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Microscale Tools for Sample Preparation, Separation and Detection of Neuropeptides
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Mikroskaliga verktyg för provpreparering, separation och detektion av neuropeptider (Swedish)
Abstract [en]

The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection.

In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities.

The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.

Place, publisher, year, edition, pages
Uppsala: Kemiska institutionen , 2005. , p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 64
Keywords [en]
Analytical chemistry, Neuropeptides, Microchip, Enhanced microdialysis, Poly(dimethylsiloxane) (PDMS), Electrospray ionization (ESI), Multidimensional separation, Electrochemical manipulation, Mass spectrometry (MS), Capillary electrophoresis (CE), Microdevice, Microfabrication, Micro total analysis system (μ-TAS)
Keywords [sv]
Analytisk kemi
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-5838ISBN: 91-554-6279-0 (print)OAI: oai:DiVA.org:uu-5838DiVA, id: diva2:166588
Public defence
2005-06-03, Room B42, BMC, Uppsala, 10:15
Opponent
Supervisors
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2011-12-08Bibliographically approved
List of papers
1. A feasibility study of solid supported enhanced microdialysis
Open this publication in new window or tab >>A feasibility study of solid supported enhanced microdialysis
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2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 6, p. 1678-1682Article in journal (Refereed) Published
Abstract [en]

For the first time, a solid supported enhanced microdialysis methodology for analysis of neuropeptides is described. The microdialysis samples were, in this study, subsequently collected in fractions, dissolved from the solid particles, dried, and resolved in a formic acid buffer in order to make them suitable for capillary liquid chromatography-mass spectrometry. Different microdialysis flow profiles were evaluated where air-gapped continuous flow was considered most suitable for the solid supported microdialysis mode. Six endogenous neuropeptides were initially used to investigate the feasibility of this enhanced microdialysis methodology. The improved relative recovery obtained from the solid supported enhanced microdialysis was varying from no effect to 10 times higher as compared to ordinary microdialysis. The most efficient enrichment was obtained for luteinizing hormone releasing hormone, which was the largest but also the most hydrophilic of the peptides. In contrast, no significant difference in recovery was observed for Leu-enkephalin being the smallest and the most hydrophobic peptide tested. These results indicate an increased flux and selective uptake of hydrophilic peptides across the membrane and enrichment on the particles in solid supported microdialysis.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93181 (URN)10.1021/ac035305l (DOI)15018567 (PubMedID)
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-14Bibliographically approved
2. Poly(dimethylsiloxane)-Based Microchip for Two-Dimensional Solid-Phase Extraction-Capillary Electrophoresis with an Integrated Electrospray Emitter Tip
Open this publication in new window or tab >>Poly(dimethylsiloxane)-Based Microchip for Two-Dimensional Solid-Phase Extraction-Capillary Electrophoresis with an Integrated Electrospray Emitter Tip
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2005 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 16, p. 5356-5363Article in journal (Refereed) Published
Abstract [en]

A microchip in poly(dimethylsiloxane) (PDMS) for in-line solid-phase extraction-capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (SPE-CE-ESI-TOF-MS) has been developed and evaluated. The chip was fabricated in a novel one-step procedure where mixed PDMS was cast over steel wires in a mold. The removed wires defined 50-um cylindrical channels. Fused-silica capillaries were inserted into the structure in a tight fit connection. The inner walls of the inserted fused-silica capillaries and the PDMS microchip channels were modified with a positively charged polymer, PolyE-323. The chip was fabricated in a two-level cross design. The channel at the lower level was packed with 5-um hyper-cross-linked polystyrene beads acting as a SPE medium used for desalting. The upper level channel acted as a CE channel and ended in an integrated emitter tip coated with conducting graphite powder to facilitate the electrical contact for sheathless ESI. An overpressure continuously provided fresh CE electrolyte independently of the flows in the different levels. Further studies were carried out in order to investigate the electrophoretic and flow rate properties of the chip. Finally, six-peptide mixtures, in different concentrations, dissolved in physiological salt solution was injected, desalted, separated, and sprayed into the mass spectrometer for analysis with a limit of detection in femtomole levels.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-74074 (URN)10.1021/ac050495g (DOI)16097780 (PubMedID)
Available from: 2005-08-26 Created: 2005-08-26 Last updated: 2017-12-14Bibliographically approved
3. A simplified multidimensional approach for analysis of complex biological samples: on-line LC-CE-MS
Open this publication in new window or tab >>A simplified multidimensional approach for analysis of complex biological samples: on-line LC-CE-MS
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2006 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 131, no 7, p. 791-798Article in journal (Refereed) Published
Abstract [en]

Information on protein expression, disease biomarkers or surrogate markers and genetic disorders can nowadays be achieved from analysis of complex biological samples by liquid separation coupled to mass spectrometric (MS) detection. This paper describes fast multidimensional separation by on-line liquid chromatography (LC) and capillary electrophoresis (CE), followed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) MS detection. This detector provides ultrahigh resolution of the detected ions, mass accuracy at the ppm-level and high sensitivity. Most of the challenge of this system lies in the development of a new interface for the on-line coupling of LC to CE. The interface developed in poly(dimethylsiloxane) provides a RSD for injection repeatability of <3.5% and surface control for unspecific binding by deactivation with a cationic polymer, PolyE-323. We have evaluated the interface, as well as the overall system, with respect to robustness and deconvolution ability. Sequence coverage for bovine serum albumin (BSA) of 93% showed a high recovery of sample in the different transfer steps through the system. The detection limit for identification is 277 ng mL−1 (or 280 nM) on average for peptides. In the future, we expect LC-CE-MS to be a novel strategy for elucidating the chemistry of biological matrices.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-80932 (URN)10.1039/b601660j (DOI)
Available from: 2006-06-29 Created: 2006-06-29 Last updated: 2017-12-14Bibliographically approved
4. On-line coupling of a microelectrode array equipped poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip to electrospray mass spectrometry
Open this publication in new window or tab >>On-line coupling of a microelectrode array equipped poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip to electrospray mass spectrometry
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In: Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-93184 (URN)
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2011-03-21
5. Sheathless electrospray from polymer microchips
Open this publication in new window or tab >>Sheathless electrospray from polymer microchips
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2003 In: Analytical Chemistry, Vol. 75, no 15, p. 3934-3940Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-93185 (URN)
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2011-03-21
6. Capillary electrophoresis coupled to mass spectrometry from a polymer modified poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip
Open this publication in new window or tab >>Capillary electrophoresis coupled to mass spectrometry from a polymer modified poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip
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2005 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 130, no 2, p. 193-199Article in journal (Refereed) Published
Abstract [en]

Hybrid capillary-poly(dimethysiloxane) (PDMS) microchips with integrated electrospray ionization (ESI) tips were directly fabricated by casting PDMS in a mould. The shapes of the emitter tips were drilled into the mould, which produced highly reproducible three-dimensional tips. Due to the fabrication method of the microfluidic devices, no sealing was necessary and it was possible to produce a perfect channel modified by PolyE-323, an aliphatic polyamine coating agent. A variety of different coating procedures were also evaluated for the outside of the emitter tip. Dusting graphite on a thin unpolymerised PDMS layer followed by polymerisation was proven to be the most suitable procedure. The emitter tips showed excellent electrochemical properties and durabilities. The coating of the emitter was eventually passivated, but not lost, and could be regenerated by electrochemical means. The excellent electrochemical stability was further confirmed in long term electrospray experiments, in which the emitter sprayed continuously for more than 180 h. The PolyE-323 was found suitable for systems that integrate rigid fused silica and soft PDMS technology, since it simply could be applied successfully to both materials. The spray stability was confirmed from the recording of a total ion chromatogram in which the electrospray current exhibited a relative standard deviation of 3.9% for a 30 min run. CE-ESI-MS separations of peptides were carried out within 2 min using the hybrid PDMS chip resulting in similar efficiencies as for fused silica capillaries of the same length and thus with no measurable band broadening effects, originating from the PDMS emitter.

National Category
Analytical Chemistry Inorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-93084 (URN)10.1039/b414592e (DOI)15665973 (PubMedID)
Available from: 2005-05-03 Created: 2005-05-03 Last updated: 2017-12-14Bibliographically approved
7. Sample pretreatment on a microchip with an integrated electrospray emitter
Open this publication in new window or tab >>Sample pretreatment on a microchip with an integrated electrospray emitter
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2006 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 27, no 11, p. 2075-2082Article in journal (Refereed) Published
Keywords
Electrospray emitter, Microchip, PDMS, Sample pretreatment
National Category
Chemical Sciences Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-95129 (URN)10.1002/elps.200500763 (DOI)
Available from: 2006-11-17 Created: 2006-11-17 Last updated: 2017-12-14Bibliographically approved

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