Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays
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, w...
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| Published in | Analytical and bioanalytical chemistry Vol. 398; no. 7-8; pp. 3023 - 3032 |
|---|---|
| Main Authors | , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Berlin/Heidelberg
Berlin/Heidelberg : Springer-Verlag
01.12.2010
Springer-Verlag Springer |
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| 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 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. |
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| AbstractList | 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. 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 (fiuorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a lightemitting 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.sub.50] values, which were comparable with those measured by a conventional plate realer 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. Keywords Miniaturization * Acetylcholine binding protein * LED-based confocal fluorescence detection * Microfluidics * Bioassay 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. 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. 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. |
| Audience | Academic |
| Author | de Kloe, Gerdien E Smit, August B Kool, Jeroen Nahar, Tariq T van Iperen, Dick de Esch, Iwan J. P Irth, Hubertus Niessen, Wilfried M. A Heus, Ferry Buijs, Joost Giera, Martin Lingeman, Henk |
| Author_xml | – sequence: 1 fullname: Heus, Ferry – sequence: 2 fullname: Giera, Martin – sequence: 3 fullname: de Kloe, Gerdien E – sequence: 4 fullname: van Iperen, Dick – sequence: 5 fullname: Buijs, Joost – sequence: 6 fullname: Nahar, Tariq T – sequence: 7 fullname: Smit, August B – sequence: 8 fullname: Lingeman, Henk – sequence: 9 fullname: de Esch, Iwan J. P – sequence: 10 fullname: Niessen, Wilfried M. A – sequence: 11 fullname: Irth, Hubertus – sequence: 12 fullname: Kool, Jeroen |
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| Keywords | 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 |
| Language | English |
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Tatosian |
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| SubjectTerms | 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) |
| Title | Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays |
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