Kwapiszewski, Radosław and Skolimowski, Maciej and Ziółkowska, Karina and Jędrych, Elżbieta and Chudy, Michał and Dybko, Artur and BrzÃ³zka, Zbigniew (2011) A microfluidic device with fluorimetric detection for intracellular components analysis. BIOMEDICAL MICRODEVICES, 13 (3). pp. 431-440. ISSN 1387-2176 (Print) 1572-8781 (Online)
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An integrated microfluidic system that coupled lysis of two cell lines: L929 fibroblasts and A549 epithelial cells, with fluorescence-based enzyme assay was developed to determine Î²-glucocerebrosidase activity. The microdevice fabricated in poly(dimethylsiloxane) consists of three main parts: aÂ chemical cell lysis zone based on the sheath flow geometry, a micromeander and an optical fibers detection zone. Unlike many methods described in literature that are designed to analyse intracellular components, the presented system enables to perform enzyme assays just after cell lysis process. It reduces the effect of proteases released in lysis process on determined enzymes. Glucocerebrosidase activity, the diagnostic marker for Gaucherâs disease, is the most commonly measured in leukocytes and fibroblasts using 4-methylumbelliferyl-Î²-D-glucopyranoside as synthetic Î²-glucoside. The enzyme cleavage releases the fluorescent product, i.e. 4-methylumbelliferone, and its fluorescence is measured as a function of time. The method of enzyme activity determination described in this paper was adapted for flow measurements in the microdevice. The curve of the enzymatic reaction advancement was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, determined Î²-glucocerebrosidase activity was recalculated with regard to 105 cells present in samples used for the tests. The obtained results were compared with a cuvette-based measurements. The lysosomal Î²-glucosidase activities determined in the microsystem were in good correlation with the values determined during macro-scale measurements.
|Uncontrolled Keywords:||Cell lysis – Sheath flow – Optical detection – β-glucocerebrosidase activity – Gaucher’s disease|
|Subjects:||Biomedical Science > Nanobiotechnology|
Biomedical Science > Nanomedicine
|Deposited On:||08 Jun 2011 11:08|
|Last Modified:||08 Jun 2011 11:08|
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