento preciso seria necessário. Para esse fim, foi fabricado numa fresadora um sistema de alinhamento em PMMA. Para que o alinhamento fosse o correto foram feitos 4 furos de alinhamento no sistema de PMMA e na PCB e para reduzir a distancia ao máximo foi feita uma bolsa na PCB da mesma área que o chip deixando a distancia do magnete aos sensores de 1 mm (0.3 mm de PCB + 0.7 mm de substrato de silício). Com isto, o alinhamento em x foi conseguido, mas para alinhar em y foi criado um trilho no sistema de PMMA onde o magnete pudesse deslizar, controlando-o pela rotação de um parafuso com passo de 0.5 mm. Para colocar o magnete na posição ideal, foi medida consecutivamente a curva de transferência do 4º sensor de uma das colunas, num campo magnético de -141 Oe a 141 Oe, até que este tivesse um campo de acoplamento efectivo (Hf) de aproximadamente 0 Oe, o que significa que a curva de transferência estaria perfeitamente centrada em zero e criaria um sinal bipolar perfeito. Após o alinhamento e posicionamento do magnete, todos os sensores foram caracterizados e, nesses resultados, podemos ver perfeitamente o efeito das componentes x e y do magnete. Com o lado longo do magnete paralelo ao lado longo das SVs e alinhado de forma que o Hf fosse o mais próximo de 0 Oe no 4º sensor de uma coluna, percebemos que a componente x (lado longo) do campo magnético criado pelo magnete tem efeitos na sensibilidade dos sensores fazendo com que esta caia à medida que nos afastamos do centro do magnete. Enquanto que a componente y tem efeitos sobre o Hf dos sensores tornando-o mais positivo à medida que medimos a 3ª, 2ª e 1ª linha de sensores e tornando-o mais negativo quando medimos a 5ª, 6ª e 7ª linha. São também apresentadas simulações dos canais microfluídicos para perceber como a velocidade das partículas afeta o sinal e qual a velocidade máxima permitida para que placa de aquisição eletrónica seja capaz de o detetar. Com estas conclusões, um novo chip foi desenhado e fabricado. Neste novo chip a distância entre as colunas de SVs foi reduzida para apenas 1 mm, o que obrigou também à alteração dos canais microfluídicos, ao tamanho do chip e da estrutura de PDMS. Também são apresentadas simulações que mostram que se um segundo magnete, alinhado com o primeiro, for colocado sobre os canais microfluídicos poderá melhorar a magnetização e a homogeneidade do campo, o que permitirá que os 4 canais tenham a mesma sensibilidade e um desvio padrão de Hf menor. Todos os antecedentes teóricos, os métodos de microfabricação e técnicas de caracterização usados são apresentados e descritos.The diagnosis, prognosis and monitoring of diseases serves for the only purpose of preserving and improving life. Being this the greatest objective of the human kind, since ever that efforts have been made to better our ways to do that. One of those, a very important component in several specialties within the clinical laboratory is the flow cytometer, a biophysical technology which uses biomolecular recognition to sort and count biological entities by suspending them in a stream of fluid and detecting them through an electronic detection apparatus. The improvement of the semiconductor and microfluidic fabrication techniques have created the chance to bring the expensive, specialized and bulky equipment out of the laboratories and generate new machines able of having the same efficiency but with smaller price, size, allowing portability and removing the need for specialized personnel. This is the concept behind the next generation of in pointof- care apparatus, the La-on-a-Chip (LOC). At INESC-MN it is understood the potential that magnetic particles (MP) have in a LOC flow cytometer and as such a real-time detection of single magnetic particles magnetoresistive based cytometer was prototyped. Demonstration of this technique for cytometer applications was accomplished by indicating that for high concentration samples it can have the same efficiency as the hemocytometer method but with lesser error. This thesis has as objective the optimization of the magnetic and microfluidic components of a LOC to allow the parallelization of measurements and enabling the real-time measurement of different particles at the same time. For this purpose, a bibliographic review of the theoretical backgrounds, of the fabrication and characterization techniques, of the different detecting principles and of the already existing magnetoresistive counting modules was made to get a deeper understanding of the optimization possibilities. The present work describes the above-mentioned platform for dynamic detection of magnetic labels with a magnetoresistive based flow cytometer, where a permanent magnet is used to magnetize the labels enabling them to trigger the sensor. Several simulations of the magnetic fields created by the permanent magnet and the microfluidic channels were done and analyzed in order to characterize the MPs signal, understand which would be the best positioning of these components and which fluid velocities would be in the range of the electronic read-out capabilities. This study led to the fabrication of a micromachined polymethylmethacrylate (PMMA) alignment system to correctly position the permanent magnet under the cytometer’s chip. This made the control over the magnet’s positioning more sensible and thus reducing the influence of its unwanted magnetic components on the chip. The approximation of the magnet to the chip enhanced the signal by optimizing the MPs magnetization and consequently the signal amplitude, the precise alignment corrected the sensors response by improving its sensitivity and removing them from saturation states. Through this new setup all the sensors in the chip became operational. Finally, using the several techniques of microfabrication also describe in this thesis, a new chip was designed and fabricated to improve even more the sensors sensitivity and consequently augment the number of the cytometer’s counting channels

On-Chip Fabry-Pérot Microcavity for Refractive Index Cytometry and Deformability Characterization of Single Cells

Une identification correcte et précise du phénotype et des fonctions cellulaires est fondamentale pour le diagnostic de plusieurs pathologies ainsi qu’à la compréhension de phénomènes biologiques tels que la croissance, les réponses immunitaires et l’évolution de maladies. Conséquemment, le développement de technologies de pointe offrant une mesure multiparamétrique à haut débit est capital. À cet égard, la cytométrie en flux est l’étalon de référence due à sa grande spécificité, sa grande sensibilité et ses débits élevés. Ces performances sont atteintes grâce à l’évaluation précise du taux d’émission de fluorophores, conjugués à des anticorps, ciblant certains traits cellulaires spécifiques. Néanmoins, sans ce précieux étiquetage, les propriétés physiques caractérisées par la cytométrie sont limitées à la taille et la granularité des cellules. Bien que la cytométrie en flux soit fondamentalement un détecteur optique, elle ne tire pas avantage de l’indice de réfraction, un paramètre reflétant la composition interne de la cellule. Dans la littérature, l’indice de réfraction cellulaire a été utilisé comme paramètre phénotypique discriminant pour la détection de nombreux cancers, d’infections, de la malaria ou encore de l’anémie. Également, les structures fluidiques de la cytométrie sont conçues afin d’empêcher une déformation cellulaire de se produire. Cependant, les preuves que la déformabilité est un indicateur de plusieurs pathologies et d’état de santé cellulaire sont manifestes. Pour ces raisons, l’étude de l’indice de réfraction et de la déformabilité cellulaire en tant que paramètres discriminants est une avenue prometteuse pour l’identification de phénotypes cellulaires. En conséquence, de nombreux biodétecteurs qui exploitent l’une ou l’autre de ces propriétés cellulaires ont émergé au cours des dernières années. D’une part, les dispositifs microfluidiques sont des candidats idéaux pour la caractérisation mécanique de cellules individuelles. En effet, la taille des structures microfluidiques permet un contrôle rigoureux de l’écoulement ainsi que de ses attributs. D’autre part, les dispositifs microphotoniques excellent dans la détection de faibles variations d’indice de réfraction, ce qui est critique pour un phénotypage cellulaire correcte. Par conséquent, l’intégration de composants microfluidiques et microphotoniques à l’intérieur d’un dispositif unique permet d’exploiter ces propriétés cellulaires d’intérêt. Néanmoins, les dispositifs capables d’atteindre une faible limite de détection de l’indice de réfraction tels que les détecteurs à champ évanescent souffrent de faibles profondeurs de pénétration. Ces dispositifs sont donc plus adéquats pour la détection de fluides ou de molécules. De manière opposée, les détecteurs interférométriques tels que les Fabry- Pérots sont sensibles aux éléments présents à l’intérieur de leurs cavités, lesquelles peuvent mesurer jusqu’à plusieurs dizaines de micromètres.----------Abstract Accurate identification of cellular phenotype and function is fundamental to the diagnostic of many pathologies as well as to the comprehension of biological phenomena such as growth, immune responses and diseases development. Consequently, development of state-of-theart technologies offering high-throughput and multiparametric single cell measurement is crucial. Therein, flow cytometry has become the gold standard due to its high specificity and sensitivity while reaching a high-throughput. Its marked performance is a result of its ability to precisely evaluate expression levels of antibody-fluorophore complexes targeting specific cellular features. However, without this precious fluorescence labelling, characterized physical properties are limited to the size and granularity. Despite flow cytometry fundamentally being an optical sensor, it does not take full advantage of the refractive index (RI), a valuable labelfree measurand which reflects the internal composition of a cell. Notably, the cellular RI has proven to be a discriminant phenotypic parameter for various cancer, infections, malaria and anemia. Moreover, flow cytometry is designed to prevent cellular deformation but there is growing evidence that deformability is an indicator of many pathologies, cell health and state. Therefore, cellular RI and deformability are promising avenues to discriminate and identify cellular phenotypes. Novel biosensors exploiting these cellular properties have emerged in the last few years. On one hand, microfluidic devices are ideal candidates to characterize single cells mechanical properties at large rates due to their small structures and controllable flow characteristics. On the other hand, microphotonic devices can detect very small RI variations, critical for an accurate cellular phenotyping. Hence, the integration of microfluidic and microphotonic components on a single device can harness these promising cellular physical properties. However, devices achieving very small RI limit of detection (LOD) such as evanescent field sensors suffer from very short penetration depths and thus are better suited for fluid or single molecule detection. In opposition, interference sensors such as Fabry-Pérots are sensitive to the medium inside their cavity, which can be several tens of micrometers in length, and thus are ideally suited for whole-cell measurement. Still, most of these volume sensors suffer from large LOD or require out-of-plane setups not appropriate for an integrated solution. Such a complex integration of high-throughput, sensitivity and large penetration depth on-chip is an ongoing challenge. Besides, simultaneous characterization of whole-cell RI and deformability has never been reported in the literature

Study of particle suspensions in microfluidics for the development of optical devices

The vision of this PhD research project is to create a microfluidic system for controlling the locations of suspended particles in order to form three dimensional (3D) objects on demand. To realize this, the author implemented a microfluidic system that can apply suitable and desired forces on particles on demand. Particles of various refractive indices were placed close to each other in order to form a media having reconfigurable and tuneable properties. Light was coupled into such well-controlled particles in order to form dynamically tuned objects suspended in liquid such as optical waveguides. The dielectrophoretic (DEP) force was used for manipulating the locations of particles as it is capable of focusing and scattering suspended particles from pre-determined locations. Additionally, when combined with hydrodynamic forces, the DEP force was able to form densely packed areas of such particles with non-turbulent boundaries. The research was implemented in three stages. In the first stage, the author utilized a platform consisting of a microfluidic system integrated with DEP microelectrodes, microfluidics and optical peripherals for the coupling of light. Light was directly coupled into densely packed silicon dioxide (SiO2) particles with diameters of 230 and 450 nm, respectively. Light was transmitted via the closely packed 230 nm particles and in contrast was significantly scattered by the 450 nm particles. The outcomes, which were resulted from this initial stage, were the first demonstration of a dynamically tuneable optical waveguide based on the DEP focused particles in microfluidics. In the second stage of his research, the author integrated a multi mode polymeric waveguide into the microfluidic system. Tungsten trioxide (WO3) and SiO2 particles with diameters of 80 and 450 nm were investigated. The findings demonstrated that the densely packed WO3 particles were able to couple light from the polymeric waveguide, while the SiO2 particles did not affect the transmission of the optical signals significantly. The investigations of the second stage platform resulted in the first demonstration of optical waveguide tuning based on DEP focused particles. Finally, in the third stage of this research, the author implemented a quasi single mode polymeric waveguide integrated with the microfluidics. The author used WO3, zinc oxide (ZnO) and SiO2 particles with diameters of 80, 50 and 72 nm, respectively. Under the DEP force, these particles were able to interact with the optical guided modes. The results show that the WO3 particles were capable of forming layers of packed particles with anti-resonant characteristics. In particular, the fundamental mode was strongly coupled to the packed WO3 particles. However, under certain particle focusing conditions, the first order mode was anti-resonant to the closely packed WO3 particles as it was largely isolated. These findings were the first demonstration of the coupling and manipulation of optical guided modes using DEP focused particles with resonant and anti-resonant behaviors

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals

Fiscal Year 1995

The mission of the Engineering Research, Development, and Technology Program at Lawrence Livermore National Laboratory (LLNL) is to develop the knowledge base, process technologies, specialized equipment, tools and facilities to support current and future LLNL programs. Engineering`s efforts are guided by a strategy that results in dual benefit: first, in support of Department of Energy missions, such as national security through nuclear deterrence; and second, in enhancing the nation`s economic competitiveness through their collaboration with US industry in pursuit of the most cost-effective engineering solutions to LLNL programs. To accomplish this mission, the Engineering Research, Development, and Technology Program has two important goals: (1) identify key technologies relevant to LLNL programs where they can establish unique competencies, and (2) conduct high-quality research and development to enhance their capabilities and establish themselves as the world leaders in these technologies. To focus Engineering`s efforts, technology thrust areas are identified and technical leaders are selected for each area. The thrust areas are comprised of integrated engineering activities, staffed by personnel from the nine electronics and mechanical engineering divisions, and from other LLNL organizations. This annual report, organized by thrust area, describes Engineering`s activities for fiscal year 1995. The report provides timely summaries of objectives methods, and key results from eight thrust areas: computational electronics and electromagnetics; computational mechanics; microtechnology; manufacturing technology; materials science and engineering; power conversion technologies; nondestructive evaluation; and information engineering

All-in-one microsystem for long-term cell culturing and real-time chip-level lensless microscopy

The study is to concept, develop and evaluate an all-in-one microsystem with combined long-term animal cell culturing and real-time chip-level lensless microscopy functions suitable for applications in cell biology studies, and at the point-of-use. The microsystem consists of a 5 megapixel CMOS image sensor, a disposable microchip for cell culture, heating elements and LED illumination. The overall size is only 40 mm x 40 mm x 50 mm. The disposable microchip for cell culture is composed of a polymer microfluidic interface and a silicon micro-cavity chip with a 1 µm thick 1 mm x 1 mm transparent Si3N4 bottom membrane, which is directly placed onto the image sensor surface. Under the collimated LED illumination, the optical resolution of the lensless imaging module is only dependent on the digital resolution of the image sensor, which amounts to 3.5 µm (double pixel pitches). The imaging quality is proven comparable to a 4x optical microscope without image computation or processing. Both the morphologies of different cell cultures (L929, A549 and T47D) and the single cells with colorimetric staining can be clearly visualized in real time. With the additional deposition of an interference filter on the image sensor surface, fluorescence spreading cells in culture are observed on the chip under a common blue LED illumination. The temperature for the incubating module is controlled at 37±0.2°C in the room environment. Mammalian cells (L929 and A549) are cultured with conventional culture medium and monitored under the time-lapse lensless microscopy by the all-in-one microsystem up to 5 days outside a laboratory incubator. Very fast operational processes, such as cell loading, passaging and staining, have been readily carried out and monitored in real-time by the platform. Besides cell cultures in monolayer, the formation of 3D clusters of L929 cells has also been demonstrated and recorded under time-lapse lensless microscopy by using the all-in-one microsystem.Das Ziel der vorliegenden Dissertation ist die Konzeption, Entwicklung und Evaluation eines All-in-One-Mikrosystems mit der Kombination aus Langzeit Kultivierung von Tierzellen und Echtzeit Linsenloser Mikroskopie Funktionen auf Chip Level, die für Anwendungen in zellbiologischen Studien sowie für Point-of-use geeignet sind. Das Mikrosystem besteht aus einem 5 Megapixel CMOS-Bildsensor, einem Einweg-Mikrochip für die Zellkultur, Heizelementen sowie einer LED-Beleuchtung Die Gesamtgröße beträgt nur 40 mm x 40 mm x 50 mm. Der Einweg-Mikrochip für die Zellkultur besteht aus einer polymeren, mikrofluidischen Grenzfläche und einem Silizium-Mikrohohlraum-Chip mit einer 1 µm dicken und 1 mm x 1 mm transparenten Si3N4-Bodenmembran, die direkt auf die Bildsensoroberfläche aufgesetzt wird. Unter der kollimierten LED-Beleuchtung ist die optische Auflösung des linsenlosen Abbildungsmoduls nur von der digitalen Auflösung des Bildsensors abhängig, was 3,5 µm beträgt (Doppelpixelabstände). Die Bildqualität ist vergleichbar mit einem 4x optischen Mikroskop ohne Bildberechnung oder Verarbeitung. Sowohl die Morphologien verschiedener Zellkulturen (L929, A549 und T47D) als auch die einzelnen Zellen mit farbmetrischer Färbung können in Echtzeit deutlich sichtbar gemacht werden. Mit der zusätzlichen Abscheidung eines Interferenzfilters auf der Bildsensoroberfläche werden fluoreszenzverteilende Zellen in Kultur auf dem Chip unter einer gemeinsamen blauen LED-Beleuchtung beobachtet. Die Temperatur für das Inkubationsmodul wird bei 37 ± 0,2 ° C in der Raumumgebung festgelegt. Säugetierzellen (L929 und A549) werden mit herkömmlichem Kulturmedium kultiviert und unter der Zeitrafferlinsenmikroskopie durch das All-in-One-Mikrosystem bis zu 5 Tage außerhalb eines Laborinkubators überwacht. Sehr schnelle operative Prozesse wie z. B. Zellbeladung, Durchfluss und Färbung wurden in Echtzeit durch die Plattform durchgeführt und überwacht. Neben den Zellkulturen in der Monoschicht wurde auch die Bildung von 3D-Clustern von L929-Zellen in Zeitraffer bei lichtempfindlicher Mikroskopie unter Verwendung des All-in-One-Mikrosystems nachgewiesen und aufgezeichnet

Microfluidics for Biosensing and Diagnostics

Efforts to miniaturize sensing and diagnostic devices and to integrate multiple functions into one device have caused massive growth in the field of microfluidics and this integration is now recognized as an important feature of most new diagnostic approaches. These approaches have and continue to change the field of biosensing and diagnostics. In this Special Issue, we present a small collection of works describing microfluidics with applications in biosensing and diagnostics

3D printed microfluidic devices for particle and cell analysis

Particle/cell analysis is crucial in many health, industrial and environmental monitoring processes. Its integration into miniaturised lab-on-a-chip systems enables a host of portable technologies. However, current lab-on-a-chip lithographical fabrication methods are costly, time-consuming and restrictive in design, impeding their widespread implementation. This has led to 3D printing being explored as an alternative in recent years, due to its ability to form devices in a single step, and its three-dimensional freedom. [Continues.

Separation and Focusing of Magnetic Beads for Agglutination Tests

Functional magnetic micro- and nanoparticles are used in bioanalytical applications as solid carriers for capture, transport and detection of biomolecules or magnetically labeled cells. Colloidal suspensions of such particles provide a large specific surface for chemical binding and therefore allow highly efficient interactions with target molecules in a sample solution. Controlled actuation and manipulation of these mobile substrates in the microfluidic format offers interesting new opportunities for on-chip bioassays with previously unmatched properties. Separation of functional magnetic particles or magnetically labeled entities is therefore a key feature for bioanalytical or biomedical applications and also an important component of lab-on-a-chip devices for biological applications. In this thesis we present two novel integrated microfluidic magnetic bead manipulation devices. The first system consists of dosing of magnetic particles, controlled release and subsequent magnetophoretic size separation with high resolution. On-chip integrated soft-magnetic microtips with different shapes provide the magnetic driving force for the bead manipulation. The system is designed to meet the requirements of specific bioassays, in particular of on-chip agglutination assays for the detection of rare analytes, in which the latter can be quantified via the counting of the particle doublets. In a second approach, magneto-microfluidic three-dimensional (3D) focusing of microparticles has been developed. In this system, magnetic microparticles from a dense plug are released into a single streamline with longitudinal inter-particle spacing. Plug formation is induced by a high-gradient magnetic field generated at the sidewall of a microchannel by a micromachined magnetic tip that is connected to an electromagnet. Controlled release of the microparticles is achieved using an exponential damping protocol of the magnetic retention force in the presence of an applied flow. Carefully balancing the relative strengths of the drag force imposed by the flow and the magnetic retention force moreover allows in-flow size separation of the microparticles. Adding subsequently a lateral sheath flow microchannel focuses the microparticles into a single stream situated within 0plusmn; 5 µm from the channel center axis. Our system for 3D focusing and in-flow separation of magnetic microparticles has been used for performing an immuno-agglutination assay on-chip. 3D focusing was of the basis of reliable in-flow counting of singlets and agglutinated doublets. We demonstrated the potential of the agglutination assay in a microfluidic format using a streptavidin/biotinylated-bovine serum albumin (bBSA) model system. A bBSA detection limit of about 400 pg/mL (6 pM) is achieved

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles