title: Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays.
abstract: One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.
authors.affiliation: BioMolecular Analysis, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Ferry
authors.initials: F
authors.lastname: Heus
authors.forename: Martin
authors.initials: M
authors.lastname: Giera
authors.forename: Gerdien E
authors.initials: GE
authors.lastname: de Kloe
authors.forename: Dick
authors.initials: D
authors.lastname: van Iperen
authors.forename: Joost
authors.initials: J
authors.lastname: Buijs
authors.forename: Tariq T
authors.initials: TT
authors.lastname: Nahar
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: Henk
authors.initials: H
authors.lastname: Lingeman
authors.forename: Iwan J P
authors.initials: IJ
authors.lastname: de Esch
authors.forename: Wilfried M A
authors.initials: WM
authors.lastname: Niessen
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002352:Carrier Proteins / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins