Cell Separation In Microfluidic Channels

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2013Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2013Bu çalışmada yaklaşık 15 mikron çapında olan MDA-MB-231 ve yaklaşık 20 micron çapında olan MCF7 insan meme kanseri hücreleri içeren karışımdan MCF7 konsantrasyonu için Dean akışından faydalanan dönel kanal tasarımı yapılmıştır. Buna göre dört farklı yükseklik x genişlik değerlerine sahip olan kanallar üretilmiştir. Bu kanallar, bir giriş ve üç çıkışa sahiplerdir. Hedef hücre olan MCF7 hücre iki farklı floresan boya ile boyanmış ve öncelikle bu boyaların hücrelerin yüzde kaçında etkili olduğunu belirlemek için akış sitometrisi ile analiz edilmiştir. Bu analizden sonra ilk önce dizilim için uygun debileri bulmak üzere deney kurulmuş ve boyalı MCF7 hücreleri ve boyanmamış MDA-MB-231 hücreleri her birinden 100 ml’de bir milyon olacak şekilde toplam iki milyon hücre içeren karışım hazırlanmıştır. Bu karışımdan her bir kanal için 20’şer ml alınarak deneyler gerçekleştirilmiştir. Bu deneyler sırasında yüksek debilerde çalışılması gerektiği anlaşılmıştır. deneylere göre gerekli debiye ulaşıldığında kanalların 2. çıkışlarında MCF7 hücreleri dizilimi olduğu görülmüş ve bu akış sitometrisi analizi ile de doğrulanmıştır. İkinci yapılan deneylerde MCF7 hücreleri ilkinden farklı bir boyayla boyanmıştır ve daha fazla sayıda debi incelenmiştir. Üretilen dönel kanallar, ucuz ve küçük boyutlu, hızlı ve daha doğru sonuç alınabilecek bir teşhis cihazı geliştirmede ön çalışma olarak kullanılabilecek bir tasarım olmuştur.In this work, Dean force coupled curved microchannels were designed for concentration of MCF7 human breast cancer cells from a suspension that contains MDA-MB-231 (~ 15 μm in diameter) and MCF7 (~ 20 μm in diameter) human breast cancer cell lines. Channels which have four different height x width dimensions were fabricated. Channels had one inlet and three outlets. Target cell –MCF7- has stained with two different fluorescent dye and effectiveness of these dyes were investigated with flow cytometry. Experiments were performed to determined apoppriate flow rates for focusing. Then, a million labeled MCF7 and a million unlabeled MDA-MB-231 cells were mixed in 100 ml solution. 20 ml cell suspension was used for each experiment. It was understood that high flow rates are required for focusing of MCF7 cells and according to first experiment there was MCF cell focusing at second outlets . Experiments showed that MCF7 enrichment at the second outlets of each channel and it was confirmed with flow cytometry analysis. Then another group of MCF7 cells were stained with another fluorescent dye for second experimental group and more flow rate values were investigated. These curved channels can be regarded as a prototype of a microfluidic diagnostic device with their fast reaction time, relatively accurate results, cost effective and miniaturized features.Yüksek LisansM.Sc

Wafer scale integration of coulomb blockade-based nanobiosensors with microfluidic channels for label-free detection of cancer biomarkers

Dans cette thèse, nous proposons et démontrons un nouveau type de nanobiocapteur pour la détection de biomolécules à haute sensibilité et leur intégration à grande échelle (plaquette de 4 pouces). Le principe du nouveau nanobiocapteur électrique est basé sur la variation de conductivité électrique à travers des nano-îlots grâce au phénomène quantique appelé « blocage de coulomb ». Les nano-îlots de nickel (5nm de diamètre) sont placés entre les nano-électrodes interdigitées (IND) (~45nm de largeur). La conductivité de ces dispositifs à jonctions tunnel multiples (MTJ) est modifiée par l’adsorption de biomarqueurs impliqués dans la tumorogènese. Les oncologues ont récemment isolé et caractérisé un nouveau fragment d’anticorps à chaine simple (scFv) qui reconnaît sélectivement la forme active de RhoA. Ce biomarqueur potentiel a été trouvé surexprimé dans diverses tumeurs. Les fragments d’anticorps ont été adsorbés, par des liaisons de coordination, sur les nano-îlots de nickel. Ces fragments sont capables de reconnaître spécifiquement la forme active de RhoA. Nous avons étudié ce biomarqueur et validé la chimie de surface à base d’îlots de nickel pour la détection sans marquage, en utilisant une microbalance à quartz (QCM). Puis, nous avons mis au point et adapté à notre dispositif une méthodologie innovatrice pour réaliser, à l’échelle d’une plaquette, des microcanaux basés sur du photoPDMS. La caractérisation électrique finale des dispositifs intégrés a été testée en temps réel et à flux biologique continu. La forme active de RhoA a été détectée en discriminant la forme inactive. En annexe, je présente mon opinion épistémologique et éthique sur la nanotechnologie ___ In this thesis we propose and implement the fabrication on 4 inch wafer of a novel type of nanobiosensor capable of high sensitivity detection. The principle of the nanobiosensor is based on the variation of electrical tunnelling conductivity through metal nanoislands due to the quantum phenomenon called coulomb blockade. Nickel nanoislands(~5nm diameter), are placed between interdigitated nanoelectrodes devices (IND) (width~45nm). Hence, the conductivity of these Multiple-Tunnel-Junction (MTJ) devices is modified by the absorption of biomarkers involved in tumourigenesis. Oncologists have recently isolated and characterised a new conformational single chain variable fragment (scFv) which selectively recognises the active form of RhoA. This potential biomarker has been found overexpressed in various tumours. Antibodies fragments (scFv) are absorbed through coordinative bonds onto nickel nanoislands. Hence the scFv are capable of recognising specifically the active RhoA conformation. We have investigated this biomarker and validated the nickel nanoilands based chemical construction for label-free biodetection using quartz crystal microbalance (QCM) before implementing the methodology to our devices. An innovative methodology to realise photoPDMS-based microchannels was also developed. Encapsulation with an etched PDMS-nanocomposite finalised the integration of the devices. The final electrical characterisation of the integrated device was tested in real time and continuous biological flow. The active form of RhoA was discriminated against its inactive conformation. In annexe, I present my epistemological and ethical opinions in nanotechnolog

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