| 1. |
- Stubberud, Karin, et al.
(författare)
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Partial filling micellar electrokinetic chromatography optimisation studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry : Part I
- 2002
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 23:4, s. 572-577
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Tidskriftsartikel (refereegranskat)abstract
- Studies have been performed to evaluate whether an on-line partial filling-micellar electrokinetic chromatography (PF-MEKC) system could be applied to a recently developed MEKC method for the separation of ibuprofen, codeine and one of the degradation products. Attempts to couple the PF-MEKC system to MS have also been performed. SDS concentration, micellar zone length and concentration of acetonitrile in the buffer were optimized using factorial design. When a small micelle zone was injected directly after the sample introduction, the results improved markedly. The MS parameters have not been optimized, but the studies show promising results for the use of PF-MEKC-mass spectrometry for identification of the degradation products.
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| 2. |
- Andersson, Helene, et al.
(författare)
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Micromachined filter-chamber array with passive valves for biochemical assays on beads
- 2001
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 22:2, s. 249-257
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Tidskriftsartikel (refereegranskat)abstract
- The filter-chamber array presented here enables a real-time parallel analysis of three different samples on beads in a volume of 3 nL, on a 1 cm(2) chip. The filter-chamber array is a system containing three filter-chambers, three passive valves at the inlet channels and a common outlet. The design enables parallel sample handling and time-controlled analysis. The device is microfabricated in silicon and sealed with a Pyrex lid to enable real-time analysis. Single nucleotide polymorphism analysis by using pyrose-quencing has successfully been performed in single filter-chamber devices. The passive valves consist of plasma-deposited octafluorocyclobutane and show a much higher resistance towards water and surface-active solutions than previous hydrophobic patches. The device is not sensitive to gas bubbles, clogging is rare and reversible, and the filter-chamber array is reusable. More complex (bio)chemical reactions on beads can be performed in the devices with passive valves than in the devices without valves.
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| 3. |
- Andersson, Helene, et al.
(författare)
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Patterned self-assembled beads in silicon channels
- 2001
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 22:18, s. 3876-3882
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Tidskriftsartikel (refereegranskat)abstract
- A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 mum wide and 50 mum deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 mum, has also been generated on the bottom of a 500 mum wide and 50 mum deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.
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| 4. |
- Curcio, M., et al.
(författare)
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Multiplex high-throughput solid-phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection
- 2002
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 23:10, s. 1467-1472
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Tidskriftsartikel (refereegranskat)abstract
- Minisequencing, solid-phase single-nucleotide primer extension reaction, is a robust method for performing multiplex single-nucleotide polymorphism (SNP) analysis. We have combined this technology with capillary gel electrophoresis in a multicapillary format, using liquid core waveguide (LCW) fluorescence detection. Polymerase chain reaction (PCR) amplification of multiple DNA targets is performed with one primer for each target biotinylated. Separation of the complementary strands, minisequencing and washing steps are carried out using streptavidin-coated magnetic beads. Dideoxynucleotides analogues labelled with different fluorophores are used for the extension of the minisequencing primers. The extended oligonucleotides, the length of which defines the position on the target and the color the identity of the polymorphism, are then separated in a gel-filled array of capillaries, coated on the outside with a layer of a fluoropolymer to provide the liquid core waveguide characteristics. The technology has a potential for extremely high throughputs when a combination of multiplex PCR-minisequencing is used together with a large array of capillaries, four-color detection and high-speed separation.
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| 5. |
- Emmer, Åsa, et al.
(författare)
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Wall deactivation with fluorosurfactants for capillary electrophoretic analysis of biomolecules
- 2001
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 22:4, s. 660-665
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes the use of fluorosurfactants as buffer additives for capillary electrophoretic separation of proteins and peptides. Due to fluorosurfactant bilayer formation at the capillary inner wall, the surface charge can be adjusted and even reversed. If the running buffer pH is kept at a level where the proteins have the same sign of charge as the wall, electrostatic repulsion will be obtained. The protein wall adsorption can therefore be reduced and the separation performance can be noticeably increased. The separation performance can also be further improved by including mixtures of different types of fluorosurfactants in the running buffer. The buffer system can accordingly be adapted for a certain separation problem. Mechanisms for the use of fluorosurfactants for wall deactivation in capillary electrophoretic protein separations is discussed in the present work and some examples of applications are also presented.
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| 6. |
- Hanning, A., et al.
(författare)
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A liquid core waveguide fluorescence detector for multicapillary electrophoresis applied to DNA sequencing in a 91-capillary array
- 2000
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 21:15, s. 3290-3304
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Tidskriftsartikel (refereegranskat)abstract
- A new laser-induced fluorescence (LIF) detector for multicapillary electrophoresis is presented. The detection principle is based on waveguiding of the emitted fluorescence from the point of illumination to the capillary ends by total internal reflection (TIR) and imaging of the capillary ends. The capillaries themselves thus act as liquid core waveguides (LCWs). At the illumination point, the capillaries are arranged in a planar array, which allows clean and efficient illumination with a line-focused laser beam. The capillary ends are rearranged into a small, densely packed two-dimensional array, which is imaged end-on with high light collection efficiency and excellent image quality. Wavelength dispersion is obtained with a single prism. Intercapillary optical crosstalk is less than 0.5%, and rejection of stray light is very efficient. The detector is applied to four-color DNA sequencing by gel electrophoresis in a 91-capillary array, with simple fluorescein and rhodamine dyes as fluorophores. Since the imaged two-dimensional array is so compact, the detector has a high potential for very large-scale multiplexing.
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| 7. |
- Larsson, Marita, et al.
(författare)
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Transient isotachophoresis for sensitivity enhancement in capillary electrophoresis-mass spectrometry for peptide analysis
- 2000
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Ingår i: Electrophoresis. - Weinheim : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 21:14, s. 2859-2865
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Tidskriftsartikel (refereegranskat)abstract
- Transient isotachophoresic (ITP) focusing was used for the on-line analysis of peptides by capillary zone electrophoresis-mass spectrometry (CZE-MS), allowing injection volumes of up to 0.9 microL. A sheath liquid electrospray interface was used with a single quadrupole mass analyzer. First, the technique was applied to the qualitative analysis of a tryptic digest of cytochrome c, resulting in low-background, high-quality spectra. Second, the linear range was investigated by selected ion monitoring (SIM) for a peptidomimetic direct thrombin inhibitor melagatran (Mr 429.5) and two endogenous peptides, substance P (Mr 1348) and calcitonin gene-related peptide (alpha-CGRP; Mr 3806).
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| 8. |
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| 9. |
- Nilsson, H., et al.
(författare)
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Microparticles for selective protein determination in capillary electrophoresis
- 2001
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 22:12, s. 2384-2390
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Tidskriftsartikel (refereegranskat)abstract
- A system for detection of trace amounts of protein was developed. Two different monoclonal antibodies against human chorionic gonadotropin (hCG) were covalently bound to latex particles. When the latex particles were mixed with a sample containing hCG, a latex-protein-latex complex (immunocomplex) was formed. The complex was separated from the single latex particles using capillary electrophoresis and detected using UV-Vis detection. Limit of detection was 8 amol hCG. The separation was also monitored in real time using laser induced fluorescence - charge coupled device (LIF-CCD) imaging detection. However, a limitation of the method is the restriction to detection of proteins for which monoclonal antibodies are available.
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| 10. |
- Seidl, Rainer, et al.
(författare)
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Deficient brain snRNP70K in patients with Down syndrome
- 2001
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Ingår i: Electrophoresis. - : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 22:1, s. 43-48
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Tidskriftsartikel (refereegranskat)abstract
- The small nuclear ribonucleoprotein 70K (snRNP 70K; U1-70 kDa) is an integral part of the spliceosome, a large RNA-protein complex catalyzing the removal of introns from nuclear pre-mRNA. snRNP is one of the best-studied essential subunits of snRNPs, is highly conserved and its inactivation was shown to result in complete inhibition of splicing. Applying subtractive hybridization, we found a sequence with 100% identity to snRNP absent in fetal Down syndrome (DS) brain. This observation made us determine snRNP-mRNA steady-state levels and protein levels in brains of adult patients with DS. snRNP-mRNA and protein levels of five individual brain regions of DS and controls each, were determined by blotting techniques. snRNP-mRNA steady state levels were significantly decreased in DS brain. Performing Western blots with monoclonal and human antibodies, snRNP protein levels were decreased in several regions of DS brain, although one monoclonal antibody did not reveal different snRNP-immunoreactivity. Although decreased snRNP-protein could be explained by decreased mRNA-steady state levels, another underlying mechanism might be suggested: snRNP is one of the death substrates rapidly cleaved during apoptosis by interleukin-1-beta-converting enzyme-like (ICE) proteases, which was well-documented by several groups. As apoptosis is unrequivocally taking place in DS brain leading to permanent cell loses, decreased snRNP-protein levels may therefore reflect decreased synthesis and increased apoptosis-related proteolytic cleavage.
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