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MP30:Kwapiszewski:A point-of-care system for laboratory diagnostics of lysosomal storage disorders

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Radoslaw Kwapiszewski, Karina Ziolkowska, Michal Chudy, Artur Dybko, Zbigniew Brzozka
Department of Microbioanalytics, Institute of Biotechnology, Warsaw University of Technology, POLAND


Due to the present development of science and technology, diagnostics and treatment of currently incurable disorders become feasible. The early identification of organism dysfunction is as crucial as development of new methods and diagnostic tools. Fast and accurate recognition of a disorder enables to start a proper therapy, and, in fact, it improves patient’s comfort and quality of life, and often prevents from early death.
Lysosomal storage disorders (LSDs) are a group of approximately 45 rare inherited metabolic diseases that result from defects in lysosomal function, i.e. Fabry, Gaucher, Niemann-Pick, Pompe, Tay-Sachs diseases or 7 types of mucopolysaccharidoses. 70% of them are caused by abnormal activity of a proper enzyme. The importance of an early and proper diagnosis has grown since first methods of treatment were found, e.g. enzyme replacement therapy, or residual enzyme activation. Currently applied methods which are based on determination of activity of a proper enzyme are inaccurate (there are many false-positive and true-negative test results), time-consuming, and are carried out only in few laboratories in the world.
The aim of this project was to design and develop a first integrated microdevice for diagnostics of LSDs. The microsystem was fabricated in poly(dimethylsiloxane) (PDMS) by the replica molding technique using a micromilled poly(methyl methacrylate) (PMMA) master. The microsystem consists of a cell lysis module based on a sheath flow geometry, a mixing microchannel and an optical detection module connected with a light source and a detector by optical fibers.
The presented microsystem has many advantages over the currently applied diagnostic methods of LSDs. Firstly, the analysis is performed directly after the cell lysis process, what reduces the effect of released proteases on determined enzymes. Secondly, integration of each steps of the procedure in one device and great sensitivity of the developed optical detection module let to reduce the total time of analysis to 10min, whereas only the incubation step in macro-scale lasts even 2 hours. Moreover, the experimental set enables to avoid termination of enzymatic reaction and sample dilution, what increases sensitivity of the method.
The usability of the microsystem was verified by performing assays of β-glucocerebrosidase (deficient in Gaucher disease) and α-galactosidase activities (deficient in Fabry’s disease) using human fibroblasts as a source of enzymes. The calculated β-glucocerebrosidase and α-galactosidase activities were 4.88±0.7 µU/106cells and 8.36±0.9 µU/106cells, respectively. Measurements were highly repeatable and correlated with cuvette-based measurements.
Future research should address the determination of enzymes’ activities characteristic for patients and carriers as well, and the validation of the analytical procedures. The microsystem is inexpensive, easy to handle, accurate, reliable and well suited to the medical context of the developing world.

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