Background: Decreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens. Methods: GALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples. Results: We present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 mu mol/l/h with a limit of detection of 0.066 mu mol/1/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples. Conclusions: A fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from beta-galactosidase.

Chapter 25 Development of Biomarker Assays for Clinical Diagnostics Using a Digital Microfluidics Platform David S. Millington, David S. MillingtonSearch for more papers by this authorRamakrishna Sista, Ramakrishna SistaSearch for more papers by this authorDeeksha Bali, Deeksha BaliSearch for more papers by this authorAllen E. Eckhardt, Allen E. EckhardtSearch for more papers by this authorVamsee Pamula, Vamsee PamulaSearch for more papers by this author David S. Millington, David S. MillingtonSearch for more papers by this authorRamakrishna Sista, Ramakrishna SistaSearch for more papers by this authorDeeksha Bali, Deeksha BaliSearch for more papers by this authorAllen E. Eckhardt, Allen E. EckhardtSearch for more papers by this authorVamsee Pamula, Vamsee PamulaSearch for more papers by this author Book Editor(s):Wenkui Li, Wenkui Li Novartis Institutes for BioMedical Research, East Hanover, NJ, USASearch for more papers by this authorMike S. Lee, Mike S. Lee Milestone Development Service, Newtown, PA, USASearch for more papers by this author First published: 13 June 2014 https://doi.org/10.1002/9781118890837.ch25Citations: 1 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary Digital microfluidics (DMF) is a novel lab-on-a-chip technology that permits the transport, mixing, and splitting of sub-microliter droplets within an array of electrodes on a disposable printed circuit board entirely under software control. We describe here the applications of this technology to perform different types of assays on extracts of dried blood spots (DBS) from newborns. In particular, a multiplexed assay for up to five different lysosomal enzymes from a single 3 mm DBS punch was developed and validated using quality control materials in addition to DBS from unaffected newborns and from those known to be affected by the enzyme deficiencies corresponding to Pompe, Fabry, Gacher, Hunter, and Hurler disease. Results from the chip compared well with those obtained from a standard benchtop fluorometric assay method. Advantages of DMF include the ability to load the samples and reagents and complete an entire analytical run consisting of 5 enzyme assays on 40 DBS samples, 4 calibrators, and 4 quality controls within 3.5 hours. The device is portable and inexpensive, consumes little energy, uses very low volumes of reagents, and is applicable to any assay based on colorimetric or fluorometric detection methods. In developing countries that have no centralized health care system for collection and analysis of DBS for newborn screening, point-of-care birth testing may be the only practical method to screen newborns for inherited metabolic conditions and for more common conditions faced by developing countries, such as infectious diseases. Citing Literature Dried Blood Spots: Applications and Techniques RelatedInformation

Objective: Easy tool for newborn screening of Gaucher and Hurler diseases.Methods: Method comparison between fluorometric enzymatic activity assay on a digital microfluidic platform and micro-titer plate bench assay was performed on normal (n = 100), Gaucher (n = 10) and Hurler (n = 7) dried blood spot samples.Results: Enzymatic activity analysis of glucocerebrosidase (Gaucher) and alpha-L-iduronidase (Hurler) revealed similar discrimination between normal and affected samples on both platforms.Conclusions: Digital microfluidics is suitable for Gaucher and Hurler newborn screening.

ABSTRA CT Software automated genomic engineering (SAGE) enables arbitrary genetic modification of bacteria on a fluidic platform that implements the multiplex automated genomic engineering (MAGE) process [1]. Electrowetting-ondielectric (EWD) digital microfluidics is well suited for SAGE because of its inherent reconfigurability, small reagent volumes, and parallel processing capability [2]. We report on the first demonstration of bulk cell transformation of E. coli by an electroporation device integrated with an EWD microfluidics system, which achieved up to 9.8% transformation efficiency (evaluated as the ratio of transformed cells to survived cells) while maintaining fluid transport capability. Toward the goal of enabling efficient MAGE cycling with real time feedback control, monitoring of cell recovery and growth was implemented via reflectance spectroscopy with a limit of detection of about 10 cells/ml. Furthermore, simulated MAGE cycles showed that bacteria remained viable for at least 90 cycles (27 days) on-chip.

Objective: Newborn screening for biotinidase deficiency can be performed using a fluorometric enzyme assay on dried blood spot specimens. As a pre-requisite to the consolidation of different enzymatic assays onto a single platform, we describe here a novel analytical method for detecting biotinidase deficiency using the same digital microfluidic cartridge that has already been demonstrated to screen for five lysosomal storage diseases (Pompe, Fabry, Gaucher, Hurler and Hunter) in a multiplex formatMethods:A novel assay to quantify biotinidase concentration in dried blood spots (DBS) was developed and optimized on the digital microfluidic platform using proficiency testing samples from the Centers for Disease Control and Prevention. The enzymatic assay uses 4-methylumbelliferyl biotin as the fluorogenic substrate. Biotinidase deficiency assays were performed on normal (n = 200) and deficient (n = 7) newborn DBS specimens.Results: Enzymatic activity analysis of biotinidase deficiency revealed distinct separation between normal and affected DBS specimens using digital microfluidics and these results matched the expected activity.Conclusions: This study has demonstrated performance of biotinidase deficiency assays by measurement of 4-methylumbelliferyl product on a digital microfluidic platform. Due to the inherent ease in multiplexing on such a platform, consolidation of other fluorometric assays onto a single cartridge may be realized. (C) 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.