title: Novel application of digital microfluidics for the detection of biotinidase deficiency in newborns.
abstract: 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 format.
  
  
  METHODS
  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.
authors.affiliation: Advanced Liquid Logic, Inc. (an Illumina Company), PO Box 14025, Research Triangle Park, NC 27709, USA.
authors.forename: Carrie
authors.initials: C
authors.lastname: Graham
authors.forename: Ramakrishna S
authors.initials: RS
authors.lastname: Sista
authors.forename: Jairus
authors.initials: J
authors.lastname: Kleinert
authors.forename: Ning
authors.initials: N
authors.lastname: Wu
authors.forename: Allen
authors.initials: A
authors.lastname: Eckhardt
authors.forename: Deeksha
authors.initials: D
authors.lastname: Bali
authors.forename: David S
authors.initials: DS
authors.lastname: Millington
authors.forename: Vamsee K
authors.initials: VK
authors.lastname: Pamula
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D028921:Biotinidase Deficiency / Q000097:blood* / Q000175:diagnosis; D059788:Dried Blood Spot Testing; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D005470:Fluorometry; D006801:Humans; D007231:Infant, Newborn; D044085:Microfluidics / Q000379:methods*; D015997:Neonatal Screening / Q000379:methods*; D014468:Umbelliferones / Q000378:metabolism
chemical_list: C000590923:N-biotinyl-7-amino-4-methylcoumarin; D014468:Umbelliferones; D001710:Biotin
keywords: 4-MU-biotin; 4-Methylumbelliferyl biotin; Biotinidase deficiency; CDC; Centers for Disease Control and Prevention; DBS; DL-dithiothreitol; DMF; DTT; Digital microfluidic; Digital microfluidics; Dried blood spot; Dried blood spots; LSD; Lysosomal storage diseases; Multiplex assay; NBS; NCSLPH; Newborn bloodspot screening; Newborn screening; North Carolina State Laboratory of Public Health; PT; Proficiency testing
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