Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays

• Published in: Analytical and bioanalytical chemistry Vol. 398; no. 7-8; pp. 3023 - 3032
• Main Authors: Heus, Ferry, Giera, Martin, de Kloe, Gerdien E, van Iperen, Dick, Buijs, Joost, Nahar, Tariq T, Smit, August B, Lingeman, Henk, de Esch, Iwan J. P, Niessen, Wilfried M. A, Irth, Hubertus, Kool, Jeroen
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.12.2010; Springer-Verlag; Springer
• Subjects: Acetylcholine binding protein; Analysis; Analytical Chemistry; Applied sciences; bioassays; Biochemistry; Biological Assay - instrumentation; Biological Assay - methods; Carrier Proteins - analysis; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatographic methods and physical methods associated with chromatography; Chromatography, Liquid - methods; Confocal; Exact sciences and technology; Flow Injection Analysis - methods; Fluorescence; Food Science; Global environmental pollution; Joining; Laboratory Medicine; LED-based confocal fluorescence detection; Liquid chromatography; Microfluidics; Microfluidics - instrumentation; Microfluidics - methods; Microscopy, Fluorescence - methods; Miniaturization; Monitoring/Environmental Analysis; Nanocomposites; Nanomaterials; Nanostructure; On-line systems; Original Paper; Other chromatographic methods; Pollution; Protein binding; Receptors; Tracers (Biology); Netherlands; Bioassay; Miniaturization; LED-based confocal fluorescence detection; Acetylcholine binding protein; Microfluidics; On line; Ligand; Mixture; Binding protein; Coupling; Flow injection; Fluid mechanics; Fluorescence detector; Biochemical analysis; Coupled method; Sample; Use; Biological indicator; Minimization; Liquid chromatography; Extracellular; Light emitting diode; Plate; Screening; Sensitivity; Volume; Analog; Reagents; Affinity; Acetylcholine; Detection; Tracers
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-010-4210-x

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 IC₅₀ 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.