title: Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides.
abstract: We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.
authors.affiliation: Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
authors.forename: Christin
authors.initials: C
authors.lastname: Herzog
authors.affiliation: Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Poehler
authors.affiliation: Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Peretzki
authors.affiliation: Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität Graz, Stremayrgasse 9/III, 8010 Graz, Austria.
authors.forename: Sergey M
authors.initials: SM
authors.lastname: Borisov
authors.affiliation: Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität Graz, Stremayrgasse 9/III, 8010 Graz, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Aigner
authors.affiliation: Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität Graz, Stremayrgasse 9/III, 8010 Graz, Austria.
authors.forename: Torsten
authors.initials: T
authors.lastname: Mayr
authors.affiliation: Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany. nagl@chemie.uni-leipzig.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Nagl
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D020100:Hydrogels; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D007526:Isoelectric Point; D056656:Lab-On-A-Chip Devices; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D010803:Physalaemin / Q000737:chemistry / Q000378:metabolism; D011492:Protein Hydrolysates / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D000076227:Stereolithography; D014357:Trypsin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D020100:Hydrogels; D010446:Peptide Fragments; D011492:Protein Hydrolysates; D011506:Proteins; D010803:Physalaemin; D014357:Trypsin