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|Title:||Integrated microfluidics for label-free leukocyte sorting and electrical phenotyping||Authors:||Petchakup, Chayakorn||Keywords:||DRNTU::Engineering::Mechanical engineering||Issue Date:||9-May-2019||Source:||Petchakup, C. (2019). Integrated microfluidics for label-free leukocyte sorting and electrical phenotyping. Doctoral thesis, Nanyang Technological University, Singapore.||Abstract:||Circulating leukocytes (white blood cells) in blood are known to orchestrate various biological processes and become activated during host defence (e.g. infection) or in pathogenesis of major diseases such as cancer, type 2 diabetes mellitus (T2DM) or cardiovascular diseases. Conventionally, fluorescence- and magnetic-activated cell sorting (FACS, MACS) techniques widely used in leukocyte studies require antibodies labelling which is expensive and time consuming. Leukocytes are also prone to activation during sample preparation which advocates the need to develop novel labelfree leukocyte sorting and analysis approaches. Microfluidic impedance cytometry is an established single-cell analysis tool based on intrinsic cellular dielectric properties. It is widely used for leukocyte enumeration and differential counting, but its application in leukocyte activation profiling remains unexplored. In this dissertation, two novel microfluidic technologies for label-free leukocyte sorting and electrical profiling towards rapid immune health profiling in type 2 diabetes mellitus are developed. In the first part of this project, a combinatorial microfluidic strategy for leukocyte phenotyping by enriching target leukocyte subtypes (neutrophils and monocytes) by Dean Flow Fractionation (DFF) prior impedance measurement is proposed. This increases the detection selectivity which is demonstrated for various applications namely monocyte activation, monocyte differentiation and monocyte subtype characterization. We also showed for the first time, that leukocyte impedance characteristics were associated with cardiovascular risk factors (lipid levels and Creactive protein (CRP)) in patients with T2DM, thus suggesting leukocyte impedance signature as novel surrogate biomarkers for diabetes testing. In the second part of the project, both cell sorting module and impedance detection module are integrated on a single chip. The integrated “sample in-answer out” platform provides several key advantages including high leukocyte separation efficiency, minimal sample manual handling, and rapid analysis (~5-15 mins). This platform was developed for direct neutrophil isolation and impedance characterization of neutrophil extracellular trap formation (NETosis), a recently discovered key defense mechanism of neutrophils. Our results showed distinct differences in impedance profiles of neutrophils undergoing NETosis and can be further developed to study neutrophil dysfunction in T2DM. Finally, by changing the dimensions of microfluidic design, it is demonstrated that it is possible to perform cancer cell sorting and electrical phenotyping for assessment of metastatic potential.||URI:||https://hdl.handle.net/10356/87338
|Appears in Collections:||MAE Theses|
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