MP21: A microfluidic system for studying cellular markers of metabolic diseases

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A microfluidic system for studying cellular markers of metabolic diseases
Radosław Kwapiszewski, Karina Ziółkowska, Michał Chudy, Artur Dybko, Zbigniew Brzózka
Department of Microbioanalytics, Institute of Biotechnology, Warsaw University of Technology, POLAND

Development of novel, effective diagnostic strategies and procedures based on intracellular components analysis is strongly related with applying micro total analysis systems (µTAS). Apart from low reagents consumption, short reaction time, integration, automation, versatility, possibility of diagnosis monitoring and stimulation of each step of the procedure, the use of miniaturized microdevices in progressive clinical medicine affects the patients’ comfort. However, there are still a few key challenges in this field to be solved before the chips can be accepted for real applications.
Currently more than 3000 congenital metabolic diseases are known (e.g. mitochondrial, peroxisomal, lysosomal). Most of them are caused by the abnormal value of enzyme activity. The problem is that there is no biochemical/enzymatic method that reliably identifies all carriers of many of these disorders. New methods and tools for medical diagnostics are still explored and in request.
The goal of this work was to design and develop a first microdevice for clinical diagnostics of lysosomal storage diseases. The main advantage of microchip presented is possibility for intracellular components analysis directly after the cell lysis process. This approach reduces the effect of proteases released in a lysis process on determined enzymes. We are carrying out researches into integration of such modules as: a microflow cytometer, a module for a chemical cell lysis process, a passive micromixer, an optical detection zone, and a temperature control system on one chip. There is still no fully integrated, easy to fabricate and relatively inexpensive microdevices adapted to cell-based assays.
The microsystem presented was fabricated in PDMS with photolithography and replica molding techniques. The usability of the microsystem was verified by performing assays of β-glucocerebrosidase activity (a marker of Gaucher’s disease) and α-galactosidase activity (a marker of Fabry’s disease) using L929 fibroblasts as a source of enzymes. The curve of the enzymatic reaction progression was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, enzyme activity determined was recalculated with regard to 105 fibroblasts present in samples (mouse fibroblasts) used for the tests (developed cell counting system lets for detection over 88% cells introduced to the microsystem). The results obtained were compared with a cuvette-based measurements. The calculated β-glucocerebrosidase activity, 95.0 ± 15.0 µU/105 cells, and α-galactosidase activity, 34.0 ± 6.0 µU/105 cells, determined in the microsystem were in good correlation with the values determined during macro-scale measurements: 110.2 ± 19.6 µU/105 cells and 40.5 ± 8.5 µU/105 cells, respectively. After the validation of the analytical procedure using fibroblasts from patients suffering from Gaucher’s and Fabry’s diseases and control group (in cooperation with the Institute of Psychiatry and Neurology in Poland) microsystem presented will be ready for preliminary tests in clinical laboratories.

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