title: Capillary electrophoresis coupled with automated fraction collection.
abstract: A fraction collector based on a drop-on-demand ink-jet printer was developed to interface capillary zone electrophoresis with a 96 well microtiter plate. We first evaluated the performance of the collector by using capillary zone electrophoresis to analyze a 1mM solution of tetramethylrhodamine; a fluorescent microtiter plate reader was then used to detect the analyte and characterize fraction carryover between wells. Relative standard deviation in peak height was 20% and the relative standard deviation in migration time was 1%. The mean and standard deviation of the tetramethylrhodamine peak width was 5 ± 1 s and likely limited by the 4-s period between droplet deposition. We next injected a complex mixture of DNA fragments and used real-time PCR to quantify the product in a CE-SELEX experiment. The reconstructed electrophoretic peak was 27 s in duration. Finally, we repeated the experiment in the presence of a 30-µM thrombin solution under CE-SELEX conditions; fractions were collected and next-generation sequencing was used to characterize the DNA binders. Over 25,000 sequences were identified with close matches to known thrombin binding aptamers.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Bonnie Jaskowski
authors.initials: BJ
authors.lastname: Huge
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Flaherty
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Oluwatosin O
authors.initials: OO
authors.lastname: Dada
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. Electronic address: Norman.J.Dovichi.1@nd.edu.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D052157:Aptamers, Nucleotide / Q000302:isolation & purification*; D004247:DNA / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism; D052156:SELEX Aptamer Technique / Q000379:methods*; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D012235:Rhodamines; C081676:thrombin aptamer; C005358:tetramethylrhodamine; D004247:DNA; D013917:Thrombin
keywords: Aptamer; CE-SELEX; Capillary electrophoresis; Fraction collection