title: Three-layer poly(methyl methacrylate) microsystem for analysis of lysosomal enzymes for diagnostic purposes.
abstract: Lysosomal storage diseases are chronic, progressive and typically have a devastating impact on the patient and the family. The diagnosis of these diseases is still a challenge, however, even for trained specialists. Accurate diagnostic methods and high-throughput tools that could be readily incorporated into existing screening laboratories are urgently required. We propose a new method for measuring the activity of lysosomal enzymes using a microfluidic device. The principle of the method is the fluorometric determination of a protonated form of 4-methylumbelliferone directly in the enzymatic mixture. Compared to the standard diagnostic protocols, the method eliminates the necessity to add alkaline buffer to stop the enzymatic reaction, and thus, the number of analytical steps is reduced. The system allows for on-chip short-term incubation of the enzymatic reagents, leading to a much simplified analytical procedure and a significantly shortened processing time. We measured the activity of β-galactosidase in RPMI-1788 human B lymphocytes and in isolated leukocytes from healthy adults. The method shows a good agreement with the standard diagnostic method. The agreement was confirmed by statistical analysis including construction of a Bland-Altman plot. The approach presented can be an alternative for the currently used diagnostic procedures. The method developed has a potential for the implementation into complex microfluidic devices thus becoming a powerful tool for a high-throughput and multiplex screening of newborns.
authors.affiliation: Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland. Electronic address: r.kwapiszewski@gmail.com.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Kwapiszewski
authors.affiliation: Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland; Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland.
authors.forename: Karina
authors.initials: K
authors.lastname: Kwapiszewska
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
authors.forename: Jörg P
authors.initials: JP
authors.lastname: Kutter
authors.affiliation: Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland.
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Brzozka
mesh_terms: D000328:Adult; D001402:B-Lymphocytes / Q000201:enzymology; D002460:Cell Line; D005260:Female; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006923:Hymecromone / Q000737:chemistry; D007962:Leukocytes / Q000201:enzymology; D016464:Lysosomal Storage Diseases / Q000175:diagnosis*; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D001616:beta-Galactosidase / Q000032:analysis*
chemical_list: D006923:Hymecromone; D019904:Polymethyl Methacrylate; D001616:beta-Galactosidase
keywords: Enzyme activity; GM1 gangliosidosis; Lab-on-a-chip; Lysosomal storage diseases; Microfluidics; Newborn screening